| 2021 | Effect of Initial Aflibercept, Laser, or Observation on Low-Contrast Visual Acuity in Eyes With Diabetic Macular Edema and Good Vision: Ancillary Study Within a Randomized Clinical Trial. | Transl Vis Sci Technol | To describe 2.5% low-contrast visual acuity (VA) among eyes with good vision despite center-involved diabetic macular edema and compare changes after initial management with aflibercept, laser, or observation.This was an ancillary study within a multicenter randomized clinical trial (DRCR Retina Network Protocol V). Participants had diabetes and 1 study eye with center-involved diabetic macular edema and a VA of 20/25 or better randomly assigned to aflibercept (n = 112), focal/grid laser (n = 146), or observation (n = 129). Eyes in the laser and observation groups received aflibercept if VA met prespecified worsening criteria.Participants had median age of 60 years, 37% were female and 70% were non-Hispanic White. At baseline, the mean ± standard deviation (SD) high-contrast VA was 85.2 ± 3.6 letters (Snellen equivalent 20/20), mean ± SD 2.5% low-contrast VA was 47.6 ± 18.9 letters (Snellen equivalent 20/125), and low-contrast VA letter score was 2 SDs or more below the age-specific normative values in 23%. At 2 years, the mean change ± SD in low-contrast VA in the aflibercept, laser, and observation groups was 2.7 ± 20.1, -2.0 ± 19.6, and -3.1 ± 20.8 letters (adjusted difference, aflibercept vs. laser, 5.3 [95% confidence interval, -0.2 to 10.8], P = 0.06; aflibercept vs. observation, 5.5 [95% confidence interval -0.2 to 11.2], P = 0.06; and laser vs. observation, 0.2 [95% confidence interval -4.6 to 5.0], P = 0.94).There was no significant difference between treatment groups in low-contrast VA change from baseline to 2 years. Considering the range of the 95% confidence intervals, however, the study may have been underpowered to detect a clinically meaningful benefit between treatment groups.Low-contrast VA, an important visual function, is decreased in eyes with diabetic macular edema. |
| 2021 | Prospective Case-Control Study of Cardiovascular Abnormalities 6 Months Following Mild COVID-19 in Healthcare Workers. | JACC Cardiovasc Imaging | The purpose of this study was to detect cardiovascular changes after mild severe acute respiratory syndrome coronavirus 2 infection.Concern exists that mild coronavirus disease 2019 may cause myocardial and vascular disease.Participants were recruited from COVIDsortium, a 3-hospital prospective study of 731 health care workers who underwent first-wave weekly symptom, polymerase chain reaction, and serology assessment over 4 months, with seroconversion in 21.5% (n = 157). At 6 months post-infection, 74 seropositive and 75 age-, sex-, and ethnicity-matched seronegative control subjects were recruited for cardiovascular phenotyping (comprehensive phantom-calibrated cardiovascular magnetic resonance and blood biomarkers). Analysis was blinded, using objective artificial intelligence analytics where available.A total of 149 subjects (mean age 37 years, range 18 to 63 years, 58% women) were recruited. Seropositive infections had been mild with case definition, noncase definition, and asymptomatic disease in 45 (61%), 18 (24%), and 11 (15%), respectively, with 1 person hospitalized (for 2 days). Between seropositive and seronegative groups, there were no differences in cardiac structure (left ventricular volumes, mass, atrial area), function (ejection fraction, global longitudinal shortening, aortic distensibility), tissue characterization (T, T, extracellular volume fraction mapping, late gadolinium enhancement) or biomarkers (troponin, N-terminal pro-B-type natriuretic peptide). With abnormal defined by the 75 seronegatives (2 SDs from mean, e.g., ejection fraction <54%, septal T >1,072 ms, septal T >52.4 ms), individuals had abnormalities including reduced ejection fraction (n = 2, minimum 50%), T elevation (n = 6), T elevation (n = 9), late gadolinium enhancement (n = 13, median 1%, max 5% of myocardium), biomarker elevation (borderline troponin elevation in 4; all N-terminal pro-B-type natriuretic peptide normal). These were distributed equally between seropositive and seronegative individuals.Cardiovascular abnormalities are no more common in seropositive versus seronegative otherwise healthy, workforce representative individuals 6 months post-mild severe acute respiratory syndrome coronavirus 2 infection. |
| 2021 | Comparison of Macular Thickness Measurements Using Swept-Source and Spectral-Domain Optical Coherence Tomography in Healthy and Diabetic Subjects. | Curr Eye Res | : To establish normative data for macular thickness in Chinese aged 30 to 80 years using the swept-source optical coherence tomography (SS-OCT) device.: The study included 290 normal eyes, 430 NDR eyes and 150 DR eyes of community residents aged 30 to 80 years in Guangzhou, China. Mean macular thicknesses in Early Treatment Diabetic Retinopathy Study (ETDRS) subfields, central point thicknesses (CPT), and macular volume was measured by SS-OCT (Triton DRI OCT, Topcon, Tokyo, Japan) and Spectral-Domain OCT (SD-OCT; Heidelberg Engineering, Heidelberg, Germany). We assessed agreement between SS-OCT and SD-OCT measurements by intraclass correlation coefficients (ICC) and Bland-Altman plots. We established a conversion equation relating central subfield (CSF), CPT and macular volume between the two OCT devices.: Macular thickness measurements in SS-OCT were significantly thinner than in SD-OCT. The mean CSF thickness in normal eyes measured by SS-OCT and SD-OCT were 227.8 ± 19.4 μm and 260.0 ± 19.7 μm ( < .0001). CSF thickness was a significantly difference between genders (SS-OCT: male 237.2 ± 18.8 μm vs female 222.0 ± 17.5 μm, < .0001). In all three groups, the agreement between SS-OCT and SD-OCT was excellent (all ICC > 0.9). The conversion equations for CSF, CPT and macular volume from SS-OCT to SD-OCT were derived, with over 95% of the predicted values fell within 10% of the actual measurements in DR and NDR eyes.: We propose SS-OCT CSF thicknesses of 275 μm for males and 260 μm for females as the minimum criteria for macular edema in Chinese aged 30 to 80 years based on 2 SDs above the mean CSF. SS-OCT measurements were significantly thinner than SD-OCT. We derived equations from converting SS-OCT measurements to SD-OCT equivalents. |
| 2021 | Safety and effectiveness of replacement with biosimilar growth hormone in adults with growth hormone deficiency: results from an international, post-marketing surveillance study (PATRO Adults). | Pituitary | To evaluate safety and effectiveness of biosimilar recombinant human growth hormone (rhGH; Omnitrope®) in adults with growth hormone deficiency (GHD), using data from the PATRO Adults study.PATRO Adults was a post-marketing surveillance study conducted in hospitals and specialized endocrinology units across Europe. The primary objective was to assess the safety of rhGH in adults treated in routine clinical practice. All adverse events (AEs) were monitored and recorded for the complete duration of Omnitrope® treatment. Effectiveness was evaluated as a secondary objective.As of January 2020, 1447 patients (50.9% male) had been enrolled from 82 centers in 9 European countries. Most patients had adult-onset GHD (n = 1179; 81.5%); 721 (49.8%) were rhGH-naïve at study entry. Overall, 1056 patients (73.0%) reported adverse events (AEs; n = 5397 events); the majority were mild-to-moderate in intensity. Treatment-related AEs were reported in 117 patients (8.1%; n = 189 events); the most commonly reported (MedDRA preferred terms) were arthralgia (n = 19), myalgia (n = 16), headache (n = 14), and edema peripheral (n = 10). In total, 495 patients (34.2%) had serious AEs (SAEs; n = 1131 events); these were considered treatment-related in 28 patients (1.9%; n = 35 events). Mean (standard deviation) IGF-I SDS increased from - 2.34 (1.47) at baseline to - 0.23 (1.65) at 12 months, and remained relatively stable thereafter (up to 3 years). Body mass index remained stable between baseline and 3 years.Data from PATRO Adults indicate biosimilar rhGH (Omnitrope) is not associated with any unexpected safety signals, and is effective in adults with GHD treated in real-world clinical practice. |
| 2021 | Comparison of porcine corneal decellularization methods and importance of preserving corneal limbus through decellularization. | PLoS One | The aim of this study is to compare the three previously applied, conventional porcine corneal decellularization methods and to demonstrate the importance of preserving the corneal limbus through decellularization.Fresh, wild-type (with or without) limbus porcine corneas were decellularized using three different methods, including (i) sodium dodecyl sulfate (SDS), (ii) hypertonic saline (HS), and (iii) N2 gas (NG). Post-treatment evaluation was carried out using histological, residual nuclear material, and ultrastructural analyses. Glycerol was used to help reduce the adverse effects of decellularization. The corneas were preserved for two weeks in cornea storage medium.All three decellularization methods reduced the number of keratocytes at different rates in the stromal tissue. However, all methods, except SDS, resulted in the retention of large numbers of cells and cell fragments. The SDS method (0.1% SDS, 48h) resulted in almost 100% decellularization in corneas without limbus. Low decellularization capacity of the NG method (<50%) could make it unfavorable. Although HS method had a more balanced damage-decellularization ratio, its decellularization capacity was lower than SDS method. Preservation of the corneoscleral limbus could partially prevent structural damage and edema, but it would reduce the decellularization capacity.Our results suggest that SDS is a very powerful decellularization method, but it damages the cornea irreversibly. Preserving the corneoscleral limbus reduces the efficiency of decellularization, but also reduces the damage. |
| 2020 | Flaxseed Cysteine Protease Exhibits Strong Anticoagulant, Antiplatelet, and Clot-Dissolving Properties. | Biochemistry (Mosc) | In this study, we purified and characterized flaxseed cysteine protease (FSCP) with strong anticoagulant, antiplatelet, and clot-dissolving properties. The enzyme was purified to homogeneity by a combination of gel permeation and ion-exchange column chromatography techniques. The purity of the enzyme was evaluated by SDS-PAGE, RP-HPLC, and MALDI-TOF. FSCP was observed as a single band of approximately 160 kDa in SDS-PAGE under reducing and non-reducing conditions. The exact molecular mass of FSCP was found to be 168 kDa by MALDI-TOF spectrometry. The CD spectra of FSCP revealed the presence of 25.6% helices, 25.8% turns, and 48% random coils with no beta-sheet structures. FSCP hydrolyzed both casein and gelatin with a specific activity of 3.5 and 4.2 unit/mg min respectively. The proteolytic activity of FSCP was completely abolished by iodoacetic acid (IAA), suggesting FSCP is a cysteine protease. The pH optimum for the proteolytic activity of FSCP was pH 6.0; the temperature optimum was 30°C. FSCP exhibited strong anticoagulant effect in both platelet-rich plasma (PRP) and platelet-poor plasma (PPP) by extending the clotting time from 222 to 1100 s and from 256 to 1210 s, respectively. FSCP degraded human fibrinogen and fibrin clots. The products of fibrinogen degradation by thrombin and FSCP were different. Furthermore, FSCP inhibited aggregation of washed platelets triggered by ADP, epinephrine, thrombin, collagen, arachidonic acid, and platelet activating factor (PAF). FSCP was found to be nontoxic as it did not damage the membrane of red blood cells (RBCs) and did not induce hemorrhage and edema in experimental mice. |
| 2020 | Preventing neuronal edema increases network excitability after traumatic brain injury. | J Clin Invest | Edema is an important target for clinical intervention after traumatic brain injury (TBI). We used in vivo cellular resolution imaging and electrophysiological recording to examine the ionic mechanisms underlying neuronal edema and their effects on neuronal and network excitability after controlled cortical impact (CCI) in mice. Unexpectedly, we found that neuronal edema 48 hours after CCI was associated with reduced cellular and network excitability, concurrent with an increase in the expression ratio of the cation-chloride cotransporters (CCCs) NKCC1 and KCC2. Treatment with the CCC blocker bumetanide prevented neuronal swelling via a reversal in the NKCC1/KCC2 expression ratio, identifying altered chloride flux as the mechanism of neuronal edema. Importantly, bumetanide treatment was associated with increased neuronal and network excitability after injury, including increased susceptibility to spreading depolarizations (SDs) and seizures, known agents of clinical worsening after TBI. Treatment with mannitol, a first-line edema treatment in clinical practice, was also associated with increased susceptibility to SDs and seizures after CCI, showing that neuronal volume reduction, regardless of mechanism, was associated with an excitability increase. Finally, we observed an increase in excitability when neuronal edema normalized by 1 week after CCI. We conclude that neuronal swelling may exert protective effects against damaging excitability in the aftermath of TBI and that treatment of edema has the potential to reverse these effects. |
| 2020 | Purification and characterization of non-enzymatic glycoprotein (NEGp) from flax seed buffer extract that exhibits anticoagulant and antiplatelet activity. | Int J Biol Macromol | The current study deals with the purification and characterization of non-enzymatic glycoprotein (NEGp) from flax seed buffer extract. Sephadex G-100 and DEAE-A25 column chromatography techniques were employed to isolate NEGp. NEGp showed single sharp band at 29 kDa region on 10% SDS-PAGE, and under reduced and non-reduced conditions revealed its monomeric nature. Besides, NEGp taken up the PAS stain at 29 kDa region reveals the presence of carbohydrate moiety. Purity of NEGp was adjudged by RP-HPLC, as it revealed a single sharp peak at the retention time of 3.4 min. The exact molecular mass of NEGp was found to be 26 kDa which was confirmed by MALDI-TOF. Circular di-chromism spectra of NEGp showed 12.0% α-helix, 24.3% α-helix turn and 63.7% random coils without beta pleated sheets. NEGp was found to exhibit anticoagulant activity by extending clotting time of both platelet rich plasma and platelet poor plasma from control 240 s to 1800 s and 280 s to 2100 s respectively at the concentration of 8 μg. NEGp inhibited the agonists such as ADP, epinephrine and arachidonic acid induced platelet aggregation in washed platelets. The percentage of inhibition was found to be 70%, 80% and 60% respectively. While, it did not interfere in thrombin, PAF and collagen induced platelet aggregation. NEGp did not hydrolyse RBC membrane, devoid of haemorrhagic and edema inducing properties in experimental mice. |
| 2020 | Paraphenylenediamine (Kala Pathar) Poisoning at the National Poison Control Center in Karachi: A Prospective Study. | Cureus | Introduction Suicide by self-poisoning is a common cause of death, especially in the younger population. More specifically, hair-dye poisoning is being increasingly used for suicide. Paraphenylenediamine (PPD), also known as "Kala pathar", is a highly toxic ingredient present in hair-dye that can cause death. Therefore, this study is designed to assess the demographics, clinical features, laboratory findings, and outcomes of PPD poisoning in patients admitted to the National Poison Control Center in Karachi, Pakistan. Materials and methods We conducted a prospective study for a period of six months at the National Poison Control Center, Karachi, Pakistan. A total of eight patients with PPD poisoning with no cardiac, liver, or renal co-morbidities were included in this study. The demographic characteristics, clinical features, laboratory findings, mode of intoxication, and route of intoxication were noted in a proforma. Furthermore, hospitalization time, tracheostomy status, mechanical ventilation status, and mortality rates were also recorded. For continuous variables, the means and SDs were calculated. Whereas for categorical data, percentages were calculated. Results In our study, the mean age of the patients was estimated at 25.38 ± 3.77 years. It was deemed that the majority of poisoning cases were intentional in nature (75%). These suicide cases were more commonly observed in young females (75%) who belonged to a low socioeconomic class (87.5%). The preferred route of administration was oral (87.5%). In 87.5% of the patients, the characteristic clinical features such as cervicofacial edema, dysphagia, dysphonia, and stridor were noted. During the later clinical stages of poisoning, clinical features such as rhabdomyolysis (62.5%), chocolate-colored urine (87.5%), hepatitis (75%), and acute renal failure (12.5%) were noteworthy. The mean ± SD of total leukocyte count (TLC), creatine phosphokinase (CPK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), serum creatinine and serum potassium were, respectively, noted at 10,500 ± 3,854.4 cells/mm, 32.87 ± 11.36 IU/L, 1,239.1 ± 1,106.2 IU/L, 776.8 ± 1,149.8 IU/L, 2.125 ± 2.275 mg/dL, and 4.9 ± 1.094 mmol/L. In our patients, the mean intensive care unit stay was 8.25 ± 3.99 days. Emergency tracheostomy was performed in 25% of patients. Mechanical ventilation was required for 50% of our patients. Overall, the mortality rate observed in our study stands at 25%. Conclusion PPD poisoning is associated with a high rate of morbidity and mortality. Therefore, it is imperative for physicians to be mindful of the clinical characteristics and treatment options in order to optimally manage such cases of poisoning. In addition, the use of hair-dyes composed of highly lethal PPD should also be banned. |
| 2020 | Rapid Neuronal Ultrastructure Disruption and Recovery during Spreading Depolarization-Induced Cytotoxic Edema. | Cereb Cortex | Two major pathogenic events that cause acute brain damage during neurologic emergencies of stroke, head trauma, and cardiac arrest are spreading depolarizing waves and the associated brain edema that course across the cortex injuring brain cells. Virtually nothing is known about how spreading depolarization (SD)-induced cytotoxic edema evolves at the ultrastructural level immediately after insult and during recovery. In vivo 2-photon imaging followed by quantitative serial section electron microscopy was used to assess synaptic circuit integrity in the neocortex of urethane-anesthetized male and female mice during and after SD evoked by transient bilateral common carotid artery occlusion. SD triggered a rapid fragmentation of dendritic mitochondria. A large increase in the density of synapses on swollen dendritic shafts implies that some dendritic spines were overwhelmed by swelling or merely retracted. The overall synaptic density was unchanged. The postsynaptic dendritic membranes remained attached to axonal boutons, providing a structural basis for the recovery of synaptic circuits. Upon immediate reperfusion, cytotoxic edema mainly subsides as affirmed by a recovery of dendritic ultrastructure. Dendritic recuperation from swelling and reversibility of mitochondrial fragmentation suggests that neurointensive care to improve tissue perfusion should be paralleled by treatments targeting mitochondrial recovery and minimizing the occurrence of SDs. |
| 2020 | Matrix Metalloproteases and Cathepsin D in Human Serum do not Cleave Prolactin to Generate Vasoinhibin. | Clin Lab | Vasoinhibin is generated in the pituitary gland and in multiple target tissues by proteolytic cleavage of prolactin by matrix metalloproteinases and cathepsin D. A dysregulation of vasoinhibin generation appears to contribute to diabetic retinopathy and diabetic macular edema, retinopathy of prematurity, peripartum cardiomyopathy, and preeclampsia. Here, we investigate whether vasoinhibin is generated by matrix metalloproteinases and cathepsin D in human serum.The abundance of matrix metalloproteinases 1, 2, 3, 8, 9, 10, 13, tissue inhibitors of metalloproteinases 1, 2, 4, and the activity of cathepsin D in serum samples were determined. Samples from healthy male (n = 3) and female (n = 2) subjects, pregnant subjects (n = 2), and patients with type 2 diabetes mellitus (n = 2) were investigated. The samples were incubated with recombinant prolactin at 37°C, under different pH, time, and buffer conditions. Prolactin and cleaved prolactin products were investigated by SDS-PAGE and western blotting.Matrix metalloproteases-1, -2, -3, -8, -9, -10, -13, TIMP-1, -2, and -4, and the activity of cathepsin D were detected in all sera. Full-length prolactin incubated with human sera, containing endogenous matrix metalloproteinases and cathepsin D, remained intact at neutral pH during a time frame from 1 to 24 hours. Partial enzymatic cleavage of prolactin resulting in the generation of a vasoinhibin-like 17 kDa peptide was observed in samples incubated at pH 3.4. Heat inactivation of the serum and the addition of an MMP inhibitor suppressed the generation of the 17 kDa peptide, indicating that its generation was MMP-mediated.Vasoinhibin generation by enzymatic cleavage of prolactin by matrix metalloproteases or cathepsin D does not occur in human serum at physiological pH. A limited proteolysis of prolactin, resulting in the generation of a vasoinhibin-like peptide with an apparent molecular weight of 17 kDa occurs in serum at acidic pH. The generation of vasoinhibin may require the cellular and tissue microenvironments. |
| Pitanguy Ligamentous Flap: A New Method to Prevent Supratip Deformity in Rhinoplasty. | J Craniofac Surg | A supratip deformity (SD) is an iatrogenic convexity that occurs in the cephalic region of the nasal tip. SD is still a major problem after rhinoplasty surgery.With the method we have described a ligamentous flap was used to create a supratip transition, with adjustable sharpness, while the refinements of the tip rotation and definition were ensured. The aim of the study is to present the results of this technique, which, to the best of our knowledge, has been described here for the first time.Our ligamentous flap technique was applied to 24 patients between August 2017 and March 2018. All of the patients were evaluated in terms of the formation of an SD, a hanging columella, tip projection, and the loss of rotation at the postoperative followups. The photos of patients were evaluated by another independent plastic surgeon and patients themselves at 3 months after the surgery.There were no early or late complications, such as an infection, excessive bleeding, or prolonged edema. Moreover, SDs, hanging columellas, tip projections, and rotational losses, which would require revisions, were not detected in any of the patients. Postoperative scores given by the patients and surgeons were significantly higher than the preoperative values (P < 0.05). Only 2 patients required minor revisions due to dorsal irregularities in the upper 1/3 of the nasal segment.The early results of this Pitanguy composite flap technique, which can be easily applied in every case with thin or thick skin in an open rhinoplasty, are promising. However, there is a need for an evaluation of the long-term results, as well as the advantages and disadvantages in a larger case series.Level IV. |
| 2020 | Effect of evidence-based nursing intervention on upper limb function in postoperative radiotherapy patients with breast cancer. | Medicine (Baltimore) | To investigate the effect of evidence-based nursing (EBN) intervention on upper limb function in postoperative breast cancer patients undergoing radiotherapy.A total of 126 breast cancer patients who had received postoperative radiotherapy in the Union Hospital affiliated with Tongji Medical College, Huazhong University of Science and Technology from September 2017 to September 2018 were randomly divided into 2 groups, namely, experimental and control groups, with 63 cases in each group. Both the control and experimental groups received routine postoperative radiotherapy followed by traditional and EBN interventions, respectively. All patients were followed up for 6 months and differences in the upper limb function after nursing intervention were compared between the 2 groups.The scores of self-rating anxiety scale (SAS), self-rating depression scale (SDS), and short form-36 survey (SF-36) in the 2 groups had no statistical significance before intervention. After the EBN intervention, the SAS and self-rating depression scale scores of patients in the experimental group were lower than that of those in the control group. In the experimental group, 90.67% of the patients had an excellent score for the University of California, Los Angeles shoulder score, which was higher than that of the control group (73.92%). The Mayo Elbow Performance Score of the experimental group (95.01) was higher than that of the control group (91.33). The total length of the sum of arm circumference in the experimental group was (128.39 cm) lower than that of the control group (143.66 cm). The scores of SF-36 in the overall health, physical pain, mental health, and physiological functions of the patients in the experimental group were higher than those of the control group. All of these parameters' differences between the 2 groups were of statistical significance (P < .05).EBN can positively influence the negative emotional state of breast cancer patients after radiotherapy. At the same time, it is helpful in reducing the degree of lymph node edema on the affected side of the upper limb, thereby improving the function of the shoulder joint, which has a positive effect on the upper limb function. |
| 2019 | Impact Effect of Methyl Tertiary-Butyl Ether "Twelve Months Vapor Inhalation Study in Rats". | Biology (Basel) | We investigated the early risk of developing cancer by inhalation of low doses (60 µL/day) of methyl tertiary butyl ether (MTBE) vapors using protein SDS-PAGE and LC-MS/MS analysis of rat sera. Furthermore, histological alterations were assessed in the trachea and lungs of 60 adult male Wistar rats. SDS-PAGE of blood sera showed three protein bands corresponding to 29, 28, and 21 kDa. Mass spectroscopy was used to identify these three bands. The upper and middle protein bands showed homology to carbonic anhydrase 2 (CA II), whereas the lower protein band showed homology with peroxiredoxin 2. We found that exposure to MTBE resulted in histopathological alterations in the trachea and the lungs. The histological anomalies of trachea and lung showed that the lumen of trachea, bronchi, and air alveoli packed with free and necrotic epithelial cells (epithelialization). The tracheal lamina propria of lung demonstrated aggregation of lymphoid cells, lymphoid hyperplasia, hemorrhage, adenomas, fibroid degeneration, steatosis, foam cells, severe inflammatory cells with monocytic infiltration, edema, hemorrhage. Occluded, congested, and hypertrophied lung arteries in addition, degenerated thyroid follicles, were observed. The hyaline cartilage displayed degeneration, deformation, and abnormal protrusion. In conclusion, our results suggest that inhalation of very low concentrations of the gasoline additive MTBE could induce an increase in protein levels and resulted in histopathological alterations of the trachea and the lungs. |
| 2019 | Evaluation of immune response to recombinant LFD1-PA4 chimeric protein. | Iran J Vet Res | Anthrax is a particularly dangerous infectious disease that affects humans and livestock. Efficacious vaccines that can rapidly induce a long-term immune response are required to prevent anthrax infection in humans. Domains 4 and 1 of the protective antigen (PA) and lethal factor (LF), respectively, have very high antigenic properties.In this experimental study, the pET28a-- expression vector was designed, constructed and transferred into BL21 (DE3) plysS.For this purpose, gene was amplified by p XbaI HindIII E. coli Expression and purification of chimeric proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting techniques. The chimera LFD1-PA4 and mixed LFD1+PA4 proteins were injected four times into mice and antibody production was relativity evaluated by - (ELISA) test.The results showed that both chimeric and mixed proteins are immunogenic, but LFD1-PA4 has a higher potential to stimulate mice immune system.LFD1-PA4 chimeric protein induced a higher immune response than LFD1+PA4 mixed protein and elicited antibody responses to LF and edema factor (EF), therefore, it holds promise to be a more effective trivalent vaccine candidate to use in anthrax prevention. |
| 2019 | Local and systemic effects caused by Crotalus durissus terrificus, Crotalus durissus collilineatus, and Crotalus durissus cascavella snake venoms in swiss mice. | Rev Soc Bras Med Trop | Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation.Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated.The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP.Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms. |
| 2020 | Spreading Depolarization during the Acute Stage of Experimental Subarachnoid Hemorrhage in Mice. | Acta Neurochir Suppl | Spreading depolarization (SD) has been suggested as a pathomechanism for delayed cerebral ischemia after subarachnoid hemorrhage (SAH). However, the role of SD during the acute phase of SAH is still unclear. The objective of this study was to investigate (a) the occurrence of SD with intrinsic optical signal (IOS) imaging, (b) the effect of ketamine on SD, and (c) the resulting brain edema (brain water content (BWC)) during the acute stage of experimental SAH in mice. SAH was elicited by the endovascular filament perforation method. After SAH or sham operation, ketamine or saline, 30 mg/kg, was given every half hour. Changes in tissue light reflectance were recorded with IOS. BWC was measured during the acute stage. Overall, 199 SDs occurred in SAH groups and 33 SDs appeared in sham groups. These SDs displayed distinct originating and spreading patterns. Compared with saline, ketamine decreased SD spread and influenced the amplitude, duration, and speed of SD. However, the occurrence of SD was not prevented by ketamine. Moreover, ketamine did not reduce BWC after SAH. These results demonstrate that SD occurs with a high incidence during the acute stage of SAH. SDs are heterogeneous in incidence, origination, and propagation. It remains unclear whether ketamine effects on SD may be viewed as therapeutically beneficial after SAH. |
| 2019 | Construction and characterization of an infectious cDNA clone of coxsackievirus A 10. | Virol J | Coxsackievirus A10 (CA10) constitutes one of the four major pathogens causing hand, foot and mouth disease in infants. Infectious clones are of great importance for studying viral gene functions and pathogenic mechanism. However, there is no report on the construction of CA10 infectious clones.The whole genome of CA10 derived from a clinical isolate was amplified into two fragments and ligated into a linearized plasmid vector in one step by In-Fusion Cloning. The obtained CA10 cDNA clones and plasmids encoding T7 RNA polymerase were co-transfected into 293 T cells to rescue CA10 virus. The rescued virus was identified by SDS-PAGE, Western blotting and transmission electron microscopic. One-day-old ICR mice were intracerebrally inoculated with the CA10 virus and clinical symptoms were observed. Multiple tissues of moribund mice were harvested for analysis of pathogenic changes and viral distribution by using H&E staining, real-time PCR and immunohistochemical staining.CA10 viruses were rescued from the constructed cDNA clone and reached a maximum titer of 10TCID/mL after one generation in RD cells. The virus exhibited similar physical and chemical properties to those of the parental virus. It also showed high virulence and the ability to induce death of neonatal ICR mice. Severe necrotizing myositis, intestinal villus interstitial edema and severe alveolar shrinkage were observed in infected mice. The viral antigen and the maximum amount of viral RNA were detected in limb skeletal muscles, which suggested that the limb skeletal muscles were the most likely site of viral replication.Infectious clones of CA10 were successfully constructed for the first time, which will facilitate the establishment of standardized neonatal mouse models infected with CA10 for the evaluation of vaccines and antiviral drugs, as well as preservation and sharing of model strains. |
| 2019 | Chemical profile and toxicity evaluation of unripe essential oil. | Toxicol Rep | (Christm.) Swingle (syn. C. MEDICA var. ACIDA Brandis) (family: Rutaceae) essential oil is one of the cheapest oils found in local markets. Although, it is generally accepted as non-toxic to vital organs and cells, majority of people are cynical about it usage. Herein, the present study reports the chemical composition and oral toxicity study of unripe essential oil found in Ghana. The toxicity of essential oil extract was investigated oral administration using two methods: The acute toxicity single dose study (SDS) and the repeated dose method. The oil exhibited no acute toxicity but in the sub-chronic studies, the effects was dose and time-dependent. Chemical profile investigation of the oil showed 9 constituent of phytochemicals (Germacrene isomers (61.2%), Pineen (14%), Linalool dimmer (2.9%), Bornane (11%), Citral (2.9%), Anethole (1.5%), Anisole (1.1%), Safrole (0.3%) and Demitol (0.6%)). Histopathological studies revealed conditions such as necrosis, edema and inflammatory reaction in the liver, spleen and kidneys. Marginal upsurge of biochemical parameters above normal and elevated levels of lymphocytes (35.20-46.40 g/dL) demonstrated mild toxicity among the 100 mg/kg and 500 mg/kg dose groups at the sub-chronic stage. Low levels of hemoglobin (13.60 to 12.70 g/dL), MCV (34.20-24.0 fL), MCH (40.20-36.40 g/dL) along with high levels of liver enzymes confirmed the mild toxicity of the oil at sub-chronic stage. These results demonstrate that, despite consideration of lime essential oil as safe, it can have mild hematotoxic, nephrotoxic and hepatotoxic effects. |
| 2019 | Biochemical and molecular characterization of the hyaluronidase from Bothrops atrox Peruvian snake venom. | Biochimie | Snake venoms are a rich source of enzymes such as metalloproteinases, serine proteinases phospholipases A2 and myotoxins, that have been well characterized structurally and functionally. However, hyaluronidases (E.C.3.2.1.35) have not been studied extensively. In this study, we describe the biochemical and molecular features of a hyaluronidase (Hyal-Ba) isolated from the venom of the Peruvian snake Bothrops atrox. Hyal-Ba was purified by a combination of ion-exchange and gel filtration chromatography. Purified Hyal-Ba is a 69-kDa (SDS-PAGE) monomeric glycoprotein with an N-terminal amino acid sequence sharing high identity with homologous snake venom hyaluronidases. Detected associated carbohydrates were hexoses (16.38%), hexosamines (2.7%) and sialic acid (0.69%). Hyal-Ba selectively hydrolyzed only hyaluronic acid (HA; specific activity = 437.5 U/mg) but it did not hydrolyze chondroitin sulfate or heparin. The optimal pH and temperature for maximum activity were 6.0 and 40 °C, respectively, and its Km was 0.31 μM. Its activity was inhibited by EDTA, iodoacetate, 2-mercaptoethanol, TLCK and dexamethasone. Na and K (0.2 M) positively affect hyaluronidase activity; while Mg, Br, Ba, Cu, Zn, and Cd reduced catalytic activity. Hyal-Ba potentiates the hemorrhagic and hemolytic activity of whole venom, but decreased subplantar edema caused by an l-amino acid oxidase (LAAO). The Hyal-Ba cDNA sequence (2020 bp) encodes 449 amino acid residues, including the catalytic site residues (Glu135, Asp133, Tyr206, Tyr253 and Trp328) and three functional motifs for N-linked glycosylation, which are conserved with other snake hyaluronidases. Spatial modeling of Hyal-Ba displayed a TIM-Barrel (α/β) fold and an EGF-like domain in the C-terminal portion. The phylogenetic analysis of Hyal-Ba with other homologous Hyals showed the monophyly of viperids. Further, Hyal-Ba studies may extend our knowledge of B. atrox toxinology and provides insight to improve the neutralizing strategies of therapeutic antivenoms. |
| 2019 | Budesonide nasal irrigation improved Lund-Kennedy endoscopic score of chronic rhinosinusitis patients after endoscopic sinus surgery. | Eur Arch Otorhinolaryngol | Budesonide improves the prognosis of chronic rhinosinusitis (CRS). However, few reports have examined whether its use for nasal irrigation, compared to normal saline, improves the prognosis of patients after endoscopic sinus surgery (ESS). We compared the effects of nasal irrigation with budesonide and normal saline in CRS patients after ESS.Sixty CRS patients who had undergone ESS were randomly divided into an experimental group (30 patients), which used budesonide nasal irrigation, and a control group (30 patients), which used normal saline nasal irrigation. All patients received regular follow-up evaluations and were assessed via questionnaires, including the Lund-Kennedy endoscopic score (LKES), the symptom visual analog scale (VAS), the 22-item Sino-Nasal Outcome Test (SNOT-22), the Short-Form 36-Item Questionnaire (SF-36), the Self-Rating Anxiety Scale (SAS), the Self-Rating Depression Scale (SDS) and a side effects scale.Scores of polyposis, mucosal edema, secretions and total score of LKES; VAS scores of nasal blockage, hyposmia and rhinorrhea; and SNOT-22 results in both groups were significantly improved 3 months after ESS. Scores of polyposis, mucosal edema, secretions and scarring and total score of LKES in experimental group were significantly better than in control group 3 months after ESS. No significant differences were observed in SF-36, SAS or SDS before or 3 months after ESS within or between the two groups. The side effects of the two groups were not significantly different.Nasal irrigation improved the prognosis of CRS patients after ESS. Budesonide nasal irrigation had a better effect than normal saline nasal irrigation. |
| 2019 | A new triple chimeric protein as a high immunogenic antigen against anthrax toxins: theoretical and experimental analyses. | Immunopharmacol Immunotoxicol | : Anthrax is a zoonotic disease caused by and it can be deadly in 6 days. Considerable efforts have been conducted toward developing more effective veterinary and human anthrax vaccines because these common vaccines have several limitations. secretes a tripartite toxin, comprising protective antigen (PA), edema factor (EF), and lethal factor (LF). Several studies have shown important role of PA in protection of anthrax. LF and EF induce production of toxin neutralizing antibodies too. PA in fusion form with LF/EF has synergistic effects as a potential subunit vaccine. : In this study, for the first time, a triple chimeric protein called ELP was modeled by fusing three different domains of anthrax toxic antigens, the N-terminal domains of EF and LF, and the C-terminal domain of PA as a high immunogenic antigen using Modeller 9.19 software. Immunogenicity of the ELP was assessed in guinea pigs using enzyme-linked immunosorbent assay (ELISA) test and MTT assay. : Theoretical studies and molecular dynamics (MD) simulation results suggest that the ELP model had acceptable quality and stability. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified ELP, its domains, and PA were matched with their molecular size and confirmed by western blotting analysis. In the immune guinea pigs, antibody was produced against all of the ELP domains. It was observed that ELP induced strong humoral response and could protect murine macrophage cell line (RAW 264.7 cells) against anthrax lethal toxin (LeTx). : ELP chimeric antigen could be considered as a high immunogenic antigen. |
| 2019 | Lectin purified from Lonchocarpus campestris seeds inhibits inflammatory nociception. | Int J Biol Macromol | Lonchocarpus campestris (tribe Dalbergieae) possess a mannose biding lectin (LCaL) purified by ion exchange chromatography on DEAE-Sephacel, HiTrap DEAE FF and TSKgel engaged in AKTA-HPLC system. LCaL agglutinates trypsinized rabbit erythrocytes and its activity was maintained after incubation in a wide range of temperature (4-100 °C) and pH (4-9). The lectin had its apparent molecular weight evaluated by size-exclusion chromatography and SDS-PAGE and presented a profile of 10 kDa and 25 kDa in denaturing and native conditions, respectively. LCaL injected by intravenous route in mice showed antinociceptive activity in the behavioral tests of Formalin and Writhing. In the formalin test LCaL inhibited the licking time by 37% in the neurogenic phase and by 73% in the inflammatory phase. In the acetic acid-induced writhing test LCaL showed inhibitory effect at 0.1 mg/kg (72%), 1 mg/kg (74%) and 10 mg/kg (70%). The lectin also inhibited the increase in vascular permeability at 10 mg/kg and leukocyte migration at 0.1, 1 and 10 mg/kg concentrations. Additionally, LCaL inhibited paw edema (mainly from 1 to 3 h by 46%) and hyperalgesia (1 h: 82%; 3 h: 63%) induced by carrageenan. In conclusion, LCaL presents an antinociceptive action mainly via inhibition of inflammation. |
| 2018 | Pain-like behaviors and local mechanisms involved in the nociception experimentally induced by Latrodectus curacaviensis spider venom. | Toxicol Lett | The present study was undertaken to characterize the behavioral manifestations of nociception and the local mechanisms involved with the nociceptive response elicited by Latrodectus curacaviensis venom (LCV) in mice. After the intraplantar LCV inoculation, spontaneous nociception, mechanical and thermal nociceptive thresholds, motor performance, edema and cytokine levels were evaluated using von Frey filaments, hot/cold plate, rota-rod, plethismometer and ELISA, respectively. Analysis of LCV was performed by SDS-PAGE and chromatography. Intraplantar injection of LCV (1-100 ng/paw) induced intense and heat-sensitive spontaneous nociception, mediated by serotonin and bradykinin receptors, TRPV1 channels, as well as by transient local inflammation. LCV (0.1-10 ng/paw) induced mechanical allodynia, which was reduced by the local pretreatment with H1 receptor or TRPV1 antagonists. Corroborating the TRPV1 involvement, in thermal nociception assays, LCV induced a similar response to that of capsaicin, a TRPV1 agonist, facilitating the response to noxious hot stimuli and inhibiting the response to cold noxious stimulation. LCV promoted mast cell degranulation, increased IL-1β paw levels, but did not produce a relevant edematogenic effect. Analysis of LCV components showed a predominance of high molecular weight proteins. This work provides the first mechanistic hypothesis to explain the local pain induced by LCV, the most frequent clinical symptom of human envenomation. |
| 2018 | Preoperative Tranexamic Acid for Treatment of Bleeding, Edema, and Ecchymosis in Patients Undergoing Rhinoplasty: A Systematic Review and Meta-analysis. | JAMA Otolaryngol Head Neck Surg | Evidence has emerged on the efficacy of tranexamic acid to control blood loss and postoperative complications after rhinoplasty.To investigate the results of tranexamic acid use to reduce intraoperative bleeding, postoperative eyelid edema, and periorbital ecchymosis in rhinoplasty.For this systematic review of randomized clinical trials, searches were performed in PubMed, Cochrane Central Register of Controlled Trials, Web of Science, SCOPUS, Science Direct, Google Scholar, OpenThesis, and ClinicalTrials.gov from inception to December 23, 2017. Key words included tranexamic acid, rhinoplasty, and nasal surgical procedures. The following elements were used to define eligibility criteria: (1) population: patients undergoing rhinoplasty surgery; (2) intervention and controls: tranexamic acid vs placebo solution or no-treatment control group; (3) outcomes: intraoperative bleeding, postoperative eyelid edema and periorbital ecchymosis, and thromboembolic events; and (4) study type: randomized clinical trials.Two reviewers extracted data and assessed study quality according to the Cochrane guidelines for randomized clinical trials. Treatment effects were defined as weighted mean difference (WMD) and 95% CIs. The strength of evidence was analyzed using the Grading of Recommendations Assessment, Development, and Evaluation rating system.Intraoperative bleeding, postoperative eyelid edema and periorbital ecchymosis. To calculate the effect sizes, means and SDs were obtained for each study group and outcome of interest.Five studies comprising 276 patients were included in the systematic review: 177 patients (64.1%) were women, and mean age was 26.8 (range, 16-42) years. Four studies comprising 246 patients estimated the amount in intraoperative bleeding as a primary outcome and were included in the meta-analysis. Eyelid edema and ecchymosis were evaluated as outcomes in 2 studies. Tranexamic acid was associated with reduced bleeding during rhinoplasty was found (WMD, -42.28 mL; 95% CI, -70.36 to -14.21 mL), with differences (P = .01) between oral (WMD, -61.70 mL; 95% CI, -83.02 to -40.39 mL; I2 = 0%) and intravenous (WMD, -23.88 mL; 95% CI, -45.19 to -2.58 mL; I2 = 56%) administration. Eyelid edema and ecchymosis scores in patients receiving tranexamic acid were significantly lower compared with the control group within the first postoperative week: lower eyelid edema, WMD, -0.76; 95% CI, -1.04 to -0.49 and lower eyelid ecchymosis, WMD, -0.94; 95% CI, -1.80 to -0.08. No cases of thromboembolic events were reported.Current available evidence suggests that preoperative administration of tranexamic acid is safe and may reduce intraoperative bleeding as well as postoperative eyelid edema and ecchymosis in patients undergoing rhinoplasty. |
| 2018 | Proteomic profile, biological activities and antigenic analysis of the venom from Bothriopsis bilineata smaragdina ("loro machaco"), a pitviper snake from Peru. | J Proteomics | In order to determine Bothriopsis bilineata smaragdina venom (BbsV) composition, proteomic approaches were performed. Venom components were analyzed by RP-HPLC, SDS- PAGE and nano LC on line with LTQ Orbitrap XL. Results showed a total of 189 identified proteins, grouped into 11 different subgroups, which include snake venom metalloproteinases (SVMPs, 54.67%), snake C-type lectins (Snaclecs, 15.78%), snake venom serine proteinases (SVSPs, 14.69%), cystein-rich secretory proteins (CRISP, 2.61%), phospholipases A (PLA, 1.14%), phosphodiesterase (PDE, 1.17%), venom endothelial growth factor (VEGF, 1.06%) 5'nucleotidases (0.33%), L-amino acid oxidases (LAAOs, 0.28%) and other proteins. In vitro enzymatic activities (SVMP, SVSP, LAAO, Hyal and PLA) of BbsV were also analyzed. BbsV showed high SVSP activity but low PLA activity, when compared to other Bothrops venoms. In vivo, BbsV induced hemorrhage and edema in mice and showed intraperitoneal median lethal dose (LD) of 92.74 (± 0.15) μg/20 g of mice. Furthermore, BbsV reduced cell viability when incubated with VERO cells. Peruvian and Brazilian bothropic antivenoms recognize BbsV proteins, as detected by ELISA and Western Blotting. Both antivenoms were able to neutralize in vivo edema and hemorrhage.In Peru, snakebite is a public health problem, especially in the rain forest, as a result of progressive colonization of this geographical area. This country is the second in Latin America, after Brazil, to exhibit the largest variety of venomous snakes. B. atrox and B. b. smaragdina snakes are sympatric species in Peruvian Amazon region and are responsible for approximately 95% of the envenomings reported in this region. B. b. smaragdina may cause a smaller share (3 to 38%) of those accidents, due to its arboreal habits, that make human encounters with these snakes less likely to happen. Despite B. b. smaragdina recognized medical importance, its venom composition and biological activities have been poorly studied. Furthermore, BbsV is not a component of the antigenic pool used to produce the corresponding Peruvian bothropic antivenom (P-BAV). Our results not only provide new insights on BbsV composition and biological activity, but also demonstrate that both P-BAV and B-BAV polyvalent antivenoms have a considerable recognition of proteins from BbsV and, more importantly, neutralized hemorrhage and edema, the main local effects of bothropic envenomation. |
| 2018 | Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum. | PLoS One | Phospholipases A2 (PLA2) are enzymes acting on the cell membrane phospholipids resulting in fatty acids and lysophospholipids and deconstructing the cell membrane. This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have natural resistance to their own venom due to protective properties in plasma, that inhibit the action of proteins present in their venom. Given that, this study aimed to purify and characterize a γPLI from Bothrops jararaca serum, named γBjPLI. PLA2 inhibitor was isolated using two chromatographic steps: an ion exchange column (DEAE), followed by an affinity column (crotoxin coupled to a CNBr-activated Sepharose resin). The purity and biochemical characterization of the isolated protein were analyzed by RP-HPLC, SEC, SDS-PAGE, circular dichroism and mass spectrometry. The ability to inhibit PLA2 was determined by enzymatic activity, neutralization of paw edema and myonecrosis. The protein purity was confirmed by RP-HPLC and SEC, whilst an apparent molecular mass of 25 kDa and 20 kDa was obtained by SDS-PAGE, under reducing and non-reducing conditions, respectively. According to mass spectrometry analysis, this protein showed 72% and 68% of coverage when aligned to amino acid sequences of two proteins already described as PLIs. Thus, the inhibitory activity of enzymatic, edema and myonecrotic activities by γBjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation. |
| 2018 | Improved Precision of Automatic Brain Volume Measurements in Patients with Clinically Isolated Syndrome and Multiple Sclerosis Using Edema Correction. | AJNR Am J Neuroradiol | The presence of edema will result in increased brain volume, which may obscure progressing brain atrophy. Similarly, treatment-induced edema reduction may appear as accelerated brain tissue loss (pseudoatrophy). The purpose of this study was to correlate brain tissue properties to brain volume, to investigate the possibilities for edema correction and the resulting improvement of the precision of automated brain volume measurements.A group of 38 patients with clinically isolated syndrome or newly diagnosed MS were imaged at inclusion and after 1, 2, and 4 years using an MR quantification sequence. Brain volume, relaxation rates (R and R), and proton density were measured by automated software.The reduction of normalized brain volume with time after inclusion was 0.273%/year. The mean SDs were 0.508%, 0.526%, 0.454%, and 0.687% at baseline and 1, 2, and 4 years. Linear regression of the relative change of normalized brain volume and the relative change of R, R, and proton density showed slopes of -0.198 ( < .001), 0.156 ( = .04), and 0.488 ( < .001), respectively. After we applied the measured proton density as a correction factor, the mean SDs decreased to 24.2%, 4.8%, 33.3%, and 17.4%, respectively. The observed atrophy rate reduced from 0.273%/year to 0.238%/year.Correlations between volume and R, R, and proton density were observed in the brain, suggesting that a change of brain tissue properties can affect brain volume. Correction using these parameters decreased the variation of brain volume measurements and may have reduced the effect of pseudoatrophy. |
| 2018 | Globus Symptoms in Patients Undergoing Thyroidectomy: Relationships with Psychogenic Factors, Thyroid Disease, and Surgical Procedure. | Thyroid | The number of patients who need thyroid surgery has increased worldwide in recent decades. Patients with thyroid disease experience globus pharyngeus as a result of direct compression and edema of the surrounding organs. Thyroid surgery is needed to improve these symptoms or as treatment for thyroid cancer. After thyroid surgery, globus symptoms may become worse and may affect the daily life of the patient for a long time. Psychogenic problems have also been thought to cause the globus sensation. A prospective analysis of globus symptoms and psychogenic factors following thyroidectomy was performed.Patients scheduled to undergo thyroid surgery between February and September 2016 completed the foreign-body sensation in the throat score (FBST; range 0-8.2) and the self-rating depression scale (SDS; range 0-100) preoperatively and three days, one month, three months, six months, and 12 months postoperatively.Long-term follow-up was completed in 616 patients (491 females). A total of 365 patients had thyroid cancer, 169 had benign tumors, and 82 had diffuse goiters with Graves' disease. The percentage of patients who complained about neck discomfort (FBST >2) was 29.4% before surgery. A preoperative high FBST showed a significant direct correlation with a high SDS, but thyroid volume did not. A postoperative high FBST was seen in 75.3% of patients at two days and 78.9% at one month after surgery, and it then gradually decreased to 49.3% at 12 months after surgery. At three days after the operation, the median FBST was significantly higher in patients who had total thyroidectomy with lateral neck dissection or total thyroidectomy only compared to those who had lobectomy only (p < 0.05). These differences were still present 12 months after surgery. A higher preoperative SDS score was also identified as an independent predictor for a high FBST at 12 months after surgery, but not at one or three months postoperatively, on multivariate analyses.Preoperative globus symptoms appear directly related to psychological factors. The area of the surgical procedure and preoperative psychological factors were related to persistent neck discomfort. |
| 2017 | Biochemical, biological and molecular characterization of an L-Amino acid oxidase (LAAO) purified from Bothrops pictus Peruvian snake venom. | Toxicon | An L-amino acid oxidase from Peruvian Bothrops pictus (Bpic-LAAO) snake venom was purified using a combination of size-exclusion and ion-exchange chromatography. Bpic-LAAO is a homodimeric glycosylated flavoprotein with molecular mass of ∼65 kDa under reducing conditions and ∼132 kDa in its native form as analyzed by SDS-PAGE and gel filtration chromatography, respectively. N-terminal amino acid sequencing showed highly conserved residues in a glutamine-rich motif related to binding substrate. The enzyme exhibited optimal activity towards L-Leu at pH 8.5, and like other reported SV-LAAOs, it is stable until 55 °C. Kinetic studies showed that the cations Ca, Mg and Mn did not alter Bpic-LAAO activity; however, Zn is an inhibitor. Some reagents such as β-mercaptoethanol, glutathione and iodoacetate had inhibitory effect on Bpic-LAAO activity, but PMSF, EDTA and glutamic acid did not affect its activity. Regarding the biological activities of Bpic-LAAO, this enzyme induced edema in mice (MED = 7.8 μg), and inhibited human platelet aggregation induced by ADP in a dose-dependent manner and showed antibacterial activity on Gram (+) and Gram (-) bacteria. Bpic-LAAO cDNA of 1494 bp codified a mature protein with 487 amino acid residues comprising a signal peptide of 11 amino acids. Finally, the phylogenetic tree obtained with other sequences of LAAOs, evidenced its similarity to other homologous enzymes, showing two well-established monophyletic groups in Viperidae and Elapidae families. Bpic-LAAO is evolutively close related to LAAOs from B. jararacussu, B. moojeni and B. atrox, and together with the LAAO from B. pauloensis, form a well-defined cluster of the Bothrops genus. |
| 2017 | Simulation of spreading depolarization trajectories in cerebral cortex: Correlation of velocity and susceptibility in patients with aneurysmal subarachnoid hemorrhage. | Neuroimage Clin | In many cerebral grey matter structures including the neocortex, spreading depolarization (SD) is the principal mechanism of the near-complete breakdown of the transcellular ion gradients with abrupt water influx into neurons. Accordingly, SDs are abundantly recorded in patients with traumatic brain injury, spontaneous intracerebral hemorrhage, aneurysmal subarachnoid hemorrhage (aSAH) and malignant hemispheric stroke using subdural electrode strips. SD is observed as a large slow potential change, spreading in the cortex at velocities between 2 and 9 mm/min. Velocity and SD susceptibility typically correlate positively in various animal models. In patients monitored in neurocritical care, the Co-Operative Studies on Brain Injury Depolarizations (COSBID) recommends several variables to quantify SD occurrence and susceptibility, although accurate measures of SD velocity have not been possible. Therefore, we developed an algorithm to estimate SD velocities based on reconstructing SD trajectories of the wave-front's curvature center from magnetic resonance imaging scans and time-of-SD-arrival-differences between subdural electrode pairs. We then correlated variables indicating SD susceptibility with algorithm-estimated SD velocities in twelve aSAH patients. Highly significant correlations supported the algorithm's validity. The trajectory search failed significantly more often for SDs recorded directly over emerging focal brain lesions suggesting in humans similar to animals that the complexity of SD propagation paths increase in tissue undergoing injury. |
| 2018 | Spreading depolarization is not an epiphenomenon but the principal mechanism of the cytotoxic edema in various gray matter structures of the brain during stroke. | Neuropharmacology | Spreading depolarization (SD) is a phenomenon of various cerebral gray matter structures that only occurs under pathological conditions. In the present paper, we summarize the evidence from several decades of research that SD and cytotoxic edema in these structures are largely overlapping terms. SD/cytotoxic edema is a toxic state that - albeit initially reversible - leads eventually to cellular death when it is persistent. Both hemorrhagic and ischemic stroke are among the most prominent causes of SD/cytotoxic edema. SD/cytotoxic edema is the principal mechanism that mediates neuronal death in these conditions. This applies to gray matter structures in both the ischemic core and the penumbra. SD/cytotoxic edema is often a single terminal event in the core whereas, in the penumbra, a cluster of repetitive prolonged SDs is typical. SD/cytotoxic edema also propagates widely into healthy surrounding tissue as short-lasting, relatively harmless events so that regional electrocorticographic monitoring affords even remote detection of ischemic zones. Ischemia cannot only cause SD/cytotoxic edema but it can also be its consequence through inverse neurovascular coupling. Under this condition, ischemia does not start simultaneously in different regions but spreads in the tissue driven by SD/cytotoxic edema-induced microvascular constriction (= spreading ischemia). Spreading ischemia prolongs SD/cytotoxic edema. Thus, it increases the likelihood for the transition from SD/cytotoxic edema into cellular death. Vasogenic edema is the other major type of cerebral edema with relevance to ischemic stroke. It results from opening of the blood-brain barrier. SD/cytotoxic edema and vasogenic edema are distinct processes with important mutual interactions. This article is part of the Special Issue entitled 'Cerebral Ischemia'. |
| 2017 | Procoagulant serine glycoprotease from Cucumis sativus L.: action on human fibrinogen and fibrin clot. | 3 Biotech | Upon examination of the fruit extract of Cucumis sativus L. for its pharmacological benefits, it was previously observed that it has potential proteolytic, fibrinogenolytic and procoagulant activities. These properties can be attributed to the presence of the protease. In this regard, the present study comprised of purification and characterization of protease. Purification of the enzyme involved ammonium sulfate precipitation followed by gel filtration and ion exchange chromatography. The purified cucumis protease (CPro) exhibits homogeneity as attested by SDS-PAGE and RP-HPLC with a retention time of 14.246 min with molecular mass ~75.3 kDa. CPro was identified as a glycoprotein and serine protease. Azocasein is the preferred substrate for CPro as it showed low K value of 0.3809 mg/ml. Purified CPro exhibits optimum activity at 37 °C and pH 8. CPro shows its involvement in hemostasis-the very first step in wound healing. CPro degrades the subunits of human fibrinogen in the order Aα > Bβ > γ. It also hydrolyzes the subunits of the partially cross-linked fibrin clot in the order α-polymer > γ-γ dimer > β-chain. CPro reduced the clotting time of citrated plasma, prothrombin time and activated partial thromboplastin time of plasma. CPro is neither hemorrhagic nor edema-inducing, thus considered to be a non-toxic protease. This work provides evidence for the use of cucumber extract in wound healing and authenticates its use in cosmetics. |
| 2017 | Preparation and neutralization efficacy of IgY antibodies raised against venom. | J Venom Anim Toxins Incl Trop Dis | The five-paced pit viper (), endemic to China and northern Vietnam, is responsible for most snakebites in the Chinese territory. Antivenom produced from horses is the main treatment for snakebites, but it may cause numerous clinical side effects and have other disadvantages involved in their production such as the welfare of animals. The present study was conducted aiming to develop an alternative antibody (IgY) from the egg yolk of leghorn chickens immunized with snake venom.IgY from the egg yolk of white leghorn chickens previously immunized intramuscularly with venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot. Finally, IgY neutralization assays to test its efficacy against hemorrhagic, edema-forming and myotoxic activities of venom were conducted on mice.For the first time, IgY antibodies against venom were raised successfully in egg yolk of chickens injected with venom multiple times. By three steps, including caprylic acid extraction, ammonium sulfate precipitation and affinity chromatography, IgY antibodies were isolated and purified from egg yolk, which exhibited a single protein band on SDS-PAGE and two bands (about 65 kDa and 35 kDa, respectively) under reducing conditions, and presented a high titer (1:40,000) tested by ELISA. Immunoblot analysis confirmed that these IgY were polyclonal antibodies since they bound to components of venom. Furthermore, immunodiffusion assay showed that anti- venom IgY cross-reacted with the venoms of and Emelianov, but did not react to the venoms of and . In the neutralizing lethal assay, the median effective dose of anti- venom IgY was 14.14 mg/kg of mouse body weight under the challenge dose (3 LD of venom). In neutralizing the hemorrhagic, edema-forming and myotoxic activities of venom, IgY showed the characteristic dose-dependent neutralization effects against all these toxic activities of venom.Anti- venom IgY antibodies with high purity and titer were for the first time raised successfully in egg yolk of chickens immunized with venom. They were effective in neutralizing the lethal effects, and the hemorrhagic, edema-forming and myotoxic acitivities of venom. IgY could be an effective source to develop a treatment against snake bites in humans or animals in the future. |
| 2017 | Geographical variability of the venoms of four populations of Bothrops asper from Panama: Toxicological analysis and neutralization by a polyvalent antivenom. | Toxicon | Bothrops asper is the medically most important venomous snake in Central America. In Panama, the country having the highest incidence of snakebites in Latin America, B. asper is widely distributed throughout the country and is responsible for the vast majority of snakebites. This study was performed to analyze whether there are variations in the toxicological profile and in some biochemical parameters between the venoms of B. asper from four different regions in Panama. The venoms showed a similar profile of lethal, hemorrhagic, in vitro coagulant, defibrinogenating, edema-forming, myotoxic and indirect hemolytic activities, with subtle quantitative variations between samples of some regions. The venoms also had similar SDS-PAGE patterns and reverse phase HPLC profiles. A polyvalent antivenom manufactured in Costa Rica, and regularly used in Panama, was effective in the neutralization of lethal activity of the venoms of the four populations, with Mean Effective Doses (ED) ranging from 5.98 to 9.72 mg venom/mL antivenom. In agreement, a widespread pattern of cross-reactivity between this antivenom and the four venoms was observed by immunoblotting. Overall, results highlight the lack of marked differences between the venoms of the various populations of B. asper in Panama, and that the antivenom from Costa Rica is effective in neutralizing lethality. |
| 2017 | The potential of aqueous extracts of Bellucia dichotoma Cogn. (Melastomataceae) to inhibit the biological activities of Bothrops atrox venom: A comparison of specimens collected in the states of Pará and Amazonas, Brazil. | J Ethnopharmacol | The effectiveness of aqueous extract of Bellucia dichotoma Cogn. (Melastomataceae) specimems collected in Santarém, PA, against some biological activities of Bothrops atrox venom (BaV) has been scientifically proven. Here, we analyzed the components and assessed the anti-snakebite potential of aqueous extracts of bark of B. dichotoma collected in Manaus, AM, (AEBd-MAO) and Santarém, PA, (AEBd-STM), both in Brazil.The phytochemical profiles of the aqueous extracts were identified using thin layer chromatography (TLC), and the concentrations of phenolics were determined by colorimetric assay. The inhibitory potential of the extracts was tested against the phospholipase A, coagulant and gelatinolytic activities of BaV in vitro and its defibrinating and edema-inducing activities in vivo. Interaction between BaV and the extracts was investigated using SDS-Page electrophoresis and Western blotting. Extract cytotoxicity and antioxidant potential were assessed using the human fibroblast cell line MRC-5 and the DPPH assay in cell culture, respectively.While there was no difference between the phytochemical profiles of the extracts, AEBd-MAO had higher concentrations of total phenolics, total tannins and hydrolysable tannins. The extracts inhibited 100% of the phospholipase and coagulant activity of BaV when pre-incubated. Without pre-incubation, however, there was no reduction in phospholipase activity, although significant inhibition of coagulant activity was observed. In the doses used in folk medicine, without pre-incubation, both extracts inhibited 100% of the coagulant activity of BaV. In vivo, the extracts were unable to inhibit the defibrinating activity of the venom but were effective in inhibiting its edema-inducing activity. In the profiles of the extracts pre-incubated with BaV, not all the protein bands revealed by SDS-PAGE and Western blot were observed. Both extracts had a high antioxidant potential and neither had a cytotoxic effect.Although the concentrations of phenolics in each extract were different, the anti-snakebite potential was similar for the concentrations of extract tested. Our findings are of importance for the quality control of this raw material, which, once tested in accordance with Brazilian National Health Surveillance Agency recommendations, may be suitable for use as a phytomedicine to complement treatment of the local effects induced by Bothrops venoms. |
| 2016 | The cutaneous secretion of the casque-headed tree frog Corythomantis greeningi: Biochemical characterization and some biological effects. | Toxicon | Corythomantis greeningi is a tree-frog endemic of the Brazilian semi-arid (Caatinga), mainly characterized by the flat, mineralized and spiny head, which is associated with phragmotic habits. It is already known that the skin secretion of this amphibian from both head and body is quite toxic and is used as an efficient chemical defence against predators. However, the biochemical characteristics and pharmacological effects of this secretion are still very little studied. We have tested the crude skin secretion, as well as the ten major fractions obtained by RP-HPLC for nociceptive and edema activity and for in vitro cytotoxicity using murine models. SDS-PAGE analyses demonstrated that the majority of proteins ranging through the gel lie between 55 and 30 kDa. LC-MS analysis showed multiple low molecular mass molecules (200-500 Da), which are consistent with masses of alkaloids and steroids. The crude skin secretion was able to induce fast and persistent edema accompanied by intense dose-dependent nociception. From the 10 tested fractions, five induced both edema and nociception, six fractions were able to induce only edema (80-170% control), and seven fractions induced only nociception (15-30 times compared to control). In addition, inhibition of cell growth (IC50) was demonstrated in murine fibroblasts and melanoma cells. From the data obtained, we confirmed that the skin secretion of C. greeningi is very toxic and is rich in compounds able to directly provoke local inflammation and nociception. Such characteristics are important as part of the chemical defensive repertory of this species. |
| Improved dissolution and anti-inflammatory activity of ibuprofen-polyethylene glycol 8000 solid dispersion systems. | Int J Pharm Investig | The purpose of this study was to develop ibuprofen (IB)-polyethylene glycol (PEG) 8000 solid dispersions (SDs) and investigate them for in vitro dissolution and in vivo anti-inflammatory activity.IB-PEG 8000 SDs were prepared by fusion method using varying combination ratios of IB and PEG 8000. Characterization based on surface morphology, particle size, absolute drug content, and Fourier transform infrared (FT-IR) spectroscopy was carried out on the SDs. The in vitro release of IB from the SDs was performed in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.4) without enzymes, whereas the anti-inflammatory activity was evaluated using egg albumin-induced rat paw edema model.Greenish brown, discrete, and irregularly shaped SDs of mean particle size range 113.5 ± 2.5-252.5 ± 1.9 μm, which were stable over 3 months, were obtained. The drug content of the SDs ranged from 73.4 ± 2.9 % to 83.5 ± 2.7%. Although the drug content increased with increased concentration of PEG 8000 in the SDs, the mean particle size decreased with increased concentration of PEG 8000 in the SDs. The FT-IR results indicate no strong chemical interaction of IB and PEG 8000 in the SDs. There was marked increase in the dissolution rate of IB from the SDs (P < 0.05) as compared to pure IB and physical mixture. The dissolution was better in SIF than in SGF. The increased dissolution rate of IB may be due to the formation of microcrystals, increased wettability and dispersibility in PEG 8000. The SDs showed good anti-inflammatory properties achieving up to 90% edema inhibition at 6 h while the pure sample of IB had 77% edema inhibition at 6 h.SDs based on IB-PEG 8000 is a good approach to enhance the dissolution rate and anti-inflammatory activity of IB, thus, encouraging further development of the SDs. |
| 2016 | Biochemical and biological characterization of Bothriechis schlegelii snake venoms from Colombia and Costa Rica. | Exp Biol Med (Maywood) | Snakebites inflicted by the arboreal viperid snake Bothriechis schlegelii in humans are characterized by pain, edema, and ecchymosis at the site of the bite, rarely with blisters, local necrosis, or defibrination. Herein, a comparative study of Bothriechis schlegelii snake venoms from Colombia (BsCo) and Costa Rica (BsCR) was carried out in order to compare their main activities and to verify the efficacy of Bothrops antivenom produced in Brazil to neutralize them. Biochemical (SDS-PAGE and zymography) and biological parameters (edematogenic, lethal, hemorrhagic, nociceptive, and phospholipase A activities) induced by BsCo and BsCR snake venoms were evaluated. The presence of antibodies in Bothrops antivenom that recognize BsCo and BsCR snake venoms by enzyme-linked immunosorbent assay and Western blotting, as well as the ability of this antivenom to neutralize the toxic activities were also verified. SDS-PAGE showed differences between venoms. Distinctive caseinolytic and hyaluronidase patterns were detected by zymography. BsCo and BsCR showed similar phospholipase A activity. Strong cross-reactivity between BsCo and BsCR was detected using Bothrops antivenom with many components located between 150 and 35 kDa. BsCR was more edematogenic and almost fourfold more hemorrhagic than BsCo, and both venoms induced nociception. BsCR (LD 5.60 mg/kg) was more lethal to mice than BsCo (LD 9.24 mg/kg). Bothrops antivenom was effective in the neutralization of lethal and hemorrhagic activities of BsCo and BsCR and was partially effective in the neutralization of edematogenic and nociceptive activities. In conclusion, geographic distribution influences the composition and activities of Bothriechis schlegelii venoms. Bothrops antivenom cross-reacted with these venoms and was partially effective in neutralizing some toxic activities of BsCo and BsCR. |
| 2016 | New insights into Clostridium perfringens epsilon toxin activation and action on the brain during enterotoxemia. | Anaerobe | Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is responsible for diseases that occur mostly in ruminants. ETX is produced in the form of an inactive prototoxin that becomes proteolytically-activated by several proteases. A recent ex vivo study using caprine intestinal contents demonstrated that ETX prototoxin is processed in a step-wise fashion into a stable, active ∼27 kDa band on SDS-PAGE. When characterized further by mass spectrometry, the stable ∼27 kDa band was shown to contain three ETX species with varying C-terminal residues; each of these ETX species is cytotoxic. This study also demonstrated that, in addition to trypsin and chymotrypsin, proteases such as carboxypeptidases are involved in processing ETX prototoxin. Once absorbed, activated ETX species travel to several internal organs, including the brain, where this toxin acts on the vasculature to cross the blood-brain barrier, produces perivascular edema and affects several types of brain cells including neurons, astrocytes, and oligodendrocytes. In addition to perivascular edema, affected animals show edema within the vascular walls. This edema separates the astrocytic end-feet from affected blood vessels, causing hypoxia of nervous system tissue. Astrocytes of rats and sheep affected by ETX show overexpression of aquaporin-4, a membrane channel protein that is believed to help remove water from affected perivascular spaces in an attempt to resolve the perivascular edema. Amyloid precursor protein, an early astrocyte damage indicator, is also observed in the brains of affected sheep. These results show that ETX activation in vivo seems to be more complex than previously thought and this toxin acts on the brain, affecting vascular permeability, but also damaging neurons and other cells. |
| 2016 | Spreading Depolarizations: A Therapeutic Target Against Delayed Cerebral Ischemia After Subarachnoid Hemorrhage. | J Clin Neurophysiol | Delayed cerebral ischemia is the most feared cause of secondary injury progression after subarachnoid hemorrhage. Initially thought to be a direct consequence of large artery spasm and territorial ischemia, recent data suggests that delayed cerebral ischemia represents multiple concurrent and synergistic mechanisms, including microcirculatory dysfunction, inflammation, and microthrombosis. Among these mechanisms, spreading depolarizations (SDs) are arguably the most elusive and underappreciated in the clinical setting. Although SDs have been experimentally detected and examined since the late 1970s, their widespread occurrence in human brain was not unequivocally demonstrated until relatively recently. We now know that SDs occur with very high incidence in human brain after ischemic or hemorrhagic stroke and trauma, and worsen outcomes by increasing metabolic demand, decreasing blood supply, predisposing to seizure activity, and possibly worsening brain edema. In this review, we discuss the causes and consequences of SDs in injured brain. Although much of our mechanistic knowledge comes from experimental models of focal cerebral ischemia, clinical data suggest that the same principles apply regardless of the mode of injury (i.e., ischemia, hemorrhage, or trauma). The hope is that a better fundamental understanding of SDs will lead to novel therapeutic interventions to prevent SD occurrence and its adverse consequences contributing to injury progression in subarachnoid hemorrhage and other forms of acute brain injury. |
| 2016 | Analysis of the intersexual variation in Thalassophryne maculosa fish venoms. | Toxicon | Gender related variation in the molecular composition of venoms and secretions have been described for some animal species, and there are some evidences that the difference in the toxin (s) profile among males and females may be related to different physiopathological effects caused by the envenomation by either gender. In order to investigate whether this same phenomenon occurs to the toadfish Thalassophryne maculosa, we have compared some biological and biochemical properties of female and male venoms. Twenty females and males were collected in deep waters of the La Restinga lagoon (Venezuela) and, after protein concentration assessed, the induction of toxic activities in mice and the biochemical properties were analyzed. Protein content is higher in males than in females, which may be associated to a higher size and weight of the male body. In vivo studies showed that mice injected with male venoms presented higher nociception when compared to those injected with female venoms, and both venoms induced migration of macrophages into the paw of mice. On the other hand, mice injected with female venoms had more paw edema and extravasation of Evans blue in peritoneal cavity than mice injected with male venoms. We observed that the female venoms had more capacity for necrosis induction when compared with male venoms. The female samples present a higher proteolytic activity then the male venom when gelatin, casein and FRETs were used as substrates. Evaluation of the venoms of females and males by SDS-PAGE and chromatographic profile showed that, at least three components (present in two peaks) are only present in males. Although the severity of the lesion, characterized by necrosis development, is related with the poisoning by female specimens, the presence of exclusive toxins in the male venoms could be associated with the largest capacity of nociception induction by this sample. |
| 2015 | Anti-inflammatory, Anti-estrogenic, and Anti-implantation Activity of Bergia suffruticosa (Delile) Fenzl. | Pharmacogn Mag | Bergia suffruticosa (Delile) Fenzl (Syn. Bergia odorata Edgew) (Elatinaceae family) is used traditionally to repair bones and is applied as a poultice on sores. It is also used for stomach troubles and as an antidote to scorpion stings. So far, very little scientific work has been reported to validate its ethnomedical uses in the alleviation of pain, bone repair, etc.This study was designed to explore the anti-inflammatory and anti-implantation potential of n-hexane extract of B. suffruticosa whole plant in mice along with identification of its chemical constituents.n-Hexane extract of B. suffruticosa whole plant was screened for acute and chronic anti-inflammatory activity followed by an anti-estrogenic activity. Eventually, n-hexane extract was tested for anti-implantation activity by exploiting markers of uterine receptivity, lipid peroxidation, and superoxide enzyme activity. The extract was administered orally at a dose of 100 mg/kg body weight in each study.Thin layer chromatography fingerprint profile of n-hexane extract revealed the presence of lupeol and β-sitosterol. The n-hexane extract reduced the edema by 80% in acute inflammation, whereas it reduced edema to 75% on the 5(th) day in chronic inflammation. The n-hexane extract reduced elevated malonaldehyde level from 6 to 2.5 nmol/g × 10(-5) and increased superoxide dismutase enzyme activity from 0 to 350 units/g in treated animals on the 5(th) day of pregnancy. Moreover, extract decreased uterine weight from 0.33 to 0.2 g in estradiol treated animals.These results indicate that n-hexane extract of B. suffruticosa is having potent anti-inflammatory, anti-estrogenic, and anti-implantation activity. This is the first report of all the pharmacological activities of B. suffruticosa mentioned above.TLC fingerprint profile of n-hexane extract of Bergia suffruticosa whole plant revealed the presence of lupeol and β-sitosteroln-Hexane extract showed in vivo anti-inflammatory activity in both acute and chronic model of inflammation in ratsn-Hexane extract possess significant anti-estrogenic activityn-Hexane extract altered the levels superoxide anion radical and superoxide dismutase enzyme activity during the blastocyst implantationAnti-implantation activity of n-hexane extract is attributed to its anti-inflammatory and anti-estrogenic potential. Abbreviations used: TLC: Thin layer chromatography; LPO: Lipid peroxidation; SOD: Superoxide dismutase; B. suffruticosa: Bergia suffruticosa; TNF-α: Tumor necrosis factor-α; NO: Nitric oxide; IL-1: Interleukin-1; LIF: Leukemia inhibitory factor; CSF-1: Colony-stimulating factor; COX: Cyclooxygenase; SDS: Sodium dodecyl sulfate; IAEC: Animal House Ethics Committee; CPCSEA: Committee for the Purpose of Control and Supervision of Experiments on Animals; HBSS: Hank's balanced salt solution; MDA: Malonaldehyde; and TBA: Thiobarbituric acid. |
| 2016 | Isolation, structural and functional characterization of a new Lys49 phospholipase A2 homologue from Bothrops neuwiedi urutu with bactericidal potential. | Toxicon | Snake venom is a complex mixture of active compounds consisting of 80-90% proteins and peptides that exhibit a variety of biological actions that are not completely clarified or identified. Of these, phospholipase A2 is one of the molecules that has shown great biotechnological potential. The objectives of this study were to isolate, biochemically and biologically characterize a Lys49 phospholipase A2 homologue from the venom of Bothrops neuwiedi urutu. The protein was purified after two chromatographic steps, anion exchange and reverse phase. The purity and relative molecular mass were assessed by SDS-PAGE, observing a molecular weight typical of PLA2s, subsequently confirmed by mass spectrometry obtaining a mass of 13,733 Da. As for phospholipase activity, the PLA2 proved to be enzymatically inactive. The analyses by Edman degradation and sequencing of the peptide fragments allowed for the identification of 108 amino acid residues; this sequence showed high identity with other phospholipases A2 from Bothrops snake venoms, and identified this molecule as a novel PLA2 isoform from B. neuwiedi urutu venom, called BnuTX-I. In murine models, both BnuTX-I as well as the venom induced edema and myotoxic responses. The cytotoxic effect of BnuTX-I in murine macrophages was observed at concentrations above 12 μg/mL. BnuTX-I also presented antimicrobial activity against gram-positive and negative bacterial strains, having the greatest inhibitory effect on Pseudomonas aeruginosa. The results allowed for the identification of a new myotoxin isoform with PLA2 structure with promising biotechnological applications. |
| 2016 | rBaltMIP, a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus (Rhinocerophis alternatus) snake, as a potential candidate to complement the antivenom therapy. | Toxicon | Phospholipase A inhibitors (PLIs) are important targets in the search and development of new drugs. This study aimed at evaluating the potential of an alpha-type phospholipase A inhibitor from Bothrops alternatus (Rhinocerophis alternatus) snake in its recombinant form (rBaltMIP) to complement the conventional antivenom therapy. Biochemical experiments showed that rBaltMIP presented pI 5.8 and molecular masses of ∼21 kDa by SDS-PAGE and 19.57 kDa by MALDI/TOF MS. After tryptic peptides sequencing, the results were compared with other PLIs available in databases, showing 100% identity between rBaltMIP and its native inhibitor BaltMIP and from 92% to 96% identity with other inhibitors. Myotoxic activities of BthTX-I and BthTX-II toxins were measured via plasma CK levels, showing myotoxic effective concentrations (EC50) of 0.1256 μg/μL and 0.6183 μg/μL, respectively. rBaltMIP neutralized the myotoxicity caused by these two toxins up to 65%, without promoting primary antibody response against itself. Nevertheless, this recombinant PLI was immunogenic when standard immunization protocol with Freud's adjuvant was used. In paw edema assays, EC50 of 0.02581 μg/μL and 0.02810 μg/μL, respectively, were observed with edema reductions of up to 40% by rBaltMIP, suggesting its use as an additional antivenom. In addition, myotoxicity neutralization experiments with the myotoxin BthTX-I showed that rBaltMIP was more effective in inhibiting muscle damage than the conventional antivenom. Thus, considering the severity of envenomations due to Bothrops alternatus (Rhinocerophis alternatus) and the low neutralization of their local effects (such as myotoxicity) by the current antivenoms, rBaltMIP is a promising molecule for the development of novel therapeutic strategies for clinical applications. |
| 2015 | Protein profiling of Haemonchus contortus found in sheep of Kashmir valley. | J Parasit Dis | Economic losses due to helminth parasites in sheep throughout the world are considerable. Haemonchus contortus is a blood sucking intestinal helminth that lives in the abomasum of small ruminants worldwide. This parasite can be devastating to producers as it causes decreased production levels due to clinical signs such as anaemia, edema and death. For isolation of the proteins of the parasite, a well defined methodology was adopted. The abomasae of sheep in which this parasite resides were collected from abattoirs of various districts and were then carried to laboratory for screening. In case of collection sites falling in far areas, the organs were screened on spot. The parasites were collected in normal saline, washed and stored in 0.05 M PBS with pH of 7.4 at 0 °C. After refrigeration, frozen nematodes were thawed, homogenized and centrifuged at 1,000-15,000 rpm for 15 min. The supernatant was thus collected as a protein mixture and stored at -20 °C. Protein concentration of the samples was estimated by Lowry method. The samples were then analyzed through PAGE and then through SDS-PAGE. Protein estimation of the samples was estimated to be 4.2 mg/ml. The processed parasite samples were then subjected to PAGE and SDS-PAGE to determine the presence of the proteins. It showed high concentration of proteins in its whole protein profile. The proteins were seen as continuous bands intermixing with each other in PAGE analysis. The present study revealed two bands of molecular weights-55 and 33 kDa in PAGE analysis. The proteins when analyzed through SDS-PAGE were mostly found in the range of 25-70 kDa. The SDS-PAGE analysis showed four prominent bands. These bands were of the molecular weights of 66, 40, 33 and 26 kDa. The present work was a challenging one since only a single study was conducted in this region on this aspect and thus obviously was a big task to peep into the field where scanty input was available. |
| 2016 | A novel N-acetyl-glucosamine lectin of Lonchocarpus araripensis attenuates acute cellular inflammation in mice. | Inflamm Res | This study had investigated the anti-inflammatory activity of a seed lectin (LAL) isolated from Lonchocarpus araripensis.LAL was purified by affinity chromatography (chitin column) and ion exchange chromatography (DEAE-Sephacel). In vitro LAL was tested for hemagglutinating activity against rabbit erythrocytes. In vivo LAL was assessed for the anti-inflammatory activity via intravenous injection (i.v.) in Swiss mice (25-30 g; n = 6/group) in models of paw edema and peritonitis.ANOVA (p < 0.05).LAL revealed two bands of 30 and 60 kDa (SDS-PAGE) and exhibited hemagglutinating activity. LAL (10 mg/kg) inhibited the paw edema (77%) and vascular permeability (26%) induced by carrageenan, and the paw edema induced by serotonin (80%), bradykinin (49%), sodium nitroprusside (83%), TNF-α (75%) and PGE2 (64%). LAL also inhibited the neutrophil migration induced by fMLP (70%) or carrageenan (69%). The intravital microscopy showed that LAL inhibited rolling (83%) and adhesion (70%) of leukocytes. LAL anti-inflammatory effect was reversed by its association with N-acetyl-glucosamine. The nine-daily treatment with LAL (10 mg/kg; i.v.) showed no toxicity.The novel N-acetyl-D-glucosamine-binding lectin isolated from L. araripensis seeds presents anti-inflammatory effect involving the lectin domain and the inhibition of 5-HT, BK, PGE2, NO, TNF-α and leukocyte rolling and adhesion. |
| 2015 | Intraocular Pressure Changes in Non-Glaucomatous Patients Receiving Intravitreal Anti-Vascular Endothelial Growth Factor Agents. | PLoS One | To study the prevalence of sustained intraocular pressure (IOP) elevation associated with intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) agents.Prospective comparative study. Non-glaucomatous patients scheduled to receive intravitreal injection of anti-VEGF therapy were recruited from an outpatient eye clinic, Songklanagarind Hospital between April 2013 and March 2014. The IOP was measured by Goldmann applanation tonometer before and at 1 hour, 1 week, 1 month, 3 months, and 6 months after injection. The IOP was compared using the repeated measures analysis. Sustained IOP elevation was defined as either an IOP > 21 mmHg or an increase from baseline ≥ 5 mmHg on two consecutive visits.Seventy eyes of 54 patients met the inclusion criteria. The most common diagnosis was diabetic macular edema (48%). The mean IOP ± standard deviation (SD) before treatment was 13.7 ± 2.8 mmHg. The means ± SDs after treatment at 1 hour, 1 week, 1 month, 3 months, and 6 months were 11.3 ± 2.6, 13.7 ± 3.6, 14.1 ± 3.3, 14.0 ± 2.3, and 13.7 ± 2.4 mmHg, respectively. A mean of IOP difference at 1 hour postinjection and at baseline was -2.36 ± 2.5 mmHg (P < 0.001). Four of 70 treated eyes (5.7%) developed sustained IOP elevation (IOP ≥ 5 mmHg from baseline on two consecutive visits). The IOP returned to baseline levels after 1 month, in three eyes. One eye had sustained IOP elevation at 3 and 6 months follow-up. Thereafter, IOP returned to baseline level. There was no need of anti-glaucoma medication.After receiving intravitreal injection of anti-VEGF agent, a small proportion of non-glaucomatous eyes developed a sustained IOP elevation without requiring IOP-lowering treatment. At 1 hour postinjection, there was a significant reduction of the mean IOP compared with the baseline level. |
| 2016 | Multifaceted roles for astrocytes in spreading depolarization: A target for limiting spreading depolarization in acute brain injury? | Glia | Spreading depolarizations (SDs) are coordinated waves of synchronous depolarization, involving large numbers of neurons and astrocytes as they spread slowly through brain tissue. The recent identification of SDs as likely contributors to pathophysiology in human subjects has led to a significant increase in interest in SD mechanisms, and possible approaches to limit the numbers of SDs or their deleterious consequences in injured brain. Astrocytes regulate many events associated with SD. SD initiation and propagation is dependent on extracellular accumulation of K(+) and glutamate, both of which involve astrocytic clearance. SDs are extremely metabolically demanding events, and signaling through astrocyte networks is likely central to the dramatic increase in regional blood flow that accompanies SD in otherwise healthy tissues. Astrocytes may provide metabolic support to neurons following SD, and may provide a source of adenosine that inhibits neuronal activity following SD. It is also possible that astrocytes contribute to the pathophysiology of SD, as a consequence of excessive glutamate release, facilitation of NMDA receptor activation, brain edema due to astrocyte swelling, or disrupted coupling to appropriate vascular responses after SD. Direct or indirect evidence has accumulated implicating astrocytes in many of these responses, but much remains unknown about their specific contributions, especially in the context of injury. Conversion of astrocytes to a reactive phenotype is a prominent feature of injured brain, and recent work suggests that the different functional properties of reactive astrocytes could be targeted to limit SDs in pathophysiological conditions. |
| 2015 | Quantitative T2 mapping after reperfusion therapy in patients with acute myocardial infarction: A comparison with late gadolinium enhancement and cine MR imaging. | Magn Reson Imaging | This study evaluates myocardial edema by quantitative T2 mapping in patients with acute myocardial infarction (AMI) and compares the lateral extent of myocardial edema with those of infarcted and dysfunctional myocardium.Cardiac magnetic resonance images (MRIs) of 31 patients (M:F=29:2, mean age: 52.5±10.8years) with AMI were reviewed. On cine-MRI, all short axis images of the left ventricle (LV) were divided into 60 sectors. The regional wall motion of each sector was calculated as follows: systolic wall thickening (SWT, %)=[(LV wall thicknessES-LV wall thicknessED)/LV wall thicknessED]*100. Dysfunctional myocardium was defined as sectors with decreased SWT lower than 40%. On LGE-images, myocardial infarction was defined as an area of hyper-enhancement more than 5 SDs from the remote myocardium. On T2 map, myocardial edema was defined as an area in which T2 values were at least 2 SDs higher than those from remote myocardium. The lateral extents of infarcted myocardium, myocardial edema, and dysfunctional myocardium were calculated as the percentage of central angles ((central angle of the involved myocardium/360)*100 (%)) and then compared.The lateral extent of myocardial edema was slightly larger than that of infarcted myocardium (37.4±13.3% vs. 35±12.9%, p<0.01). The lateral extent of dysfunctional myocardium (50.6±15.3%) was significantly larger than that of infarcted myocardium or myocardial edema (p<0.001).The lateral extent of myocardial edema beyond the infarcted myocardium might be narrow, but the dysfunctional myocardium could be significantly larger than myocardial edema, suggesting stunned myocardium without edema. |
| 2015 | eNOS uncoupling in the cerebellum after BBB disruption by exposure to Phoneutria nigriventer spider venom. | Toxicon | Numerous studies have shown that the venom of Phoneutria nigriventer (PNV) armed-spider causes excitotoxic signals and blood-brain barrier breakdown (BBBb) in rats. Nitric oxide (NO) is a signaling molecule which has a role in endothelium homeostasis and vascular health. The present study investigated the relevance of endothelial NO synthase (eNOS) uncoupling to clinical neurotoxic evolution induced by PNV. eNOS immunoblotting of cerebellum lysates processed through low-temperature SDS-PAGE revealed significant increased monomerization of the enzyme at critical periods of severe envenoming (1-2 h), whereas eNOS dimerization reversal paralleled to amelioration of animals condition (5-72 h). Moreover, eNOS uncoupling was accompanied by increased expression in calcium-sensing calmodulin protein and calcium-binding calbindin-D28 protein in cerebellar neurons. It is known that greater eNOS monomers than dimers implies the inability of eNOS to produce NO leading to superoxide production and endothelial/vascular barrier dysfunction. We suggest that transient eNOS deactivation and disturbances in calcium handling reduce NO production and enhance production of free radicals thus contributing to endothelial dysfunction in the cerebellum of envenomed rats. In addition, eNOS uncoupling compromises the enzyme capacity to respond to shear stress contributing to perivascular edema and it is one of the mechanisms involved in the BBBb promoted by PNV. |
| 2015 | Reliability of Ischemic Index Grading in Common Retinal Vascular Diseases. | Ophthalmic Surg Lasers Imaging Retina | Retinal nonperfusion is closely associated with vision-threatening complications such as neovascularization and macular edema. The purpose of this study is to investigate the reliability of a calculated ischemic index (ISI) by means of intergrader and intragrader agreement on ultrawide-field fluorescein angiography (UWFFA) in common retinal vascular diseases.Eight trained graders evaluated 15 UWFFA images provided digitally and re-graded on a different day. They included five eyes with diabetic retinopathy (DR), five with branch retinal vein occlusion (BRVO), and five with central retinal vein occlusion (CRVO). To assess intergrader and intragrader agreement and variability among different diseases, the replicate inter- and intragrader standard deviations (SDs) and coefficients of variation (CVs) were calculated.Mean ISI was 46% for images of DR, 26% for images of BRVO, and 61.3% for images of CRVO. Combined intragrader and intergrader replicate SDs were 17.8% for DR, 3.8% for BRVO and 13.0% for CRVO. Combined intragrader and intergrader replicate coefficients of variation were 38.6% (percent of mean ISI) for DR, 14.7% for BRVO, and 21.2% for CRVO.Intergrader and intragrader variability was high when assessing DR. This may be due to the chronic nature of DR progression, which can lead to patchy areas of ischemia. Intergrader and intragrader variability was better for CRVO and best for BRVO. This may be due to the acute or subacute nature of retinal vein occlusions. |
| 2015 | Bee Pollen-Induced Anaphylaxis: A Case Report and Literature Review. | Allergy Asthma Immunol Res | Bee pollen is pollen granules packed by honey bees and is widely consumed as natural healthy supplements. Bee pollen-induced anaphylaxis has rarely been reported, and its allergenic components have never been studied. A 40-year-old male came to the emergency room with generalized urticaria, facial edema, dyspnea, nausea, vomiting, abdominal pain, and diarrhea 1 hour after ingesting one tablespoon of bee pollen. Oxygen saturation was 91%. His symptoms resolved after injection of epinephrine, chlorpheniramine, and dexamethasone. He had seasonal allergic rhinitis in autumn. Microscopic examination of the bee pollen revealed Japanese hop, chrysanthemum, ragweed, and dandelion pollens. Skin-prick with bee pollen extracts showed positive reactions at 0.1 mg/mL (A/H ratio > 3+). Serum specific IgE to ragweed was 25.2, chrysanthemum 20.6, and dandelion 11.4 kU/L; however, Japanese hop, honey-bee venom and yellow-jacket venom were negative (UniCAP®, Thermo Fisher Scientific, Uppsala, Sweden). Enzyme-linked immunosorbent assay (ELISA) confirmed serum specific IgE to bee-pollen extracts, and an ELISA inhibition assay for evaluation of cross-allergenicity of bee pollen and other weed pollens showed more than 90% of inhibition with chrysanthemum and dandelion and ~40% inhibition with ragweed at a concentration of 1 μg/mL. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and IgE-immunoblot analysis revealed 9 protein bands (11, 14, 17, 28, 34, 45, 52, 72, and 90 kDa) and strong IgE binding at 28-34 kDa, 45 and 52 kDa. In conclusion, healthcare providers should be aware of the potential risk of severe allergic reactions upon ingestion of bee pollen, especially in patients with pollen allergy. |
| 2015 | A heterodimer of a VHH (variable domains of camelid heavy chain-only) antibody that inhibits anthrax toxin cell binding linked to a VHH antibody that blocks oligomer formation is highly protective in an anthrax spore challenge model. | J Biol Chem | Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from "pre-pore" to its SDS and heat-resistant "pore" conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes. |
| 2015 | Proteomic and functional analyses of the venom of Porthidium lansbergii lansbergii (Lansberg's hognose viper) from the Atlantic Department of Colombia. | J Proteomics | The venom of the Lansberg's hognose pitviper, Porthidium lansbergii lansbergii, a species found in the northern region of Colombia, is poorly known. Aiming to increase knowledge on Porthidium species venoms, its proteomic analysis and functional evaluation of in vitro and in vivo activities relevant to its toxicity were undertaken. Out of 51 protein components resolved by a combination of RP-HPLC and SDS-PAGE, 47 were assigned to 12 known protein families. In similarity with two previously characterized venoms from species within this genus, Porthidium nasutum and Porthidium ophryomegas, that of P. lansbergii lansbergii was dominated by metalloproteinases, although in lower proportion. A common feature of the three Porthidium venoms appears to be a high content of disintegrins. Proteins not previously observed in Porthidium venoms belong to the vascular endothelium growth factor, phosphodiesterase, and phospholipase B families. P. lansbergii lansbergii venom showed relatively weak lethal activity to mice, and induced a moderate local myotoxicity, but considerable hemorrhage. Its isolated VEGF component showed potent edema-inducing activity in the mouse footpad assay. Significant thrombocytopenia, but no other major hematological changes, were observed in envenomed mice. In vitro, this venom lacked coagulant effect on human plasma, and induced a potent inhibition of platelet aggregation which was reproduced by its purified disintegrin components. Phospholipase A2 and proteolytic activities were also demonstrated. Overall, the compositional and functional data herein described for the venom of P. lansbergii lansbergii may contribute to a better understanding of envenomings by this pitviper species, for which specific clinical information is lacking.Porthidium lansbergii lansbergii is estimated to be responsible for nearly 20% of snakebite envenoming cases at the Atlantic Department of Colombia, but the identity and functional properties of its venom components are largely unknown. This study provides the first combined proteomic and functional analyses of the venom of this pitviper, which may contribute to a better understanding of the features of envenomings by this species. |
| Prognostic factors in outcome of angioedema in the emergency department. | Allergy Asthma Proc | Angioedema is a transient, localized swelling caused by two distinct mechanisms, mediated by histamine and bradykinin, respectively, although a proportion of cases remain idiopathic. Studies that characterize undifferentiated angioedema presenting in emergency departments (EDs) are limited. This study investigates the presentation patterns of undifferentiated angioedema in the ED based on the presumed mechanism of swelling. Medical records from all ED visits to two tertiary care hospitals from July 2007 to March 2012 were electronically reviewed. Records with documented visible swelling on general inspection and/or fiberoptic laryngoscopy and a diagnostic code for anaphylactic shock, angioneurotic edema, allergy unspecified, defects in the complement system, or unspecified drug adverse effects were included. Demographic, clinical, and outcome data were collected via a standardized form. Data were analyzed descriptively, including frequencies and percentages for categorical data and means and SDs for continuous data. Predictors for admission were identified using multivariate logistic regression models. ED records from 527 visits for angioedema by 455 patients were included in the study. Annual rate of angioedema was 1 per 1000 ED visits. Urticaria was associated with peripheral (p = 0.008) and lip angioedema (p = 0.001), and the absence of urticaria correlated with tongue angioedema (p = 0.001) and trended toward correlation with pharyngeal angioedema (p = 0.056). Significant predictors of admission included nonsteroidal anti-inflammatory drug-induced angioedema (odds ratio [OR], 15.3), epinephrine treatment (OR, 8.34), hypotension (OR, 15.7), multiple-site angioedema (OR, 4.25), and pharyngeal (OR, 1.23) and tongue angioedema (OR, 4.62). Concomitant urticaria was associated with a significant longer stay in the ED (p < 0.001). The presence of urticaria correlated with the location of angioedema, need for airway management, length of ED visit, and recurrence. A detailed drug and family history, screening blood work for C1 esterase inhibitor deficiency when indicated, and prompt management of angioedema based on presumed mechanism of swelling are crucial steps in managing undifferentiated angioedema in ED. |
| 2015 | Temporal change of enhancement after gadolinium injection on contrast-enhanced CMR in reperfused acute myocardial infarction. | J Cardiol | A recent report demonstrated that early enhancement on contrast-enhanced cardiac magnetic resonance (CE-CMR) correlated with myocardial edema detected by T2-weighted CMR in reperfused acute myocardial infarction (AMI). However, the time at which the enhancement in salvaged myocardium disappears is yet to be determined. We aimed to examine the time course of the enhancement with the use of different quantification techniques and to compare the extent of enhancement with the myocardial edema.CE-CMR was performed at 2-20 min after gadolinium administration in 32 AMI patients. The extent of enhancement (% myocardium) was quantified by manual delineation and the threshold methods of 2-5 SDs above remote myocardium. In subendocardial infarct, the enhancement was greatest at 2 min regardless of the quantification techniques and decreased with time, particularly in the first 6 min. In transmural infarct, the change in the size of enhancement was modest although the time course of enhancement varied according to the quantification techniques. The sizes of enhancement were not significantly different between 15 and 20 min regardless of the techniques and infarct transmurality. The best agreement with myocardial edema was found at 2 min with average differences of 0.5% and -1.2% and limits of agreement of ±20.2% and ±21.2% for the manual and 2-SD techniques, respectively.The optimal timing for delineation of salvaged myocardium on CE-CMR is at 2min when the manual or 2-SD technique was employed. Imaging needs to be completed in a short time (ideally within a minute) because of rapid reduction of enhancement in salvaged myocardium. |
| 2014 | A comparison of the ability of Bellucia dichotoma Cogn. (Melastomataceae) extract to inhibit the local effects of Bothrops atrox venom when pre-incubated and when used according to traditional methods. | Toxicon | Bellucia dichotoma Cogn. (Melastomataceae) is one of various plant species used in folk medicine in the west of the state of Pará, Brazil, to treat snake bites. Many studies have been carried out to evaluate the effectiveness of anti-snake bite plants, but few of these use the same preparation methods and doses as those traditionally used by the local populations. This study therefore compared inhibition of the main local effects of B. atrox venom (BaV) by aqueous extract of B. dichotoma (AEBd) administered according to traditional methods and pre-incubated with BaV). The concentrations of phenolic compounds (tannins and flavonoids) in AEBd were determined by colorimetric assays. The effectiveness of AEBd in inhibiting the hemorrhagic and edematogenic activities of BaV was evaluated in mice in four different experimental in vivo protocols: (1) pre-incubation (venom:extract, w/w); (2) pre-treatment (p.o.); (3) post-treatment (p.o.); and (4) AEBd (p.o.) in combination with Bothrops antivenom (BA) (i.v.). To assess in vitro inhibition of BaV phospholipase A₂ activity, the pre-incubation method or incorporation of AEBd or BA in agarose gels were used. The effect of AEBd on BaV was determined by SDS-PAGE, zymography and Western blot. Colorimetric assays revealed higher concentrations of (condensed and hydrolyzable) tannins than flavonoids in AEBd. Hemorrhagic activity was completely inhibited using the pre-incubation protocol. However, with pre-treatment there was no significant inhibition for the concentrations tested, and with the post-treatment only the 725 mg/kg dose of AEBd was able to inhibit 40.5% (p = 0.001) of the hemorrhagic activity of BaV. Phospholipase A₂ activity was only inhibited when AEBd was pre-incubated with BaV. BaV-induced edema was completely inhibited with pre-incubation (p < 0.05) and significantly reduced (p < 0.05) with pre- and post-treatment (p.o.) for the concentrations tested. The reduction in local edema was even greater when AEBd was administered in combination with BA. The SDS-PAGE profiles showed that several of the BaV protein (SDS-PAGE) and enzyme (zymography) bands were not detected when the venom was pre-incubated, and Western blot revealed that this was not caused by the AEBd enzymes observed in the zymogram. The "pseudo inhibition" observed after pre-incubation in this study may be due to the presence of tannins in the extract, which could act as chelating agents, removing metalloproteins and Ca²⁺ ions and thus inhibiting hemorrhagin and PLA₂ activity. However, when administered according to traditional methods, B. dichotoma extract was effective in blocking BaV-induced edematogenic activity and had an additional effect on inhibition of this activity by BA. |
| 2013 | Using a Caesalpinia echinata Lam. protease inhibitor as a tool for studying the roles of neutrophil elastase, cathepsin G and proteinase 3 in pulmonary edema. | Phytochemistry | Acute lung injury (ALI) is characterized by neutrophil infiltration and the release of proteases, mainly elastase (NE), cathepsin G (Cat G) and proteinase 3 (PR3), which can be controlled by specific endogenous inhibitors. However, inhibitors of these proteases have been isolated from different sources, including plants. For this study, CeEI, or Caesalpinia echinata elastase inhibitor, was purified from C. echinata (Brazil-wood) seeds after acetone fractionation, followed by ion exchange and reversed phase chromatographic steps. Characterization with SDS-PAGE, stability assays, amino acid sequencing and alignment with other protein sequences confirmed that CeEI is a member of the soybean Kunitz trypsin inhibitor family. Like other members of this family, CeEI is a 20 kDa monomeric protein; it is stable within a large pH and temperature range, with four cysteine residues forming two disulfide bridges, conserved amino acid residues and leucine-isoleucine residues in the reactive site. CeEI was able to inhibit NE and Cat G at a nanomolar range (with K(i)s of 1.9 and 3.6 nM, respectively) and inhibited PR3 within a micromolar range (K(i) 3.7 μM), leading to hydrolysis of specific synthetic substrates. In a lung edema model, CeEI reduced the lung weight and pulmonary artery pressure until 180 min after the injection of zymosan-activated polymorphonuclear neutrophils. In experiments performed in the presence of a Cat G and PR3, but not an NE inhibitor, lung edema was reduced only until 150 min and pulmonary artery pressure was similar to that of the control. These results confirm that NE action is crucial to edema establishment and progression. Additionally, CeEI appears to be a useful tool for studying the physiology of pulmonary edema and provides a template for molecular engineering and drug design for ALI therapy. |
| 2013 | Suppression of severe lesions, myonecrosis and hemorrhage, caused by Protobothrops flavoviridis venom with its serum proteins. | Toxicon | Protobothrops flavoviridis serum proteins precipitated with ammonium sulfate were chromatographed on a DEAE-Toyopearl 650M column at pH 7.5 with stepwise increase or with linear gradient of NaCl concentration. Peaks 3 and 4 serum proteins, obtained by linear gradient elution and named Fr(de3) and Fr(de4), contained Habu serum factors (HSF) and phospholipase A2 (PLA2) inhibitors (PfPLI), respectively. The serum proteins eluted at 0.2 M NaCl by stepwise elution, named Fr(0.2NaCl), effectively suppressed myonecrosis and hemorrhage caused by P. flavoviridis venom in rat or mouse thigh muscles. The Fr(0.2NaCl) were fractionated by HPLC and the fractions, after SDS-PAGE, underwent far-western blot analysis with PLA2 ([Asp(49)]PLA2) and BPI ([Lys(49)]PLA2) as the probes. Four PfPLIs, namely, PfαPLI-A, PfαPLI-B, PfγPLI-A and PfγPLI-B, were identified together with their selective binding specificities to PLA2 species. In addition, a new 9 kDa protein, which is specifically bound to BPI, was found. Suppression of P. flavoviridis venom-induced severe lesions, such as myonecrosis, hemorrhage and edema, with its serum proteins was histopathologically observed in the present work for the first time. The cooperative use of P. flavoviridis antivenom and its serum proteins as medication for P. flavoviridis snake bites is discussed. |
| 2013 | Acute and delayed protective effects of pharmacologically induced hypothermia in an intracerebral hemorrhage stroke model of mice. | Neuroscience | Hemorrhagic stroke, including intracerebral hemorrhage (ICH), is a devastating subtype of stroke; yet, effective clinical treatment is very limited. Accumulating evidence has shown that mild to moderate hypothermia is a promising intervention for ischemic stroke and ICH. Current physical cooling methods, however, are less efficient and often impractical for acute ICH patients. The present investigation tested pharmacologically induced hypothermia (PIH) using the second-generation neurotensin receptor (NTR) agonist HPI-201 (formerly known as ABS-201) in an adult mouse model with ICH. Acute or delayed administrations of HPI-201 (2mg/kg bolus injection followed by 2 injections of 1mg/kg, i.p.) were initiated at 1 or 24h after ICH. HPI-201 induced mild hypothermia within 30 min and body and brain temperatures were maintained at 32.7 ± 0.4°C for at least 6h without causing observable shivering. With the 1-h delayed treatment, HPI-201-induced PIH significantly reduced ICH-induced cell death and brain edema compared to saline-treated ICH animals. When HPI-201-induced hypothermia was initiated 24h after the onset of ICH, it still significantly attenuated brain edema, cell death and blood-brain barrier breakdown. HPI-201 significantly decreased the expression of matrix metallopeptidase-9 (MMP-9), reduced caspase-3 activation, and increased Bcl-2 expression in the ICH brain. Moreover, ICH mice received 1-h delayed HPI-201 treatment performed significantly better in the neurological behavior test 48 h after ICH. All together, these data suggest that systemic injection of HPI-201 is an effective hypothermic strategy that protects the brain from ICH injury with a wide therapeutic window. The protective effect of this PIH therapy is partially mediated through the alleviation of apoptosis and neurovascular damage. We suggest that pharmacological hypothermia using the newly developed neurotensin analogs is a promising therapeutic treatment for ICH. |
| 2014 | Biochemical and biological analysis of Philodryas baroni (Baron's green racer; Dipsadidae) venom: relevance to the findings of human risk assessment. | Hum Exp Toxicol | Philodryas baroni--an attractively colored snake--has become readily available through the exotic pet trade. Most people consider this species harmless; however, it has already caused human envenomation. As little is known about the venom from this South American opisthoglyphous "colubrid" snake, herein, we studied its protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as its effects on the hemostatic system. Both reducing and nonreducing SDS-PAGE analysis demonstrated that the venom exhibits greatest complexity in the range of 50-80 kDa. The venom displayed proteolytic activity toward azocollagen, with a specific activity of 75.5 U mg⁻¹, and rapidly hydrolyzed the Aα-chain of fibrinogen, exhibiting lower activity toward the Bβ- and γ-chains. The venom from P. baroni showed no platelet proaggregating activity per se, but it inhibited collagen- and thrombin-induced platelet aggregation. Prominent hemorrhage developed in mouse skin after intradermal injection of the crude venom, and its minimum hemorrhagic dose was 13.9 μg. When injected intramuscularly into the gastrocnemius of mice, the venom induced local effects such as hemorrhage, myonecrosis, edema, and leucocyte infiltration. Due to its venom toxicity shown herein, P. baroni should be considered dangerous to humans and any medically significant bite should be promptly reviewed by a qualified health professional. |
| 2013 | Comparison of venom composition and biological activities of the subspecies Crotalus lepidus lepidus, Crotalus lepidus klauberi and Crotalus lepidus morulus from Mexico. | Toxicon | The rock rattlesnakes Crotalus lepidus comprise a group (lepidus, klauberi, morulus and maculosus) of poorly known mountain cold-tolerant snakes in Mexico. In particular, Crotalus lepidus morulus is a snake endemic of the northeast of Mexico, whereas Crotalus lepidus klauberi and C. l. lepidus are distributed in some regions of the north and central Mexico and southern U. S. Until now very little data are available from C. lepidus subspecies from Mexico, as the terrain inhabited by these snakes is generally steep and rugged. In this work, we have determined some biochemical and biological properties of C. l. morulus, C. l. klauberi and C. l. lepidus crude venoms. Some minor differences in venoms were noted in SDS-PAGE, HPLC profile and MALDI-TOF mass spectrometry analysis. Partial sequences of metalloproteinases, phospholipases A₂ (PLA₂) and galactose-specific lectins were identified in the venoms. Venoms of C. l. klauberi and C. l. lepidus had significantly higher hemorrhagic and lethal activities than C. l. morulus venom. Proteolytic activity in azocasein was higher in C. l. morulus venom, whereas gelatin hydrolysis was higher in C. l. klauberi. Fibrinogenolytic and PLA₂ activities were very similar in all venoms tested. The histological observations in the gastrocnemius muscle damaged by venoms from all the subspecies confirmed myonecrotic and hemorrhagic activities (at 3 and 24 h), which resulted in a poor regenerative response after 14 days. However, C. l. lepidus and C. l. klauberi venom induced a higher increase in the plasma activity of creatine kinase (CK), evidencing higher myotoxicity, whereas paw edema-inducing activity was higher in C. l. lepidus venom. The results indicate that the venoms from the three subspecies have similar protein profiles in electrophoresis, HPLC and molecular weight determinations. However, differences were found in the biological activities in mice. Notably, the venoms of C. l. lepidus and C. l. klauberi present higher toxicity (lower LD₅₀) and hemorrhagic activity than C. l. morulus venom. |
| 2013 | Biochemical and immunological characteristics of Peruvian Loxosceles laeta spider venom: neutralization of its toxic effects by anti-loxoscelic antivenoms. | Toxicon | This manuscript describes the general biochemical properties and immunological characteristics of Peruvian spider Loxosceles laeta venom (PLlv), which is responsible for the largest number of accidents involving venomous animals in Peru. In this work, we observed that the venom of this spider is more lethal to mice when compared with L. laeta venom from Brazil (BLlv). The LD₅₀ of PLlv was 1.213 mg/kg when the venom was intradermally injected. The venom displayed sphingomyelinase activity and produced dermonecrotic, hemorrhagic and edema effects in rabbits. 2-D SDS-PAGE separation of the soluble venoms resulted in a protein profile ranging from 20 to 205 kDa. Anti-PLlv and anti-BLlv sera produced in rabbits and assayed by ELISA showed that rabbit antibodies cross-reacted with PLlv and BLlv and also with other Brazilian Loxosceles venoms. Western blotting analysis showed that bands corresponding to 25-35 kDa are the proteins best recognized in every Loxosceles spp venoms analyzed. The immunized rabbits displayed protective effect after challenge with PLlv and BLlv. In vitro assays with horse anti-loxoscelic antivenoms produced in Brazil and Peru demonstrated that these commercial antivenoms were efficient to inhibit the sphingomyelinase activity of PLlv and BLlv. |
| 2013 | Isolation and in vitro partial characterization of hemolytic proteins from the nematocyst venom of the jellyfish Stomolophus meleagris. | Toxicol In Vitro | Jellyfish venom contains various toxins and can cause itching, edema, muscle aches, shortness of breath, blood pressure depression, shock or even death after being stung. Hemolytic protein is one of the most hazardous components in the venom. The present study investigated the hemolytic activity of the nematocyst venom from jellyfish Stomolophus meleagris. Anion exchange chromatography, DEAE Sepharose Fast Flow, and gel filtration chromatography, Superdex200 had been employed to isolate hemolytic proteins from the nematocyst venom of jellyfish S. meleagris. Hemolysis of chicken red blood cells was used to quantify hemolytic potency of crude nematocyst venom and chromatography fractions during the purification process. Native-PAGE profile displayed one protein band in the purified hemolytic protein (SmTX); however, two protein bands with apparent molecular weights of ≈ 45 kDa and 52 kDa were observed in the reducing SDS-PAGE analysis. Approximately 70 μg/mL of SmTX caused 50% hemolysis (HU50) of the erythrocyte suspension. The hemolytic activity of SmTX was shown to be temperature and pH dependent, with the optimum temperature and pH being 37°C and pH 5.0. The present study is the first report of isolation and partial characterization of hemolytic proteins from the nematocyst venom of the jellyfish S. meleagris. The mechanism of the hemolytic activity of SmTX is not clear and deserves further investigation. |
| 2013 | Unmasking snake venom of Bothrops leucurus: purification and pharmacological and structural characterization of new PLA2 Bleu TX-III. | Biomed Res Int | Bleu TX-III was isolated from Bothrops leucurus snake venom on one-step analytical chromatography reverse phase HPLC, was homogeneous on SDS-PAGE, and was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry in 14243.8 Da. Multiple alignments of Bleu TX-III show high degree of homology with basic PLA2 myotoxins from other Bothrops venoms. Our studies on local and systemic myotoxicity "in vivo" reveal that Bleu TX-III is myotoxin with local but not systemic action due to the decrease in the plasmatic CK levels when Bleu TX-III is administrated by intravenous route in mice (dose 1 and 5 μg). And at a dose of 20 μg myotoxin behaves like a local and systemic action. Bleu TX-III induced moderate marked paw edema, evidencing the local increase in vascular permeability. The inflammatory events induced in the mice (I.M.) were investigated. The increase in the levels of IL-1, IL-6, and TNF-α was observed in the plasma. It is concluded that Bleu TX-III induces inflammatory events in this model. The enzymatic phospholipid hydrolysis may be relevant to these phenomena. Bothrops leucurus venom is still not extensively explored, and the knowledge of its toxins separately through the study of structure/function will contribute for a better understanding of its action mechanism. |
| 2012 | Retinal thickness in people with diabetes and minimal or no diabetic retinopathy: Heidelberg Spectralis optical coherence tomography. | Invest Ophthalmol Vis Sci | To evaluate macular thickness in people with diabetes but minimal or no retinopathy using Heidelberg Spectralis optical coherence tomography (OCT).In a multicenter, cross-sectional study of mean retinal thickness, on Spectralis OCT in the nine standard OCT subfields, spanning a zone with 6-mm diameter, center point, and total retinal volume were evaluated. Central subfield (CSF) thickness was evaluated for association with demographic and clinical factors. Stratus OCT scans also were performed on each participant.The analysis included 122 eyes (122 participants) with diabetes and no (n = 103) or minimal diabetic retinopathy (n = 19) and no macular retinal thickening on clinical exam. Average CSF thickness was 270 ± 24 μm. Central subfield thickness was significantly greater in males relative to females (mean 278 ± 23 μm vs. 262 ± 22 μm, P < 0.001). After adjusting for gender, no additional factors were found to be significantly associated with CSF thickness (P > 0.10). Mean Stratus OCT CSF thickness was 199 ± 24 μm.Mean CSF thickness is approximately 70 μm thicker when measured with Heidelberg Spectralis OCT as compared with Stratus OCT among individuals with diabetes in the absence of retinopathy or with minimal nonproliferative retinopathy and a normal macular architecture. CSF thickness values ≥ 320 μm for males and 305 μm for females (~2 SDs above the average for this normative cohort) are proposed as gender-specific thickness levels to have reasonable certainty that diabetic macular edema involving the CSF is present using Spectralis measurements. |
| 2012 | The mode of action of isocyanide in three aquatic organisms, Balanus amphitrite, Bugula neritina and Danio rerio. | PLoS One | Isocyanide is a potential antifouling compound in marine environments. In this study, we investigated its mode of action in three aquatic organisms. Two of them, the bryozoan Bugula neritina and the barnacle Balanus amphitrite, are major marine fouling invertebrates, and the other organism is the non-target species zebrafish Danio rerio. In the swimming larvae of B. neritina, isocyanide did not affect the total attachment rate (≤50 µg ml(-1)), but it did change the attachment site by increasing the percentage of attachment on the bottom of the container rather than on the wall or air-water inter-surface. Isocyanide binds several proteins in B. neritina as identified via SDS-PAGE-LC-MS/MS: 1) a 30 kD protein band containing two proteins similar to voltage dependent anion channels (VDAC), which control the direct coupling of the mitochondrial matrix to the energy maintenance of the cytosol and the release of apoptogenic factors from mitochondria of mammalian cells; and 2) an unknown 39 kD protein. In B. amphitrite cyprids, the isocyanide binding protein were 1) a protein similar to NADH-ubiquinone oxidoreductase, which is the "entry enzyme" of oxidative phosphorylation in mitochondria; and 2) cytochrome P450. In Danio rerio embryos, isocyanide caused "wavy" notochords, hydrocephalus, pericardial edema, poor blood circulation, and defects in pigmentation and hematopoiesis, which phenocopied copper deficiency. This is the first report on isocyanide binding proteins in fouling organisms, as well as the first description of its phenotype and potential toxicology in zebrafish. |
| 2012 | Purification and biological activities of Abelmoschus esculentus seed lectin. | Protein J | The Abelmoschus esculentus (Malvaceae) plant originated in Africa and has spread across a number of tropic countries, including northeastern Brazil. The plant has been used to treat various disorders, such as cancer, microbial infections, hypoglycemia, constipation, urine retention and inflammation. The lectin of A. esculentus (AEL) was isolated by precipitation with ammonium sulfate at a saturation level of 30/60 and purified by ion exchange chromatography (Sephacel-DEAE). The electrophoresis (SDS-PAGE) profile of the AEL showed two protein bands of apparent molecular mass of approximately 15.0 and 21.0 kDa. The homogenity of the protein was confirmed by electrospray mass spectrometry (ESI-MS), which revealed the presence of a 10.29-kDa monomer and a 20.58-kDa dimer. The AEL exhibits agglutinating activity against rabbit (74.41 UH/mP) and human type ABO erythrocytes (21.00 UH/mP). This activity does not require the presence of divalent cations and is specifically inhibited by lactose, fructose and mannose. The intravenous treatment with 0.01, 0.1 and 1 mg/kg of AEL inhibited the paw edema elicited by carrageenan by approximately 15, 22 and 44 %, respectively, but not that induced by dextran. In addition, treatment with 0.1, 1 and 10 mg/kg of AEL also inhibited the abdominal writhing induced by acetic acid by approximately 52, 57 and 69 %, respectively. In conclusion, AEL is a new lectin with a molecular mass of 20.0 kDa, which is -composed of a 10.291-Da monomer and a 20.582-kDa dimer, that exhibits anti-inflammatory, antinociceptive and hemagglutinating activities. In addition, the lectin hemagglutinating property is both metallo-independent and associated with the lectin domain. |
| 2012 | Small molecule inhibitors of Bacillus anthracis protective antigen proteolytic activation and oligomerization. | J Med Chem | Protective antigen (PA), lethal factor, and edema factor, the protein toxins of Bacillus anthracis , are among its most important virulence factors and play a key role in infection. We performed a virtual ligand screen of a library of 10000 members to identify compounds predicted to bind to PA and prevent its oligomerization. Four of these compounds slowed PA association in a FRET-based oligomerization assay, and two of those protected cells from intoxication at concentrations of 1-10 μM. Exploration of the protective mechanism by Western blot showed decreased SDS-resistant PA oligomer on cells and, surprisingly, decreased amounts of activated PA. In vitro assays showed that one of the inhibitors blocked furin-mediated cleavage of PA, apparently through its binding to the PA substrate. Thus, we have identified inhibitors that can independently block both PA's cleavage by furin and its subsequent oligomerization. Lead optimization on these two backbones may yield compounds with high activity and specificity for the anthrax toxins. |
| 2012 | Improved dissolution and anti-inflammatory effect of ibuprofen by solid dispersion. | Front Med | The purpose of this study was to improve the dissolution rate and anti-inflammatory effect of ibuprofen by a solid dispersion (SD) method. Initial screening was developed based on drug solubility in carriers in the liquid state to select a suitable water-soluble carrier system for the preparation of SDs. The dissolution of ibuprofen in urea was higher than in PEG4000 or mannitol. Thus, urea was selected as the carrier for the preparation of SDs. SDs were characterized in terms of dissolution, differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) spectroscopy. Solid dispersion-based (SDBT) and conventional (CT) tablets were prepared by the wet granulation method. The anti-inflammatory effect of SDBT was evaluated using the mouse ear edema test with xylene. In vitro release results indicated that the ibuprofen dissolution rate was improved by the SD. SD characterization results suggested that ibuprofen partly precipitates in crystalline and amorphous forms after SD preparation and that ibuprofen and urea do not interact. SDBT displayed more significant anti-inflammatory effects than CT. The dissolution rate and anti-inflammatory effect of ibuprofen were significantly enhanced by the ibuprofen-urea SD. |
| 2012 | Antinociceptive and anti-inflammatory effects of a lectin-like substance from Clitoria fairchildiana R. Howard seeds. | Molecules | Lectins are proteins that have the ability to bind specifically and reversibly to carbohydrates and glycoconjugates, without altering the structure of the glycosyl ligand. They are found in organisms such as viruses, plants and humans, and they have been shown to possess important biological activities. The objective of this study was to purify and characterize lectins in the seeds of Clitoria fairchildiana, as well as to verify their biological activities. The results indicated the presence of a lectin (CFAL) in the glutelin acid protein fraction, which agglutinated native rabbit erythrocytes. CFAL was purified by column chromatography ion-exchange, DEAE-Sephacel, which was obtained from a peak of protein retained in the matrix by applying 0.5 M NaCl using the step-wise method. Electrophoretic analysis of this lectin in SDS-PAGE indicated a two band pattern protein molecular mass of approximately 100 and 116 kDa. CFAL proved to be unspecific to all carbohydrates/glycoconjugates in common use for the sugar inhibition test. This lectin showed no significant cytotoxicity to human red blood cells. It was observed that CFAL has anti-inflammatory activity in the paw edema induced by carrageenan model, in which a 64% diminution in edema was observed. Antinociceptive effects were observed for CFAL in the abdominal writhing test (induced by acetic acid), in which increasing doses of the lectin caused reduction in the number of contortions by up to 72%. It was concluded that the purified and characterized lectin from the seeds of Clitoria fairchildiana has anti-inflammatory and antinociceptive activity, and is not cytotoxic to human erythrocytes. |
| 2012 | Site-specific chemical modification of human serum albumin with polyethylene glycol prolongs half-life and improves intravascular retention in mice. | Biol Pharm Bull | Human serum albumin (HSA) is used as an important plasma volume expander in clinical practice. However, the infused HSA may extravasate into the interstitial space and induce peripheral edema in treating the critical illness related to marked increase in capillary permeability. Such poor intravascular retention also demands a frequent administration of HSA. We hypothesize that increasing the molecular weight of HSA by PEGylation may be a potential approach to decrease capillary permeability of HSA. In the present study, HSA was PEGylated in a site-specific manner and the PEGylated HSA carrying one chain of polyethylene glycol (PEG) (20 kDa) per HSA molecule was obtained. The purity, PEGylated site and secondary structure of the modified protein were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), thiol group blockage method and circular dichroism (CD) measurement, respectively. In addition, the pharmacokinetics in normal mice was investigated, vascular permeability of the PEGylated HSA was evaluated in lipopolysaccharide (LPS)-induced lung injury mouse model and the pharmacodynamics was investigated in LPS-induced sepsis model with systemic capillary leakage. The results showed that the biological half-life of the modified HSA was approximately 2.3 times of that of the native HSA, PEG-HSA had a lower vascular permeability and better recovery in blood pressure and haemodilution was observed in rats treated with PEG-HSA. From the results it can be inferred that the chemically well-defined and molecularly homogeneous PEGylated HSA is superior to HSA in treating capillary permeability increase related illness because of its longer biological half-life and lower vascular permeability. |
| 2011 | Purification of a lectin from Arisaema erubescens (Wall.) Schott and its pro-inflammatory effects. | Molecules | The monocot lectin from the tubers of Arisaema erubescens (Wall.) Schott has been purified by consecutive hydrophobic chromatography and ion exchange chromatography methods. The molecular weight of this A. erubescens lectin (AEL) was determined to be about 12 kDa by high performance liquid chromatography (HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) methods. AEL could agglutinate rabbit erythrocytes. The haemagglutination activity of AEL was only inhibited by asialofetuin, while monosaccharide did not react. Rat paw edema and neutrophil migration models were used to investigate the pro-inflammatory activity of AEL. AEL (100 and 200 μg/paw) could induce significant rat paw edema. In addition, AEL (100, 200 and 300 μg/mL/cavity) could induce significant and dose-dependent neutrophil migration in the rat peritoneal cavities. Besides, AEL at doses ranging from 100 to 300 μg/mL/cavity could significantly increase the concentration of nitric oxide (NO), prostaglandin E(2 )(PGE(2)) and tumor necrosis factor alpha (TNF-α) in peritoneal fluid. As compared with control animals, 75% depletion in the number of resident cells following peritoneal lavage did not reduce the AEL-induced neutrophil migration. However, pre-treatment with 3% thioglycollate which increased the peritoneal macrophage population by 201%, enhanced the neutrophil migration induced by AEL (200 μg/mL/cavity) (p < 0.05). Reduction of peritoneal mast cell population by chronic treatment of rat peritoneal cavities with compound 48/80 (N-methyl-p-methoxyphenethylamine with formaldehyde) did not modify AEL-induced neutrophil migration. The results provided the basis for identifying the toxic components of A. erubescens and AEL could be a new useful tool for pro-inflammatory research. |
| 2011 | Purification and partial characterization of low molecular weight vicilin-like glycoprotein from the seeds of Citrullus lanatus. | Protein J | The watermelon (Citrullus lanatus) seeds are highly nutritive and contain large amount of proteins and many beneficial minerals such as magnesium, calcium, potassium, iron, phosphorous, zinc etc. In various parts of the world, C. lanatus seed extracts are used to cure cancer, cardiovascular diseases, hypertension, and blood pressure. C. lanatus seed extracts are also used as home remedy for edema and urinary tract problems. In this study, we isolated protein fraction of C. lanatus seeds using various protein separation methods. We successfully purified a low molecular weight vicilin-like glycoprotein using chromatographic methods followed by SDS-PAGE and MALDI-TOF/MS identification. This is the first report of purification of a vicilin like polypeptide from C. lanatus seeds. In next step, we extracted mRNA from immature seeds and reverse transcribed it using suitable forward and reverse primers for purified glycoprotein. The PCR product was analysed on 1% agarose gel and was subsequently sequenced by Dideoxy DNA sequencing method. An amino acid translation of the gene is in agreement with amino acid sequences of the identified peptides. |
| 2011 | Isolation and functional characterization of proinflammatory acidic phospholipase A2 from Bothrops leucurus snake venom. | Comp Biochem Physiol C Toxicol Pharmacol | In the present study, an acidic PLA(2), designated Bl-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000Da and pI was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-α, IL-1β and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA(2) induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism. |
| 2011 | Structural and functional characterization of a γ-type phospholipase A2 inhibitor from bothrops jararacussu snake plasma. | Curr Top Med Chem | Phospholipases A2 (PLA2s) from snake venoms comprise a group of 14-18 kDa proteins, responsible for several toxic effects induced by the whole venom. Considering this, studies aiming at the search for natural inhibitors of these proteins are very important. The present work had as objectives the isolation and functional/structural characterization of a γ-type phospholipase A2 inhibitor (PLI) from Bothrops jararacussu snake plasma, named γBjussuMIP. This acidic glycoprotein was isolated in a high purity level through affinity chromatography on CNBr-Sepharose 4B coupled with BthTXII, showing a pI ∼ 5.5 and molecular weight of 23,500 for the monomer (determined by SDS-PAGE), and 160,000 for the oligomer (determined by molecular exclusion chromatography on Sephacryl S-200). The interaction between γBjussuMIP (MIP) and Phospholipase A2 (PLA2) was confirmed using circular dichroism (CD) and emission fluorescence techniques. The helical content of the 1:1 molar mixture was higher than that calculated for the addition of the spectra of the unbound proteins indicating binding. The emission fluorescence experiments pointed that Trp residues in PLA2 participate in proteins interaction as blue shift of 4 nm was observed. The γBjussuMIP cDNA, obtained by PCR of the liver of B. jararacussu snake, revealed 543 bp codifying for a mature protein of 181 amino acid residues. Alignment of its amino acid sequence with those of other snake γPLIs showed 89-94% of similarity. γBjussuMIP mainly inhibited the pharmacological properties of Asp49 PLA2s, such as phospholipase, anticoagulant, myotoxic, edema inducing, cytotoxic, bactericidal and lethal activities. In addition, it showed to be able to supplement Bothrops antivenom, potentiating its antimyotoxic effect. The aspects broached in this work will be able to provide complementary information on possible mechanisms of action, relating structure and function, which could result in a better understanding of the inhibitory effects induced by γBjussuMIP. |
| 2011 | Hypothalamic Obesity following Craniopharyngioma Surgery: Results of a Pilot Trial of Combined Diazoxide and Metformin Therapy. | Int J Pediatr Endocrinol | Objective. To assess the effect of combined diazoxide-metformin therapy in obese adolescents treated for craniopharyngioma. Design. A prospective open-label 6-month pilot treatment trial in 9 obese subjects with craniopharyngioma. Diazoxide (2 mg/kg divided b.i.d., maximum 200 mg/day) and metformin (1000 mg b.i.d.). Whole body insulin sensitivity index (WBISI) and area-under-the-curve insulin (AUC(ins)) were calculated. Results. Seven subjects completed: 4M/3F, mean ± SD age 15.4 ± 2.9 years, weight 99.7 ± 26.3 kg, BMI 35.5 ± 5.6 kg/m(2), and BMI SDS 2.3 ± 0.3. Two were withdrawn due to vomiting and peripheral edema. Of participants completing the study, the mean ± SD weight gain, BMI, and BMI SDS during the 6 months were reduced compared to the 6 months prestudy (+1.2 ± 5.9 versus +9.5 ± 2.7 kg, P = .004; -0.3 ± 2.3 versus +2.2 ± 1.5 kg/m(2), P = .04; -0.04 ± 0.15 versus +0.11 ± 0.08, P = .021, resp.). AUC(ins) correlated with weight loss (r = 0.82, P = .02) and BMI decrease (r = 0.96, P = .009). Conclusion. Combined diazoxide-metformin therapy was associated with reduced weight gain in patients with hypothalamic obesity. AUC(ins) at study commencement predicted effectiveness of the treatment. |
| 2011 | Long term clinical management of girls with Turner syndrome at a center of pediatric endocrinology. | Exp Clin Endocrinol Diabetes | To evaluate clinical management of patients with Turner syndrome in one center over a long period.Retrospective analysis of 89 patients cared for between 1974 and 2004. Assessment of age and height at diagnosis, indications for karyotyping, induction of puberty and final height attainment.Average age at diagnosis was 8.21 years, with a significant decline over the observation period. Mean height SDS at diagnosis was -2.86. Main reasons for karyotyping were edema in youngest ages and growth retardation in ages older than 6 years. Puberty induction was started at a mean age of 13.93 years, with a significant decline over the observation period. Mean duration until menarche was 2.51 years. An appropriate clinical response with changes in Tanner stages was observed. Mean final height after GH therapy was 151.81 cm, height SDS for TS was increased by +1.82.In recent study years, Turner Syndrome is being diagnosed at younger ages and at heights closer to normal heights. The clinical spectrum warrants karyotyping at an early age. In spite of late diagnoses, puberty induction was started within a physiological age in recent years and was, just as GH therapy, successful to mimic physiological progress in most instances. |
| 2011 | Biochemical and pharmacological characterization of PhTX-I a new myotoxic phospholipase A2 isolated from Porthidium hyoprora snake venom. | Comp Biochem Physiol C Toxicol Pharmacol | This paper reports the biochemical and pharmacological characterization of a new myotoxic PLA(2) (EC 3.1.1.4) called PhTX-I, purified from Porthidium hyoprora venom by one step analytical chromatography reverse phase HPLC. The homogeneity of the PhTX-I fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14.249Da and constituted of a single polipeptidic chain. Amino acid sequence was determined by "de novo sequencing," in tandem mass spectrometry, belonging to D49-PLA(2) enzyme class and exhibiting high identity (44-90%) with other myotoxics PLA(2) from snake venoms. The enzymatic investigation showed maximal activity at pH 8 and 35-45°C. This activity was dependent on Ca(2+), other cations (Mg(2+), Mn(2+), Cd(2+) and Zn(2+)) reduced notably the enzymatic activity, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca(2+). Ex vivo, whole venom and PhTX-I PLA(2) caused blockade of the neuromuscular transmission in young chick biventer cervicis preparations similar to other isolated snake venom toxins from the Bothrops genus. In vivo, both induced local myotoxicity and systemic interleukin-6 response upon intramuscular injection, additionally, induced moderate footpad edema. In vitro, both induced low cytotoxicity in skeletal muscle myoblasts, however PhTX-I PLA(2) was able to lyse myotubes. |
| 2011 | Purification and characterization of a metalloproteinase, Porthidin-1, from the venom of Lansberg's hog-nosed pitvipers (Porthidium lansbergii hutmanni). | Toxicon | Porthidium lansbergii hutmanni is a small pit viper found on Margarita Island, Venezuela. Local tissue damage is one of the most obvious characteristics of P. l. hutmanni envenomation, which can lead to diverse pathological effects, such as hemorrhage, edema, blistering, necrosis, lymphatic vessel damage and degradation of extracellular matrix. Metalloproteinases are one of the major components in venoms responsible for these effects. To date, very little is known or has been reported on P. l. hutmanni venom. Crude P. l. hutmanni venom had a LD(50) of 2.5 mg/kg and was considered very hemorrhagic (minimal hemorrhagic dose [MHD]: 0.98 μg) when compared to other hemorrhagic (Bothrops) venoms in Venezuela. Crude P. l. hutmanni venom also inhibited ADP-induced platelet aggregation. A metalloproteinase, Porthidin-1, from this venom was isolated by three chromatography steps (Sephadex G100, Superose 12 HR10/30 and Bioscale Q2). Porthidin-1 falls in the SVMP P-I class having a molecular weight of 23 kDa, verified by both SDS-PAGE and mass spectrometry. High-resolution mass spectrometry and a database search identified a peptide from Porthidin-1 (YNGDLDK) belonging to the SVMP family of proteins. Porthidin-1 contained hemorrhagic, fibrino(geno)lytic, caseinolytic and gelatinolytic activities, and these activities were capable of being neutralized by metalloproteinase inhibitors but not serine proteinase inhibitors. The peptide YNGDLDK shared similarities with five venom proteins with a BLAST e-value of <1. This work details the biochemical and pathophysiological effects that can result from envenomations, and highlights the importance and significance for characterizing unknown or poorly documented venoms from different geographical regions. |
| 2011 | Fibrinogenolytic and anticoagulant activities in the tissue covering the stingers of marine stingrays Dasyatis sephen and Aetobatis narinari. | J Thromb Thrombolysis | Stingray envenomation is one of the major problems in the marine and freshwater ecosystem. Accidents in human cause immediate, local and intense pain, erythema, edema, hemorrhage, tissue necrosis and secondary bacterial infection are also common. To determine the effect of two marine stingray species Dasyatis sephen and Aetobatis narinari venom extract on coagulation, fibrin(ogen)olytic, proteolytic activities. Plasma coagulation, Thrombin catalyzed fibrinocoagulation, Fibrin plate assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), substrate SDS-PAGE and thrombin like activity by using chromogenic substrate were used to determine the effect of venom on plasma coagulation, its fibrin(ogen)olytic and proteolytic activity. The results show the presence of fibrin(ogen)olytic, anticoagulant and gelatinolytic activity in both stingray venom extracts. D. sephen venom delays coagulation of citrated plasma more significantly than A. narinari upon using increasing concentration of the venom. The same results were obtained in the fibrinocoagulation assays. SDS-PAGE analysis of fibrinogen and fibrin after incubation with D. sephen and A. narinari venom show fibrin(ogen)olytic activity. Through SDS-PAGE analysis it is confirmed that the delaying in coagulation process by stingray venom is due to its fibrin(ogen)olytic activity and fibrinolytic activity also confirmed through fibrin plate assay. Zymogram analysis shows the presence of array of gelatinolytic and fibrinogenolytic enzymes above 43-276 kDa in the D. sephen and A. narinari venom respectively. Protease inhibitor studies show the serine and metallo proteases are responsible for these activities. From the results, fibrinogenolytic, proteolytic activity of the stingray venom is confirmed, but it has no thrombin like activity and these activities may aid in hemorrhages, tissue necrosis and secondary bacterial infections at the site of envenomation. |
| 2011 | Purification and partial characterization of a new pro-inflammatory lectin from Bauhinia bauhinioides Mart (Caesalpinoideae) seeds. | Protein Pept Lett | A new galactose-specific lectin, named BBL, was purified from seeds of Bauhinia bauhinioides by precipitation with ammonium sulfate, followed by two steps of ion exchange chromatography. BBL haemagglutinated rabbit erythrocytes (native and treated with proteolytic enzymes) showing stability even after exposure to 60 °C for an hour. The lectin haemagglutinating activity was optimum between pH 8.0 and 9.0 and inhibited after incubation with D-galactose and its derivatives, especially α-methyl-D-galactopyranoside. The pure protein possessed a molecular mass of 31 kDa by SDS-PAGE and 28.310 Da by mass spectrometry. The lectin pro-inflammatory activity was also evaluated. The s.c. injection of BBL into rats induced a dose-dependent paw edema, an effect that occurred via carbohydrate site interaction and was significantly reduced by L-NAME, suggesting an important participation of nitric oxide in the late phase of the edema. These findings indicate that BBL can be used as a tool to better understand the mechanisms involved in inflammatory responses. |
| 2010 | Characterization of biological activity of Scatophagus argus venom. | Toxicon | Scatophagus argus of the family Scatophagidae inflicts painful wounds in fishermen while handling it. The venom induces prominent local tissue damage characterized by pain, edema and necrosis. The pathogenesis of acute muscle damage in gastrocnemius muscle induced by S. argus venom was studied in mice. The inflammatory response induced by S. argus venom in the mice hind paw was studied measuring paw edema. Intramuscular injection of S. argus venom induced motoxicity. The effect of S. argus venom on the cellular components of inflammatory response was investigated. Venom from S. argus were quantitatively analyzed for enzymic and biochemical activity. The biochemical changes induced by the sublethal concentration of S. argus venom and histopathological studies of effect of venom on mice were carried out. Venom induced a rapid increment in serum creatine kinase (CK) and lactate dehydrogenase (LDH) showing the myotoxicity of venom. Concomitant with this a reduction of muscle CK and LDH activity was observed, where as no increment in muscle lactate was detected. Our findings showed that the edematic activity was dose dependent and remained significantly elevated over 48 h after injection. Administration of S. argus venom caused a significant cell accumulation of neutrophils in to peritoneal cavity as well as foot pad up to 24h with maximal being at 4-6h. The venom components analyzed showed the presence of phosphodiesterase, acid phosphatases, alkaline phosphatases, proteinase, and caseinolytic activity. SDS PAGE revealed the presence of major and minor protein bands between 6.5 and 68 kDa. The biochemical changes induced by the sublethal concentration of S. argus venom showed reversible changes in the hematological (blood cell count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin and platelet count) parameters which were significantly altered at 6 and 24h (GLM repeated measures p<0.05). Serum enzymes such as AST, ALT, ACP, ALP, LDH and urea were altered significantly which in turn confirmed the damage of vital organ tissue. |
| Irritancy potential of 17 detergents used commonly by the Indian household. | Indian J Dermatol Venereol Leprol | Detergents are used by almost every household in the developed and developing world. Soap and most detergents are anionic surfactants and attack the horny layer of the skin and increase its permeability with little or no inflammatory change and may result in hand eczema, which is very distressing and incapacitating.To evaluate the irritant potential of common household detergents (laundry and dish wash) used by the Indian population using a 24-hour patch test and to convincingly educate the patients on the detergents less likely to cause irritation in the particular individual.Seventeen commonly used detergents found in Indian market were included in the study, of which, 12 were laundry detergents (powders--seven, bar soap--five) and five were dish wash detergents (powder--one, liquid--one, bar soap--three). The irritant potential of the 17 detergents were evaluated in 30 volunteers. Thirty microliters of each of the detergent bar solutions, distilled water (negative control), and 20% SDS (positive control) were applied to Finn chambers with a micropipette and occluded for 24 hours. Erythema, scaling, and edema were graded in comparison to the reaction at the negative control site (distilled water) for each volunteer separately. The scoring of erythema/dryness and wrinkling on a 0-4 point scale and edema on another 0-4 point scale was based on the Draize scale. The pH of each of the detergent solutions was determined using litmus papers (Indikrom papers from Qualigens fine chemicals).The difference between detergents (F value) was significant for erythema/dryness and wrinkling (F = 3.374; p = 0.000), but not significant for edema (F = 1.297; p = 0.194). [Table 2] lists the means for erythema/dryness and wrinkling, and edema. The F value of the totals of the means for erythema/dryness and wrinkling and edema was significant (F = 2.495; p = 0.001). The pH of all the detergents was found to be alkaline except Pril utensil cleaner which tested acidic (pH 6). The positive control, 20% SDS also tested acidic (pH 6).Similar to patch testing in allergic contact dermatitis, 24-hour patch testing with detergent solutions (8% w/v), will educate the patient on what detergent to avoid. This may bring down the total medication requirement and frequent hospital consultations for these patients. |
| 2010 | Neurotoxic, myotoxic and cytolytic activities of the new basic PLA(2) isoforms BmjeTX-I and BmjeTX-II isolated from the Bothrops marajoensis (Marajó Lancehead) snake venom. | Protein J | The BmjeTX-I and BmjeTX-II isoforms of PLA(2) were purified from Bothrops marajoensis venom by ion-exchange chromatography and reverse phase HPLC. Both isoforms showed a molecular mass of 13808.89 Da (BmjeTX-I) and 13863.97 Da (BmjeTX-II) determined by based on the determined primary structures and SDS-PAGE and confirmed experimentally by MALDI-TOF mass spectrometry. Multiple alignment of BmjeTX-I and BmjeTX-II isoforms of PLA(2) show high degree of homology with basic PLA(2) myotoxins from other Bothrops venoms. Ex vivo, both isoforms caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In chick preparations, contractures to exogenous acetylcholine (55 and 110 microM) or KCl (13.4 mM) were unaltered after complete blockade for the both isoforms BmjeTX-I and BmjeTX-II of PLA(2). These results, which strongly suggested a presynaptic mechanism of action for these toxins. In mice, both isoforms induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Both isoforms BmjeTX-I and BmjeTX-II of PLA(2) also induced moderate marked paw edema, evidencing the local increase in vascular permeability. Since both isoforms of PLA(2) exert a strong proinflammatory effect, the enzymatic hydrolysis of phospholipids might be relevant for this phenomenon and produced cytotoxicity in murine skeletal muscle C2C12 myoblasts and myotubes. |
| 2009 | Biochemical and biological characterization of a PLA2 from crotoxin complex of Crotalus durissus cumanensis. | Toxicon | A new PLA2 (Cdcum6) from crotoxin complex of Colombian Crotalus durissus cumanensis rattlesnake was purified using molecular exclusion chromatography and RP-HPLC. The molecular mass of Cdcum6 was determined by SDS-PAGE approximately 14 KDa and confirmed by MALDI-TOF (14321.98 Da). The enzyme showed Km 6.0 mM, Vmax 3.44 nmol/min, optimum pH was 8.0 and temperature was between 30 and 45 degrees C, and it had a strict requirement of Ca2+ for its activity. The N-terminal sequence of PLA2 was SLVQF EKMIK EVAGK NGVPWY. Comparison of amino acid sequence data with other PLA2 from South American Crotalus durissus rattlesnakes showed that Cdcum6 shares the highest sequence identity with Cdr13 an isoform PLA2 from Crotalus durissus ruruima, nevertheless, Cdcum6 showed high content of basic and hydrophobic amino acids. In mice, Cdcum6 presented higher LD50 than crotoxin complex from C d. cumanensis. Additionally, Cdcum6 induced a conspicuous local myotoxic effect and moderate footpad edema; in vitro, it was antigoagulant in doses as low as 0.5 microg/l ml, and it was not cytotoxic on myoblast but Cdcum6 was able to lyse myotubes. |
| Neutralization of Bothrops mattogrossensis snake venom from Bolivia: experimental evaluation of llama and donkey antivenoms produced by caprylic acid precipitation. | Toxicon | Polyspecific bothropic/crotalic and bothropic/lachesic antivenoms were produced in Bolivia by immunizing two donkeys with the venoms of Bothrops mattogrossensis and Crotalus durissus terrificus and one llama with the venoms of B. mattogrossensis and Lachesis muta. These antivenoms are currently being used for snakebite envenomation in Bolivia. The rationale for using these animals is that donkeys and llamas are better adapted than horses to the high altitudes in South America and constitute good alternatives for antivenom production in these regions. Plasma was fractionated by caprylic acid precipitation of non-immunoglobulin plasma proteins, to obtain whole IgG preparations. Donkey-derived antivenom showed one band of 150 kDa when analyzed by SDS-PAGE, whereas llama antivenom presented two immunoglobulin bands, of 170 kDa and 120 kDa, the latter corresponding to the heavy-chain antibodies present in camelid sera. The effectiveness of these antivenoms to neutralize lethal, hemorrhagic, myotoxic, edema-forming, and defibrinogenating activities of the venom of B. mattogrossensis from Bolivia, a species formerly known as Bothrops neuwiedii, was assessed at the experimental level. Although llama antivenom has a total protein concentration four times lower than donkey antivenom, both preparations have similar neutralizing capacity against all toxic activities assessed. Llama and donkey IgG-based antivenoms are effective in the neutralization of B. mattogrossensis venom and represent valuable alternatives for antivenom manufacture in highland regions of South America. |
| 2009 | [Construction of attenuated Salmonella choleraesuis vaccine strain expressing recombinant antigen of Shiga-like toxin Escherichia coli]. | Wei Sheng Wu Xue Bao | To construct an attenuated Salmonella choleraesuis vaccine strain expressing the gene SLT-IIeB and FedF of Shiga-like toxin Escherichia coli (SLTEC) O138 with balanced lethal system.The gene of SLT-IIeB and FedF of SLTEC O138 was amplified, and then recombined with a vector pYA3493 (Asd+). The recombinant was electroporated into attenuated Salmonella choleraesuis C500 (Asd-). The expression of SLT-IIeB and FedF was analyzed by SDS-PAGE. The stability of the vaccine strains was studied by generation culture in vitro.The stable attenuated Salmonella choleraesuis vaccine strain expressing SLT-IIeB and FedF of SLTEC O138 was constructed with balanced lethal system. The expressed products with protein quality 37000 could react with the antibody of FedF and SLT-IIeB.The attenuated Salmonella choleraesuis vaccine strain could probably serve as a vaccine against edema disease and piglets paratyphoid. |
| 2009 | Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress. | J Neurochem | Hypoxic (low oxygen) and reperfusion (post-hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood-brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O(2), 60 min), Hx (6% O(2), 60 min), or hypoxia/reoxygenation (H/R, 6% O(2), 60 min followed by 21% O(2), 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent-free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro-octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non-reducing and reducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis/western blot of PFO-solubilized occludin revealed that occludin oligomeric assemblies co-localizing with 'TJ-associated' raft domains contained a high molecular weight 'structural core' that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO-solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non-covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide-bonded inner core, and dispersal of these non-covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains. |
| 2008 | [Reconstruction tissue-engineered corneal epithelium using xenogeneic acellular corneal stroma as scaffold]. | Zhonghua Yan Ke Za Zhi | To compare two methods for preparing acellular corneal stroma and evaluate the possibility of culturing corneal epithelium on xenogeneic acellular corneal stroma.Experimental study, applying completely randomized design method. Dispase followed by Triton-X-100 detergent and sodium chloride SDS detergent followed by trypsinase were applied respectively to treat the rabbit cornea. The characteristics of corneal stroma and acellular status after treatment were examined with slit lamp, optical microscope and transmission electron microscope. The rabbit limbal cells were then cultured on the acellular porcine Bowman's membrane/stroma. Rabbit corneal epithelium lamella reconstructed in vitro and evaluated in morphology, histopathology and immunohisto-chemistry.Acellular corneal stroma prepared by two different methods is quite similar in morphology, being gray and opaque with visible edema and soft texture. Collagen fibers of the stroma were regularly in histopathology and ultrastructure. But the one prepared by the NaCl-SDS-Trypsinase method retained a amounts of cell debris, while there was none in the other did by Dispase-Triton-X-100 method. The limbal cells began to shift out at 24 hours after being inoculated on xenogeneic acellular corneal stroma, then attached on it, formed a confluent monolayer containing normal-appearing in 7 days. The tissue engineering corneal epithelium was cryosectioned and characterized immunohistochemically at 14 days after inoculation. Meanwhile, epithelium associated antigen CK3 in endochylema was stained.Dispase-Triton-X-100 was proved better in obtaining acellular corneal stroma. It is possible to reconstruction tissue-engineered rabbit's corneal epithelium on acellular porcine corneal Bowman's membrane/stroma. |
| 2009 | Development of a standardized definition for Hirschsprung's-associated enterocolitis: a Delphi analysis. | J Pediatr Surg | The reported incidence of Hirschsprung's-associated enterocolitis (HAEC) is extremely variable. A standardized definition would permit comparison of different studies and provide an interpretable outcome measure for future prospective studies in patients with Hirschsprung's disease.The Delphi method is a technique for achieving consensus among a panel of experts. A list of 38 potential criteria from the history, physical examination, radiologic studies, and pathologic specimens was made available to pediatric surgeons and gastroenterologists who have contributed to the literature on Hirschsprung's disease. Each expert ranked the diagnostic importance of each item using a Likert scale. In subsequent surveys, the same process was used, but the means and SDs from previous rounds were included as a way of influencing the experts toward consensus. Cronbach's alpha was used after each round to measure variability among the experts. Once consensus was reached, an overall "HAEC score" was developed by assigning a value of 1 or 2 to each item that was considered important by the expert panel. The score was then validated by circulating 10 clinical cases to the panel and asking if each represented HAEC or not.Twenty-seven experts completed the survey. Cronbach's alpha increased from 0.93 after the first round to 0.97 after the second. Criteria receiving the highest scores were diarrhea, explosive stools, abdominal distension, and radiologic evidence of bowel obstruction or mucosal edema. Eighteen items were included in the score. During the validation process, the score agreed with the experts in 9 of the 10 case scenarios.The most important clinical diagnostic criteria for HAEC were identified from a larger pool of potential diagnostic items through a consensus approach using the Delphi method. A score was developed and validated and can now be used as a standardized and reproducible outcome measure for future studies in children with Hirschsprung's disease. |
| 2009 | BthMP: a new weakly hemorrhagic metalloproteinase from Bothrops moojeni snake venom. | Toxicon | In this work, a new weakly hemorrhagic metalloproteinase (BthMP) was purified from Bothrops moojeni snake venom. This enzyme was homogeneous by native and SDS-PAGE. It showed a polypeptide chain of 23.5kDa, pI=7.1, and N-terminal blocked. BthMP is comprised of high proteolytic activity on casein, fibrin and bovine fibrinogen, with no coagulating, esterase or phospholipase A(2) activities; it was inhibited by EDTA, EGTA and 1,10-phenanthroline and maintained its activity on pH from 7.0 to 9.0 and temperature from 5-40 degrees C. Assays with metal ions showed that Ca(2+) is an activator, whereas Zn(2+) and Hg(2+) inhibited about 50 and 80% of its activity, respectively. The edema evidenced the important role of the toxin in the inflammatory activity of the venom. BthMP also caused unclotting, and provoked histological alterations in the gastrocnemius muscle of mice inducing hemorrhage, necrosis and leukocytic infiltrate. The molecular mass and the inhibition assays suggest that the metalloproteinase BthMP belongs to class P-I of SVMPs. |
| 2009 | Toxicity of phospholipases A2 D49 (6-1 and 6-2) and K49 (Bj-VII) from Bothrops jararacussu venom. | Cell Biol Toxicol | Purified phospholipase A2 (PLA2) enzymes from Bothrops jararacussu snake venom were examined to evaluate NIH 3T3 and COS7 fibroblast cytotoxicity, as well as muscle myotoxic and inflammatory activities. Separation of fractions Bj-VII (from BthTX-I; a Lys49 PLA2 homolog) and 6-1 and 6-2 (from BthTX-II; an Asp49 PLA2) from B. jararacussu snake venom by SDS-PAGE in tricine buffer in the absence and presence of dithiothreitol revealed a homodimer with an estimated molecular mass of approximately 30 kDa (monomer mass approximately 15 kDa). This finding indicates that these toxins form dimeric complexes-a previously reported tendency among PLA2s. These toxins were assayed for viability with the MTT assay, which is used to examine the effects of phospholipases on the mitochondrial viability of cells. The toxins were also assayed for cytolysis of the fibroblast cell lines NIH 3T3 and COS7 by quantification of lactate dehydrogenase released into the medium. The results indicate that the PLA2s 6-1, 6-2 and the Bj-VII PLA2 homolog studied here induce moderate footpad edema and local myotoxicity. Moreover, exposure to these phospholipases led to a reduction in fibroblast viability; at the 1 muM dose of PLA2 tested, a reduction of 50% in cell viability was observed. The present findings indicate that the inflammatory activity observed in envenomation may be correlated with the cytotoxicity observed in fibroblasts. |
| 2008 | Snake venomics of the Lesser Antillean pit vipers Bothrops caribbaeus and Bothrops lanceolatus: correlation with toxicological activities and immunoreactivity of a heterologous antivenom. | J Proteome Res | The venom proteomes of the snakes Bothrops caribbaeus and Bothrops lanceolatus, endemic to the Lesser Antillean islands of Saint Lucia and Martinique, respectively, were characterized by reverse-phase HPLC fractionation, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venoms contain proteins belonging to seven ( B. caribbaeus) and five ( B. lanceolatus) types of toxins. B. caribbaeus and B. lanceolatus venoms contain phospholipases A 2, serine proteinases, l-amino acid oxidases and zinc-dependent metalloproteinases, whereas a long disintegrin, DC-fragments and a CRISP molecule were present only in the venom of B. caribbaeus, and a C-type lectin-like molecule was characterized in the venom of B. lanceolatus. Compositional differences between venoms among closely related species from different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. The venoms of these two species differed in the composition and the relative abundance of their component toxins, but they exhibited similar toxicological and enzymatic profiles in mice, characterized by lethal, hemorrhagic, edema-forming, phospholipase A 2 and proteolytic activities. The venoms of B. caribbaeus and B. lanceolatus are devoid of coagulant and defibrinogenating effects and induce only mild local myotoxicity in mice. The characteristic thrombotic effect described in human envenomings by these species was not reproduced in the mouse model. The toxicological profile observed is consistent with the abundance of metalloproteinases, PLA 2s and serine proteinases in the venoms. A polyvalent (Crotalinae) antivenom produced in Costa Rica was able to immunodeplete approximately 80% of the proteins from both B. caribbaeus and B. lanceolatus venoms, and was effective in neutralizing the lethal, hemorrhagic, phospholipase A 2 and proteolytic activities of these venoms. |
| 2008 | Toxic activities of Brazilian centipede venoms. | Toxicon | Centipedes have a venom gland connected to a pair of forceps, which are used to arrest preys. Human victims bitten by centipedes usually manifest burning pain, paresthesia and edema, which may develop into superficial necrosis. The aim of this work was to characterize and compare toxic activities found in venoms of three species of Brazilian centipedes-Otostigmus pradoi, Cryptops iheringi and Scolopendra viridicornis. By SDS-PAGE (4-20%), important differences were noticed among venoms (between 7 and 205kDa). Few bands showed feeble caseinolytic, fibrinogenolytic and gelatinolytic activities by zymography, but strong hyaluronidase activity was observed in S. viridicornis and O. pradoi venoms. In addition, such activities could be inhibited by o-phenanthroline, indicating that these enzymes are metalloproteinases. All venoms induced nociception, edema and myotoxicity in mice, but only S. viridicornis induced mild hemorrhagic activity. No coagulant activity was detected in centipede venoms. Low phospholipase A(2) activity was observed exclusively in S. viridicornis and O. pradoi venoms, but these venoms had intense direct hemolytic activity on human erythrocytes. Cross-reactivity among venoms was observed using species-specific sera raised in rabbits. Differences were noticed among centipede venoms, but S. viridicornis is indeed the most toxic venom and thereby it could induce a more severe envenomation. |
| 2008 | [Studies on immunomodulation effect of recombinant Sj16 from Schistosoma japonicum on inflammation response of host]. | Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi | To study the immunomodulation effect of the recombination Sj16 from Schistosoma japonicum (reSj16) on inflammation response of host.reSj16 expressed in pGEX-4T-1 was purified by GST purification kit and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectra (MS). The immunomodulation effect of reSj16 was observed by means of dimethylbenzene inducing mouse ear edema model, carrageenan inducing rat voix pedis swell model and acetic acid inducing mouse experiment peritonitis model.The soluble protein of reSjl6 was obtained and identified by SDS-PAGE and MS. reSjl6 1.0, 5.0, 10.0 microg/kg evidently suppressed the mouse ear edema induced by dimethyl-benzene, significantly mitigated the rat voix pedis swelling induced by carrageenan, and remarkably suppressed the increase of the capillary permeability of abdominal cavity in experiment peritonitis mouse model.The results further prove that Sj16 may be a potential immunosuppressive molecule and may have a notable effect on immunomodulation. |
| 2007 | [Expression of GST-3B fusion protein of Escherichia coli of Ee strain producing SLT-IIe toxin and study on its biological activities and immunogenicity]. | Wei Sheng Wu Xue Bao | Three copies of DNA fragment encoding the truncated SLT-IIeB of Ee strain which was responsible for the edema disease in piglets in Hubei province were fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pK3 B. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-3B fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against diseases of edema of swine. The fusion protein was further purified and used as an antigen for receptor-binding inhibition assay. The receptor-binding inhibition assay showed GST-3B fusion protein had more strong biological activities than GST-B. The fusion protein of GST-3B or GST-B was purified and emulsified with Freund' s incomplete adjuvant in equal volumes to get subunit bacterin. Groups of SPF KM mice were vaccinated subcutaneously at 0 week with 25 micrograms and at 2 weeks with 25 micrograms of purified GST-3B or GST-B and challenged intraperitoneally with volume of 5 x OD50 Ee strain. Serological tests were performed one week interval with ELISA. The IgG titres against SLT-IIeB in the sera collected at the same period from the Group GST-3B were higher than in the Group GST-B and the immune protection rate against Ee strain was respectively 60% and 40%. These results show the fusion protein GST-3B had more strong biological activities, immunogenicity and better protection against Ee strain, which built a good foundation for the further research of high efficacy vaccine against porcine edema disease. |
| 2007 | 'Partitagin' a hemorrhagic metalloprotease from Hippasa partita spider venom: role in tissue necrosis. | Biochimie | The poisonous bite by Hippasa partita, a funnel web spider from the Indian subcontinent has been demonstrated to give rise to severe dermo- and myonecrosis. In this work a hemorrhagic metalloprotease, Partitagin was purified from H. partita venom by successive chromatography on Sephadex G-100, DEAE Sephadex A-50 and Biosep DEAE columns. SDS-PAGE, reversed phase HPLC on a C(4) column, N-terminal amino acid sequencing and MALDI-TOF mass spectrometry confirmed the homogeneity. Partitagin was assayed using fat free casein as substrate. EDTA, 1,10-phenanthroline and cyanide, inactivated it irreversibly while, EGTA, PMSF, leupeptin, pepstatin and aprotinin did not inhibit. The presence of Zn(+2) was confirmed by atomic absorption spectrometry. Partitagin caused hemorrhage when tested in a mouse model. Light microscopy of skin tissue sections at the site of injection revealed extensive damage of extracellular matrix (ECM) in which the basement membrane surrounding blood vessels and capillaries showing signs of extensive destruction and also loss of vessel wall integrity. Similar intense damage was also noticed in the ECM of muscle tissue sections but with no damage caused to myocytes. Partitagin showed specificity of action on the components of ECM and degraded collagen type-IV and fibronectin but not collagen type-I. Partitagin was devoid of edema, myotoxicity and lethality. This is the first report on the isolation and characterization of a toxin from spider venom in the Indian subcontinent. |
| 2007 | Biochemical, pharmacological and structural characterization of two PLA2 isoforms Cdr-12 and Cdr-13 from Crotalus durissus ruruima snake venom. | Protein J | Cdr-12 and Cdr-13 isoforms of PLA2, a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on micro-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA2 was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry. The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA2. The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13 This sequence shows high identity values when compared to other D49 PLA2s isolated from venoms of crotalics snakes. Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA2 isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. In vitro, Cdr-12 and Cdr-13 isoforms of PLA2, caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and produced cytotoxicity in murine C2C12 skeletal muscle myotubes and lack cytolytic activity upon myoblasts in vitro. Thus, the combined structural and functional information obtained identify Cdr-12 and Cdr-13 isoforms as members of the PLA2 family, which presents the typical bioactivities described for such proteins. |
| 2006 | Purification and biological effects of Araucaria angustifolia (Araucariaceae) seed lectin. | Biochem Biophys Res Commun | This paper describes the purification and characterization of a new N-acetyl-d-glucosamine-specific lectin from Araucaria angustifolia (AaL) seeds (Araucariaceae) and its anti-inflammatory and antibacterial activities. AaL was purified using a combination of affinity chromatography on a chitin column and ion exchange chromatography on Sephacel-DEAE. The pure protein has 8.0kDa (SDS-PAGE) and specifically agglutinates rabbit erythrocytes, effect that was independent of the presence of divalent cations and was inhibited after incubation with glucose and N-acetyl-d-glucosamine. AaL showed antibacterial activity against Gram-negative and Gram-positive strains, shown by scanning electron microscopy. AaL, intravenously injected into rats, showed anti-inflammatory effect, via carbohydrate site interaction, in the models of paw edema and peritonitis. This lectin can be used as a tool for studying bacterial infections and inflammatory processes. |
| 2006 | Venom from spiders of the genus Hippasa: biochemical and pharmacological studies. | Comp Biochem Physiol C Toxicol Pharmacol | The venoms from female spiders of the genus Hippasa namely H. partita, H. agelenoides and H. lycosina are compared for biochemical and pharmacological properties. SDS-PAGE pattern revealed varied protein composition. Marked variability is seen with casein hydrolyzing enzymes in SDS-PAGE zymogram. H. partita venom was the only venom that hydrolyzed gelatin while the other two venoms did not. The venoms shared similar hyaluronidase activity, showing a single activity band in SDS-PAGE zymogram. The PLA2 activity varied as H. partita>H. agelenoides>H. lycosina venoms. Marked differences were noted in the ability to induce edema, cytotoxicity, myotoxicity and neurotoxicity, while hemorrhage was associated exclusively with H. partita venom. |
| 2006 | Self-association of the transmembrane domain of an anthrax toxin receptor. | J Mol Biol | Protective antigen (PA), lethal factor (LF) and edema factor (EF) are secreted individually by Bacillus anthracis. These components of anthrax toxin must then assemble into complexes to intoxicate mammalian cells. Toxin assembly initiates when molecules of PA bind mammalian receptors ANTXR1/2 and are cleaved by surface proteases into 20 kDa and 63 kDa fragments. After PA20 dissociates, receptor-bound PA63 homo-oligomerizes into heptamers. Oligomeric PA63 binds EF and LF and these complexes are internalized into an acidic compartment where the two enzymatic components are translocated across the membrane by a channel formed by heptameric PA63. Since oligomerization of PA63 is required to bind and translocate the enzymatic components, we sought to determine whether interactions between toxin receptors could facilitate the assembly process. In the present work, we performed a co-immunoprecipitation experiment to demonstrate that ANTXR1 is oligomeric in mammalian cells. Computer modeling predicted the self-association of the ANTXR1 transmembrane domain and we detected oligomerization of ANTXR1 transmembrane domain peptides in the membrane-mimetic environment of SDS micelles using fluorescence resonance energy transfer. Furthermore, the ANTXR1 transmembrane domain mediated oligomerization of a reporter protein construct in a bacterial membrane. In both assays, mutations that disrupted the interaction were consistent with the interaction being mediated through an asymmetric binding interface. Mutations that impaired self-association of the transmembrane domain reduced the rate of PA63 heptamer formation on the mammalian cell surface. Our findings indicate that ANTXR1 transmembrane domains self-associate and that these interactions may stabilize intermediate oligomerization states of ANTXR1-PA63 complexes. |
| 2006 | A neurotoxic phospholipase A2 variant: isolation and characterization from eastern regional Indian cobra (Naja naja) venom. | Toxicon | CM-Sephadex C-25 column chromatography profile of Indian cobra (Naja naja) venom from eastern region showed a distinct and a dominant phospholipase peak, peak-10, while it was not seen in either southern or western venom samples. Peak-10 was subjected to CM-Sephadex C-25 and Sephadex G-50 column chromatography to isolate NN-X-PLA(2). NN-X-PLA(2) is a single chain protein with the relative molecular weight of 10kDa by SDS-PAGE. It was toxic to mice with an LD(50) value 0.098 mg/kg body weight (i.p.) and the mice exhibited acute neurotoxic symptoms. Upon indirect stimulation, it inhibited the twitching of frog's gastrocnemius muscle in a dose dependent manner. NN-X-PLA(2) was weakly anticoagulant and devoid of cytotoxicity, myotoxicity, hemorrhage, edema inducing, and directlytic activities and effects on platelet aggregation process. Upon chemical modification independently with p-bromophenacyl bromide and acetic anhydride, NN-X-PLA(2) lost both enzymatic and toxic properties. |
| 2006 | Matrix metalloproteinase-9 contributes to brain extravasation and edema in fulminant hepatic failure mice. | J Hepatol | Fulminant hepatic failure (FHF) can be dreadful. When coma sets in, brain edema develops taking FHF into a lethal course. Mechanisms of brain extravasation leading to brain edema remain incompletely understood. Matrix metalloproteinase (MMP)-9 is implicated in various brain injuries. We hypothesized that MMP-9 contributes to brain edema in FHF.MMP-9 and its proform were assayed using SDS-PAGE and in situ gelatin zymographies. Brain extravasation was assessed with Evans blue. Brain water was determined by specific gravity and astrocytic endfoot swelling by electron microscopy. FHF in mice was induced by azoxymethane. MMP inhibitor GM6001 and MMP-9 monoclonal antibody were used.Active MMP-9 was significantly increased at the onset of coma and brain extravasation in FHF mice. Blocking MMP-9 with either GM6001 or MMP-9 monoclonal antibody significantly attenuated brain extravasation, astrocytic endfoot swelling, and brain edema. Brains of FHF mice did not show MMP-9 activity. In contrast, livers of these animals showed marked up-regulation of MMP-9 activity.Our findings suggest that MMP-9 contributes to the pathogenesis of brain extravasation and edema in FHF. The necrotic liver is the source of MMP-9 in FHF. Inhibition of MMP-9 may protect against the development of brain edema in FHF. |
| 2005 | Proteolytic, edematogenic and myotoxic activities of a hemorrhagic metalloproteinase isolated from Bothrops alternatus venom. | Toxicon | A hemorrhagic metalloproteinase has been isolated from Bothrops alternatus venom from specimens that inhabit the north-east region of Argentina. The present study aimed at evaluating the proteolytic, hemorrhagic, edematogenic and myotoxic activities of the purified metalloproteinase, in order to consider its participation on the phatophysiology of the intoxication by Bothrops alternatus venom. The hemorrhagic metalloproteinase was isolated by a combination of DEAE-Cellulose chromatography and gel filtration on Sephadex G-75. The enzyme showed a molecular mass around 55k Da, it exhibited a hemorrhagic activity with a minimal hemorrhagic dose of 1.9 microg, almost two fold minor than the whole venom (3.6 microg). The enzyme showed a weak proteolytic activity on casein (18.72 U/mg enzyme), similar to the one exhibited by the whole venom (20 U/mg venom). Besides, the ability to degrade casein could be detected by SDS-PAGE; beta-casein was the fraction that showed the higher degradation, followed by alphas(1)-casein and kappa-casein degradation. The hemorrhagic metalloproteinase rapidly hydrolysed the A alpha-chain of fibrinogen, followed by B beta-chain degradation and leaving the gamma-chain unaffected. Proteolytic activities were inhibited by EDTA whereas they were not inhibited by benzamidine and PMSF. The metalloproteinase showed several polypeptides chains after autocatalytic processing, including a chain of 28k Da, it could be the processed disintegrin-like and cysteine-rich domains. The isolated enzyme exhibited myotoxic activity with high CK levels at 6h, due to local ischemia resulting of its hemorrhagic activity, and a significant edema-inducing effect (MED=1.3 microg), corroborated both results by the histological observations of samples of gastrocnemius muscle. These findings showed that this hemorrhagic metalloproteinase, possesses high edematogenic and myotoxic activities and, in despite of exhibiting a weak proteolytic activity, it is able to degrade fibrinogen. So, this enzyme would contribute markedly to the phatophysiology of the bothropic envenomation. |
| 2005 | Anticoagulant and antifibrinogenolytic properties of the aqueous extract from Bauhinia forficata against snake venoms. | J Ethnopharmacol | The aqueous extract from aerial parts of Bauhinia forficata was able to neutralize the clotting activity induced by Bothrops and Crotalus crude venoms. The clotting time, upon human plasma, induced by B. moojeni venom was significantly prolonged. Clotting and fibrinogenolytic activities induced by isolated thrombin-like enzyme from Bothrops jararacussu were totally inhibited after incubation at different ratios. The extract was not able to neutralize the hemorrhagic activity induced by an Bothrops venoms, but it efficiently inhibited the edema induced by Crotalus durissus terrificus venom and isolated PLA2s. In addition, it did not inhibited the phospholipase A2 activity of Bothrops snake venoms. Interaction studies between Bauhinia forficata extract and snake venoms, when analyzed by SDS-PAGE, did not reveal any apparent degradation of the venom proteins. This extract is a promising source of natural inhibitors of serine-proteases involved in blood clotting disturbances induced by snake venoms. |
| 2005 | Enzymatic characterization, antigenic cross-reactivity and neutralization of dermonecrotic activity of five Loxosceles spider venoms of medical importance in the Americas. | Toxicon | Loxosceles spiders have a wide distribution in the temperate and tropical regions of the world. Loxoscelism is characterized by necrotic skin ulceration at the bite site and, less commonly, a systemic illness that may be fatal. The purpose of this study was to characterize and compare aspects of the major medically important Loxosceles spider venoms in a standardized manner, particularly considering their neutralization by two Brazilian antivenoms. By SDS-PAGE (12% acrylamide), Loxosceles deserta, Loxosceles gaucho, Loxosceles intermedia, Loxosceles laeta and Loxosceles reclusa venoms had similar electrophoretic profiles, with the major protein bands of 32-35 kDa. All venoms exhibited gelatinolytic, caseinolytic and fibrinogenolytic activities in vitro with a large array of proteases, mainly between 18.1 and 31.8 kDa. Most of these enzymes were metalloproteases as this activity was abolished by 1,10-phenanthroline. Hyaluronidase activity was detected in a protein band of approximately 44 kDa in all venoms. Sphingomyelinase activity was demonstrated in all five venoms. Antigenic cross-reactivity, by Western blotting, was also observed among all venoms studied using commercial equine antivenoms produced in Brazil (Institute Butantan and CPPI). These antivenoms recognized mainly components between 25 and 40 kDa in all venoms with several minor components of >89 kDa. Strong cross-reactivity was also seen among all venoms through the ELISA technique (titre range: 64,000-512,000). All venoms (5 microg doses) induced a similar local reaction when injected intradermally into the flank of rabbits, demonstrating dermonecrosis, hemorrhage, vasoconstriction, edema, and erythema. However, no reaction was observed when each venom was pre-incubated (1 h, 37 degrees C) with Brazilian commercial sera prior to injection. The antivenoms also abolished the sphingomyelinase activity in vitro, suggesting the venoms of the major medically important Loxosceles spider species have generally similar toxic and enzymatic characteristics. Thus, as Brazilian commercial antivenoms are able to neutralize the dermonecrosis induced by Loxosceles venoms of diverse geographical origin, clinical studies should be undertaken on the potential for a single global Loxosceles antivenom. |
| 2005 | Important biological activities induced by Thalassophryne maculosa fish venom. | Toxicon | The accidents caused by Thalassophryne maculosa fish venoms are frequent and represent a public health problem in some regions of Venezuela. Most accidents occur in the fishing communities and tourists. The clinical picture is characterized by severe pain, dizziness, fever, edema, and necrosis. Due to the lack of efficient therapy it may take weeks, or even months for complete recovery of the victims. The investigations presented here were undertaken to assess the eletrophoretical profile and principal biological properties of the T. maculosa venom. Venom obtained from fresh captured specimens of this fish was tested in vitro or in animal models for a better characterization of its toxic activities. In contrast to other fish venoms, T. maculosa venom showed relative low LD50. The injection of venom in the footpad of mice reproduced a local inflammatory lesion similar to that described in humans. Significant increase of the nociceptive and edematogenic responses was observed followed within 48 h by necrosis. Pronounced alterations on microvascular hemodynamics were visualized after venom application. These alterations were represented by fibrin depots and thrombus formation followed by complete venular stasis and transient arteriolar contraction. T. maculosa venom is devoid of phospholipase A2 activity, but the venom showed proteolytic and myotoxic activities. SDS-Page analysis of the crude venom showed important bands: one band located above 97 M(w), one band between 68 and 97 M(w), one major band between 29 and 43 M(w) and the last one located below 18.4 M(w) Then, the results presented here support that T. maculosa venom present a mixture of bioactive toxins involved in a local inflammatory lesion. |
| 2004 | Bactericidal and neurotoxic activities of two myotoxic phospholipases A2 from Bothrops neuwiedi pauloensis snake venom. | Toxicon | Two basic myotoxic PLA(2)s, namely BnpTX-I and II, were isolated from Bothrops neuwiedi pauloensis snake venom through three chromatographic steps: ion-exchange chromatography on CM-Sepharose, gel filtration on Sephadex G-50 and reverse phase HPLC on a C18 column. Both PLA(2)s showed a M(r) around 14,000 for the monomer and 28,000 for the dimer (as estimated by SDS-PAGE), pI approximately 7.8 and approximately 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with Asp49 basic myotoxic PLA(2)s from other snake venoms. The catalytic and anticoagulant activities of BnpTX-I were higher than those of BnpTX-II. Both were able to induce cytotoxicity in vitro, as well as, myotoxicity, edema and lethality in mice. BnpTX-I also induced neurotoxic effect on mouse neuromuscular preparations and bactericidal activity on Eschericia coli and Staphylococcus aureus. After chemical modification of BnpTX-I with BPB or incubation with EDTA or Mn(2+) ions, the catalytic activity was completely abolished, while the toxic and pharmacological activities were partially reduced. Interaction with heparin inhibited the cytotoxic and bactericidal effects. Anti-BthTX-I, anti-BthTX-II and anti-115-129-C terminal antibodies strongly recognize both BnpTX-I and II. It is shown that the neurotoxic effect induced by B. neuwiedi pauloensis venom is due to the presence of myotoxic PLA(2)s. The data also corroborate the hypothesis of a partial dissociation between toxic and enzymatic domains. In addition, BnpTX-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects. |
| 2003 | Ontogenetic variability of Bothrops atrox and Bothrops asper snake venoms from Colombia. | Toxicon | The lancehead snakes Bothrops asper and Bothrops atrox inflict 70-90% of the 3000 bites reported every year in Colombia. In this work, the venoms of B. atrox from Meta (Villavicencio, 33 specimens) and B. asper from Antioquia (San Carlos, 45 specimens), all of them born in captivity, were obtained at different ages (0-6 months; 1, 2 and 3-years old) and compared in terms of their pharmacological and immunochemical characteristics. A conspicuous ontogenetic variability was observed in venom samples from both species. Venoms from newborn and juvenile specimens showed higher lethal, hemorrhagic, edema-forming and coagulant activities, whereas venoms from 3-year old specimens showed higher indirect hemolytic, i.e. phospholipase A2 activity, being more significant in the case of B. asper. SDS-polyacrylamide gel electrophoresis of whole venom for both species evidenced a predominance of high mol. mass bands in the venoms from specimens of <1 year of age, with a change towards bands having lower mol. mass as snakes aged. Gel filtration chromatography showed five peaks in the venoms of B. asper of <6 months and in those from 3-year old specimens. Venom of adult specimens showed a higher number of peaks with indirect hemolytic activity than venom of newborn specimens. Polyvalent antivenom produced in Costa Rica recognized all the bands of both venoms from specimens at all ages tested, when assayed by Western blotting. |
| Characterization of dominant-negative forms of anthrax protective antigen. | Mol Med | Certain mutations within the protective antigen (PA) moiety of anthrax toxin endow the protein with a dominant-negative (DN) phenotype, converting it into a potent antitoxin. Proteolytically activated PA oligomerizes to form ring-shaped heptameric complexes that insert into the membrane of an acidic intracellular compartment and promote translocation of bound edema factor and/or lethal factor to the cytosol. DN forms of PA co-oligomerize with the wild-type protein and block the translocation process. We prepared and characterized 4 DN forms: a single, a double, a triple, and a quadruple mutant. The mutants were made by site-directed mutation of the cloned form of PA in Escherichia coli and tested by various assays conducted on CHO cells or in solution. All 4 mutant PAs were competent for heptamerization and ligand binding but were defective in the pH-dependent functions: pore formation, ability to convert to the SDS-resistant heptamer, and ability to translocate bound ligand. The single mutant (F427K) showed less attenuation than the others in the pH-dependent functions and lower DN activity in a CHO cell assay. The quadruple (K397D + D425K + F427A + 2beta2-2beta3) deletion showed the most potent DN activity at low concentrations but also gave indications of low stability in a urea-mediated unfolding assay. The double mutant (K397D + D425K) and the triple (K397D + D425K + F427A) showed strong DN activity and slight reduction in stability relative to the wild-type protein. The properties of the double and the triple mutants make these forms worthy of testing in vivo as a new type of antitoxic agent for treatment of anthrax. |
| 2003 | Use of proteomics methodology to evaluate inflammatory protein expression in tendinitis. | Biomed Sci Instrum | In previous studies we established a rat model of acute tendinitis including functional and mechanical measures of healing. Achilles' tendinitis was induced by injection of collagenase, an enzyme that produces localized fiber digestion and edema formation. As quantitative measures of tissue inflammation, hypercellularity and edema were evaluated in injured tendons in comparison with controls. Using the rat tendinitis model, we have applied isotope-coded affinity tag analysis (ICAT) methodology to indicate localized tendon healing by quantitating protein expression. This novel proteomics method allows detection of subtle differences in protein levels that provide a detailed picture of tendinitis healing. The method involves a new class of chemical linkers used to differentially label cysteine residues from similar peptides in control and treated protein samples with heavy (deuterium off of backbone) and light (hydrogen off of backbone) ICAT reagents that are otherwise chemically identical. Proteins were extracted under liquid nitrogen from control untreated or injured Achilles' tendons 72 hours after collagenase-injection. These proteins were digested with endoproteinase Glu-C and trypsin and the resulting peptide mixtures were evaluated using reverse-phase C18 HPLC and Tristricine SDS-polyacrylamide gel electrophoresis. The two ICAT-modified peptide populations were mixed, affinity-purified and analyzed using microcapillary liquid chromatography and electrospray ionization tandem mass-spectroscopy. The process resulted in relative abundance and charge-to-mass ratio data used in conjunction with database searching to identify proteins expressed differentially in the two treatment groups. By analyzing different time periods in the healing process, an accurate model of the healing rat tendon can be made. |
| 2000 | [Cloning and expression of Shiga-like toxin type II variant B gene of E. coli]. | Wei Sheng Wu Xue Bao | A structure sequence and a DNA fragment including the signal peptide sequence and structure sequence of Shiga-like toxin II variant B subunit gene were amplified from E. coli strain O138 by PCR. After digested with restriction endonuclease EcoRI and BamHI, the two genes were orientally inserted into the polycloning site of expression vector pYA3334 (asd+) respectively. Recombinant plasmids pB0 and pB1 were constructed and amplified in E. coli X6212 (asd-). pB0 and pB1 were then introduced into avirulent Salmonella typhimurium vaccine strain X4550 (asd-) by serial transformation through intermediate strain X3730 (asd-) to construct recombinant SLT-IIvB strain. Results of nucleotide sequencing of the cloned fragments in pB0 and pB1 revealed that they were in correct ORF of SLT-IIvB. The results of SDS-PAGE and Western-blot showed that 7.6 kD protein of SLT-IIvB antigen was expressed at pretty high level in recombinant strain X4550(pB0). The results of mice immunization indicated X4550(pB0) could initiate the host to produce specific antibodies to SLT-IIvB and LPS-O antigen of X4550. So the recombinant strain X4550 (pB0) is worth considering as a candidate vaccine strain against porcine edema disease and Salmonella typhimurium infections. |
| 2002 | Purification and characterization of a glycoprotein inhibitor of toxic phospholipase from Withania somnifera. | Arch Biochem Biophys | A phospholipase inhibitor (WSG) has been purified from Withania somnifera using gel-filtration and ion-exchange chromatographies. The WSG is an acidic glycoprotein. Its molecular mass as determined by SDS-PAGE was 27kDa. It neutralized the enzyme activity and pharmacological properties such as cytotoxicity, edema, and myotoxicity of a multi-toxic Indian cobra venom phospholipase (NNXIa-PLA) but failed to neutralize the neurotoxicity. The glycan part of the molecule does not appear to be involved in any of the pharmacological properties studied. The results suggest that the neutralization of the pharmacological effects of the toxic phospholipase is brought about by inhibition of the enzyme activity by formation of a complex between the WSG and the toxic phospholipase. We report the purification and characterization of a glycoprotein phospholipase A inhibitor from Withania somnifera, medicinal plant. |
| 2003 | Purification and characterization of an anticoagulant phospholipase A(2) from Indian monocled cobra (Naja kaouthia) venom. | Toxicon | An anticoagulant, non-toxic phospholipase A(2) was isolated from the venom of Indian monocled cobra (Naja kaouthia) by a combination of ion-exchange chromatography on CM-Sephadex C-50 and gel filtration on Sephadex G-50. This purified protein named NK-PLA(2)-I, had a subunit molecular mass of 13.6 kDa and migrated as a dimer under non-reduced condition in SDS-PAGE. NK-PLA(2)-I was a highly thermostable protein requiring basic pH optima for its catalytic activity and showed preferential hydrolysis of phosphotidylcholine. This protein exhibited higher anticoagulant, indirect hemolysis, liver and heart tissue damaging activity but exerted less toxicity, direct hemolysis, edema and lung tissue damaging activity as compared to whole venom. Treatment of NK-PLA(2)-I with rho-BPB, TPCK, PMSF, antivenom and heating had almost equal effect on PLA(2), and other pharmacological properties except in vitro tissue damaging activity. Current investigation provides a fairly good indication that NK-PLA(2)-I induces various pharmacological effects by mechanisms, which are either dependent or independent of its catalytic activity. |
| 2002 | Characteristics of capsules in enterotoxemic Escherichia coli O139:K12 strains causing swine edema disease. | Microbiol Res | The characteristics of the capsule of the enterotoxemic Escherichia coli (ETEEC) O139:K12 strains that strongly adhere to Hep-2 cells were examined. Electron microscopic studies using the freeze-substitution technique revealed that ETEEC strains had a capsule of approximately 25 nm. These strains show hydrophobic surface properties and strong adherence to human polymorphonuclear leukocytes (PMNs). In contrast, ETEEC strains RK-O139 and ED-1 show weak adherence to HEp-2 cells and fail to express the capsule layer on the cell surface. These ETEEC strains possess hydrophilic surface properties and also adhere to PMNs. The lipopolysaccharide (LPS) analysis by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that ETEEC strains had the same LPS profile and long O-side chains of LPS. Furthermore, all strains were resistant to serum killing activity. These results suggest that the capsule of ETEEC strains does not contribute as an antiphagocytic factor, but as an adherence factor to host cells. |
| 2002 | Characterization and cloning of a novel phospholipase A(2) from the venom of Trimeresurus jerdonii snake. | Toxicon | A phospholipase A(2) (PLA(2)), called jerdoxin, was isolated from Trimeresurus jerdonni snake venom and partially characterized. The protein was purified by three chromatographic steps. SDS-polyacrylamide gel electrophoresis in the presence or absence of dithiothreitol showed that it had a molecular mass of 15 kDa. Jerdoxin had an enzymatic activity of 39.4 micro mol/min/mg towards egg yolk phosphatidyl choline (PC). It induced edema in the footpads of mice. In addition, jerdoxin exhibited indirect hemolytic activity. About 97% hemolysis was observed when 2 micro g/ml enzyme was incubated for 90 min in the presence of PC and Ca(2+). No detectable hemolysis was noticed when PC was not added. Ca(2+) was necessary for jerdoxin to exert its hemolytic activity, since only 52% hemolysis was seen when Ca(2+) was absent in the reaction mixture. Furthermore, jerdoxin inhibited ADP induced rabbit platelet aggregation and the inhibition was dose dependent with an IC(50) of 1.0 micro M. The complete amino acid sequence of jerdoxin deduced from cDNA sequence shared high homology with other snake venom PLA(2)s, especially the D 49 PLA(2)s. Also, the residues concerned to Ca(2+) binding were conserved. This is the first report of cDNA sequence of T. jerdonii venom PLA(2). |
| 2002 | [Catabolism of brain natriuretic peptide (BNP) in normal and nephrotic rat kidney]. | Nihon Jinzo Gakkai Shi | The major metabolic change that characterizes nephrotic syndrome (NS) is hypoalbuminemia. Edema, which is a well-recognized clinical feature, often results in disorder of peripheral circulation and congestive heart failure. We previously reported that the albumin content of kidney lysosomal proteins was increased more than ten-fold in nephrotic rats compared to control rats, suggesting that kidney lysosomes play an important role in protein degradation in NS. The present study was carried out to elucidate the role of catabolism of BNP, which is one of the functional protein-natriuretic peptides. We injected puromysin aminonucleoside(PAN) to induce the nephrotic rat state and isolated kidney lysosomes from normal and nephrotic rat kidney cortex by our methods. Immunoblot analysis of tricine-SDS polyacrylamide gel electrophoresis of rat kidney lysosomal proteins revealed the presence of an immuno-reactive peptide band, corresponding to the endogenous BNP. In addition, this was reduced as compared with the normal control. These result suggest that kidney lysosomes play an important role in BNP metabolism to maintain body fluid homeostasis and regulate blood pressure levels. |
| 2002 | Multicenter trial of low-dose paclitaxel in patients with advanced AIDS-related Kaposi sarcoma. | Cancer | Treatment options are limited for patients with advanced acquired immunodeficiency syndrome (AIDS)-related Kaposi sarcoma (AIDS-KS) whose disease has progressed after receiving therapy with liposomal anthracyclines or combination chemotherapy with doxorubicin (Adriamycin), bleomycin, and vincristine (ABV). This study was performed to assess the safety and efficacy of a novel dose and schedule of paclitaxel in patients with AIDS-KS who failed to respond to previous systemic chemotherapy.This was an open-label, multicenter Phase II study. Eligible patients had advanced AIDS-KS consisting of at least 25 mucocutaneous lesions, visceral disease, or lymphedema, and had failed to respond to at least one previous systemic chemotherapy regimen. Patients were treated with paclitaxel at a dose of 100 mg/m(2) given intravenously over 3 hours, every 2 weeks. Primary efficacy end points were tumor response, time to progression, time to treatment failure, and survival. Quality of life and adverse events were evaluated using the Symptom Distress Scale (SDS) and the World Health Organization Toxicity Criteria, respectively.One hundred and seven male patients with advanced AIDS-KS were enrolled from nine participating sites. The median entry CD4+ lymphocyte count was 41/mm(3) (range 0-1139). Previous treatment regimens included ABV in 52, liposomal daunorubicin in 49, and liposomal doxorubicin in 40 patients. Forty-one patients (38%) received two or more previous chemotherapy regimens. Protease inhibitor use during the study was reported by 82 (77%) patients overall; 47 patients (44%) were receiving a protease inhibitor before study entry. Complete or partial response was documented in 60 patients (56%). The median duration of response was 8.9 months. Major response rate was similar when comparing patients not on a protease inhibitor at the time of response (59%) with patients on a protease inhibitor at time of response (54%). However, protease inhibitor use had a significant impact on survival (P = 0.04). Grade 4 neutropenia was reported in 35% of patients; other life-threatening side effects were uncommon. Significant improvements were seen in the total quality of life scores measured by the SDS, including significant improvement in KS-related symptoms such as facial disease, tumor-associated edema, and pulmonary involvement.Paclitaxel given every 2 weeks induces major tumor regression in the majority of patients with advanced KS who failed to respond to previous systemic chemotherapy. Paclitaxel is associated with significant improvement in quality of life with acceptable toxicity and should be considered as an effective treatment option for patients with advanced KS. |
| Severe tomato allergy (Lycopersicon esculentum). | Allergy Asthma Proc | Although tomatoes are a commonly consumed food, severe allergic reactions to tomatoes are unusual or rarely reported. Previously reported allergic manifestations to tomato include urticaria/angioedema, dermatitis, oral allergy syndrome, rhinitis, and abdominal pain. The aim of this study was to report two patients with significant immediate hypersensitivity reactions to tomato and characterize the responsible allergen. We reviewed the history and documentation of tomato-specific immunoglobulin E (IgE) of two patients with adverse symptoms after ingesting tomato. Fresh tomato extracts prepared from the skin, seeds, and flesh of red, ripe tomatoes were evaluated for total protein content and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to characterize the tomato protein. IgE enzyme-linked immunosorbent assay (ELISA) using the patients' serum against the various tomato extracts was accomplished and IgE immunoblot was performed. Percutaneous skin tests or radioallergosorbent tests (RASTs) were positive to tomato in both patients. Both adults experienced laryngeal edema and one had anaphylaxis. Similar total protein contents were found in each of the tomato extracts and gel electrophoresis revealed similar protein profile for skin and seed extracts with protein bands discernible at molecular weights of 21, 33, and 43 kDa. One patient reacted specifically to a 43-kDa protein band on IgE immunoblot. The two cases show that severe allergic reactions to tomato occur in adults and one is associated with IgE binding to a 43-kDa protein. |
| 2002 | A dynamic shift of VEGF isoforms with a transient and selective progesterone-induced expression of VEGF189 regulates angiogenesis and vascular permeability in human uterus. | Proc Natl Acad Sci U S A | A key mechanism underlying physiological angiogenesis of the human endometrium is its ability to regenerate the vascular capillary network and to perform vascular remodeling (i.e., development of spiral arteries). Vascular endothelial growth factor (VEGF) is associated with angiogenesis and capillary permeability in this tissue. VEGF is expressed as several spliced variants, its main human isoforms contain 121 and 165 aa; 17beta-estradiol (E(2)) increases endometrial VEGF, possibly in all isoforms. Here we show that progesterone (P) selectively increases the expression of the VEGF(189) (V(189)) isoform in the human uterus. V(189) is identified in the conditioned medium of stromal cells treated with E(2) + P; its presence in this in vitro model of decidual stromal cells is detected after 6-8 days, using ELISA, and after 8-10 days, using Western blot analysis with different antibodies, including one specific for V(189). The secretion pattern of V(189) parallels that of the decidual protein IGFBP-1. V(189) is secreted as a native isoform, as compared with the migration of recombinant V(189) by SDS/PAGE. In situ hybridization and immunocytochemistry(,) performed on the same biopsies, suggest that decidual cells express V(189) during the mid-late secretory phase of the menstrual cycle and early gestation. Finally, using an in vivo permeability assay, we show that native V(189) increases capillary permeability. These observations demonstrate that P regulates V(189) expression in decidual cells, which could have important implications for understanding uterine vascular remodeling and implantation, and may be relevant in a range of disease states such as edema and irregular bleeding. |
| 2002 | Variations in biochemical and pharmacological properties of Indian cobra (Naja naja naja) venom due to geographical distribution. | Mol Cell Biochem | Indian cobra (Naja naja naja) venom obtained from three different geographical regions was studied in terms of electrophoretic pattern, biochemical and pharmacological activities. SDS-PAGE banding pattern revealed significant variation in the protein constituents of the three regional venoms. The eastern venom showed highest indirect hemolysis and hyaluronidase activity. In contrast, western and southern venoms were rich in proteolytic activity. All the three regional venoms were devoid of p-tosyl-L-arginine methyl ester hydrolysing activity. The eastern venom was found to be most lethal among the three regional venoms. The lethal potency varied as eastern > western > southern regional venoms. In addition, all the three regional venoms showed marked variations in their ability to induce symptoms/signs of neurotoxicity, myotoxicity, edema and effect on plasma coagulation process. Polyclonal antiserum prepared against the venom of eastern region cross-reacted with both southern and western regional venoms, but varied in the extent of cross-reactivity by ouchterlony immunodiffusion and ELISA. |
| 2002 | Corneal toxicity of intraocular hyaluronidase. | J Ocul Pharmacol Ther | The purpose of this study was to examine the corneal toxicity of different preparations of intraocular hyaluronidase. SDS-PAGE analysis of bovine testicular hyaluronidase (Wydase) and chromatographically purified hyaluronidase (Sigma) was performed. These two preparations were injected into the anterior chamber of rabbits in amounts ranging from 1.5-150 IU (Wydase) and 1.5-300 IU (Sigma). A third set of rabbit eyes received Wydase vehicle alone or in combination with Sigma hyaluronidase. Treated control eyes were injected with saline. Slit lamp examination and indirect ophthalmoscopy were performed preoperatively and on postoperative days 1 and 7. Light microscopy of the corneas was performed. SDS-PAGE of Wydase revealed numerous protein impurities, while Sigma demonstrated one protein band consistent with mammalian hyaluronidase. Persistent corneal edema, severe anterior chamber fibrin, and endothelial necrosis, were seen in the majority of eyes injected with Wydase in amounts of 50 IU and greater (n = 11). Thirty percent (30%) of the eyes injected with the Sigma preparation (n = 11) had localized corneal opacity similar to 50% of eyes injected with saline (n = 2). Of the rabbit eyes injected with the Wydase vehicle (n = 19), 68% had toxic changes. Intracameral injection of Wydase is toxic to the rabbit cornea in amounts of 50 IU and greater. A chromatographically purified preparation showed only transient local toxicity. Toxicity of Wydase may be due to protein impurities and the thimerosal-containing vehicle. |
| 2002 | Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin. | J Neurochem | Corticosteroids provide an effective treatment to reduce edema for conditions in which the blood-brain or blood-retinal barrier is compromised. However, little is known about the mechanism by which these hormones affect endothelial cell function. We hypothesized that hydrocortisone would reduce transport of water and solutes across bovine retinal endothelial cell (BREC) monolayers coincident with changes to the tight junction protein occludin. Treatment of BREC with 103 nm hydrocortisone for two days significantly decreased water and solute transport across cell monolayers. Immunoblot analysis of occludin extracted in SDS or urea based buffers revealed a 1.65- or 2.57-fold increase in content, respectively. A similar two-fold increase in occludin mRNA was observed by real-time PCR. Immunocytochemistry revealed hydrocortisone dramatically increased both occludin and ZO-1 staining at the cell border. Additionally, 4 h of hydrocortisone treatment significantly reduced occludin phosphorylation. To our knowledge, this is the first example of a regulated decrease in occludin phosphorylation associated with increased barrier properties. In conclusion, hydrocortisone directly affects retinal endothelial cell barrier properties coincident with changes in occludin content, phosphorylation and tight junction assembly. Localized hydrocortisone therapy may be developed as a treatment option for patients suffering from retinal edema due to diabetes. |
| 2001 | Venom of the crotaline snake Atropoides nummifer (jumping viper) from Guatemala and Honduras: comparative toxicological characterization, isolation of a myotoxic phospholipase A(2) homologue and neutralization by two antivenoms. | Comp Biochem Physiol C Toxicol Pharmacol | A comparative study was performed on the venoms of the crotaline snake Atropoides nummifer from Guatemala and Honduras. SDS-polyacrylamide gel electrophoresis, under reducing conditions, revealed a highly similar pattern of these venoms, and between them and the venom of the same species from Costa Rica. Similar patterns were also observed in ion-exchange chromatography on CM-Shephadex C-25, in which a highly basic myotoxic fraction was present. This fraction was devoid of phospholipase A(2) activity and strongly reacted, by enzyme-immunoassay, with an antiserum against Bothrops asper myotoxin II, a Lys-49 phospholipase A(2) homologue. A basic myotoxin of 16 kDa was isolated to homogeneity from the venom of A. nummifer from Honduras, showing amino acid composition and N-terminal sequence similar to those of Lys-49 phospholipase A(2) variants previously isolated from other crotaline snake venoms. Guatemalan and Honduran A. nummifer venoms have a qualitatively similar toxicological profile, characterized by: lethal; hemorrhagic; myotoxic; edema-forming; coagulant; and defibrinating activities, although there were significant quantitative variations in some of these activities between the two venoms. Neutralization of toxic activities by two commercially-available antivenoms in the region was studied. Polyvalent antivenom produced by Instituto Clodomiro Picado was effective in the neutralization of: lethal; hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A(2) activities, but ineffective against edema-forming activity. On the other hand, MYN polyvalent antivenom neutralized: hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A(2) activities, albeit with a lower potency than Instituto Clodomiro Picado antivenom. MYN antivenom failed to neutralize lethal and edema-forming activities of A. nummifer venoms. |
| 2000 | Isolation and characterization of myrmexins, six isoforms of venom proteins with anti-inflammatory activity from the tropical ant, Pseudomyrmex triplarinus. | Toxicon | Venom from the tropical ant, Pseudomyrmex triplarinus, has anti-inflammatory properties demonstrated by the carrageenin-induced edema animal mode. A multi-protein complex that inhibits edema was isolated from the venom and was further characterized by high performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and amino acid sequencing. Although the complex exhibited a single band in SDS-PAGE electrophoresis, six proteins (isoforms) were resolved and purified to homogeneity and were designated myrmexin I-VI. They have very similar molecular masses between 6998 and 7142 Da. Each myrmexin is a heterodimer consisting of a small subunit (SS1 or SS2 or SS3) disulfide-linked to a larger, quite structurally unrelated subunit (LS1 or LS2). Thus, the myrmexin complex consists of six isoforms of venom proteins: myrmexin I (SS1/LS2), myrmexin II (SS1/LS1), myrmexin III (SS2/LS2), myrmexin IV (SS3/LS2), myrmexin V (SS2/LS1), myrmexin VI (SS3/LS1). Subunit SS1 is highly homologous to SS2 (96% of identity) and SS3 (87% of identity) and LS1 is highly homologous to LS2 (79% of identity). Our study suggests that myrmexins may represent a new class of anti-inflammatory proteins. |
| 2000 | Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema. | Biochim Biophys Acta | A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LlTI inhibits plasmin (K(i) 3.2 x 10(-10) M), human plasma kallikrein (K(i) 6.3 x 10(-9) M), trypsin (K(i) 2.5 x 10(-8) M) and chymotrypsin (K(i) 1.4 x 10(-8) M). Factor XIIa activity is inhibited but K(i) was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LlTI. The action of LlTI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats. |
| 2000 | Structural and functional characterization of myotoxin I, a Lys49 phospholipase A(2) homologue from Bothrops moojeni (Caissaca) snake venom. | Arch Biochem Biophys | Myotoxin-I (MjTX-I) was purified to homogeneity from the venom of Bothrops moojeni by ion-exchange chromatography on CM-Sepharose. Its molecular weight, estimated by SDS-PAGE, was 13,400 (reduced) or 26, 000 (unreduced). The extinction coefficient (E(1.0 mg/ml)(1.0 cm)) of MjTX-I was 1.145 at lambda = 278 nm, pH 7.0, and its isoelectric point was 8.2 at ionic strength mu = 0.1. When lyophilized and stored at 4 degrees C, dimeric, trimeric, and pentameric forms of the protein were identified by SDS-PAGE. This "heterogeneous" sample could be separated into three fractions by gel filtration on Sephadex G-50. The fractions were analyzed by isoelectric focusing, immunoelectrophoresis, and amino acid composition, which indicated that heterogeneity was the result of different levels of self-association. Protein sequencing indicated that MjTX-I is a Lys49 myotoxin and consists of 121 amino acids (M(r) = 13,669), containing a high proportion of basic and hydrophobic residues. It shares a high degree of sequence identity with other Lys49 PLA(2)-like myotoxins, but shows a significantly lower identity with catalytically active Asp49 PLA(2)s. The three-dimensional structure of MjTX-I was modeled based on the crystal structures of three highly homologous Lys49 PLA(2)-like myotoxins. This model showed that the amino acid substitutions are conservative, and mainly limited to three structural regions: the N-terminal helix, the beta-wing region, and the C-terminal extended random coil. MjTX-I displays local myotoxic and edema-inducing activities in mice, and is lethal by intraperitoneal injection, with an LD(50) value of 8.5 +/- 0.8 mg/kg. In addition, it is cytotoxic to myoblasts/myotubes in culture, and disrupts negatively charged liposomes. In comparison with the freshly prepared dimeric sample, the more aggregated forms showed significantly reduced myotoxic activity. However, the edema-inducing activity of MjTX-I was independent of molecular association. Phospholipase A(2) activity on egg yolk, as well as anticoagulant activity, were undetectable both in the native and in the more associated forms. His, Tyr, and Trp residues of the toxin were chemically modified by specific reagents. Although the myotoxic and lethal activities of the modified toxins were reduced by these treatments, neither its edema-inducing or liposome-disrupting activities were significantly altered. Rabbit antibodies to native MjTX-I cross-reacted with the chemically modified forms, and both the native and modified MjTX-I preparations were recognized by antibodies against the C-terminal region 115-129 of myotoxin II from B. asper, a highly Lys49 PLA(2)-homologue with high sequencial similarity. |
| 1999 | Isolation and characterization of an arginine ester hydrolase from Bothrops jararacussu venom which induces contractions of the isolated rat uterus. | Biochem Mol Biol Int | The isolation and partial characterization of a serine protease with arginine ester hydrolase activity from Bothrops jararacussu snake venom are described. The purification procedure consisted of a gel filtration of the crude venom on Sephadex G-75 followed by an ion-exchange chromatography of the active fraction on DEAE-cellulose and a rechromatography on Bio-Rex 70 resin. The esterase fraction (DI-III), M(r) = 25,000 by SDS-PAGE, showed proteolytic activity on fibrinogen and casein. After 2 hr incubation, the A alpha and B beta chains of fibrinogen were intensely hydrolysed, while the gamma chain kept apparently intact, even after 20 hr of incubation. In spite of that, DI-III did not clot fibrinogen. DI-III induced edema in the rat paw. Although unable to release bradykinin, it induced contractions of the isolated rat uterus. DI-III did not catalyse the hydrolysis of bradykinin. Its arginine ester hydrolase activity was completely inhibited by diisopropyl fluorophosphate after 1 hr incubation, but not by phenylmethylsulfonyl fluoride under the same conditions. |
| 1998 | Characterization of membrane translocation by anthrax protective antigen. | Biochemistry | Solving the crystallographic structure of the ring-shaped heptamer formed by protective antigen (PA), the B moiety of anthrax toxin, has focused attention on understanding how this oligomer mediates membrane translocation of the toxin's A moieties. We have developed an assay for translocation in which radiolabeled ligands are bound to proteolytically activated PA (PA63) at the surface of CHO or L6 cells, and translocation across the plasma membrane is induced by lowering the pH. The cells are then treated with Pronase E to degrade residual surface-bound material, and protected ligands are quantified after fractionation by SDS-PAGE. Translocation was most efficient (35%-50%) with LFN, the N-terminal PA binding domain of the anthrax lethal factor (LF). Intact LF, edema factor (EF), or fusion proteins containing LFN fused to certain heterologous proteins [the diphtheria toxin A chain (DTA) or dihydrofolate reductase (DHFR)] were less efficiently translocated (15%-20%); and LFN fusions to several other proteins were not translocated at all. LFN with different N-terminal residues was found to be degraded according to the N-end rule by the proteasome, and translocation of LFN fused to a mutant form of DHFR with a low affinity for methotrexate (MTX) protected cells from the effects of MTX. Both results are consistent with a cytosolic location of protected proteins. Evidence that a protein must unfold to be translocated was obtained in experiments showing that (i) translocation of LFNDTA was blocked by introduction of an artificial disulfide into the DTA moiety, and (ii) translocation of LFNDHFR and LFNDTA was blocked by their ligands (MTX and adenine, respectively). These results demonstrate that the acid-induced translocation by anthrax toxin closely resembles that of diphtheria toxin, despite the fact that these two toxins are unrelated and form pores by different mechanisms. |
| 1998 | Comparative study of the venoms of three subspecies of Lachesis muta (bushmaster) from Brazil, Colombia and Costa Rica. | Toxicon | A comparative study was performed on the pharmacology and biochemistry of venoms from three subspecies of Lachesis muta (L. m. stenophrys, L. m. muta and L. m. rhombeata) from Brazil, Colombia and Costa Rica. All venoms induced lethal, hemorrhagic, edema-forming, myotoxic, coagulant and defibrinating effects, showing also proteolytic and indirect hemolytic activities. The venoms of L. m. stenophrys from Costa Rica and L. m. muta from Cascalheira, Brazil, had the highest lethal and hemorrhagic activities and the venom of L. m. rhombeata showed the highest coagulant activity, whereas no significant differences were observed in myotoxic and edema-forming activities at most of the time intervals studied. In addition, venoms showed similar electrophoretic patterns on SDS-polyacrylamide gel electrophoresis. In conclusion, despite quantitative differences in toxic and enzymatic activities, together with subtle variations in electrophoretic patterns, our results indicate that experimental envenomation by these venoms induce a qualitatively similar pathophysiological profile. |
| 1998 | Decorin and biglycan of normal and pathologic human corneas. | Invest Ophthalmol Vis Sci | Corneas with scars and certain chronic pathologic conditions contain highly sulfated dermatan sulfate, but little is known of the core proteins that carry these atypical glycosaminoglycans. In this study the proteoglycan proteins attached to dermatan sulfate in normal and pathologic human corneas were examined to identify primary genes involved in the pathobiology of corneal scarring.Proteoglycans from human corneas with chronic edema, bullous keratopathy, and keratoconus and from normal corneas were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), quantitative immunoblotting, and immunohistology with peptide antibodies to decorin and biglycan.Proteoglycans from pathologic corneas exhibit increased size heterogeneity and binding of the cationic dye alcian blue compared with those in normal corneas. Decorin and biglycan extracted from normal and diseased corneas exhibited similar molecular size distribution patterns. In approximately half of the pathologic corneas, the level of biglycan was elevated an average of seven times above normal, and decorin was elevated approximately three times above normal. The increases were associated with highly charged molecular forms of decorin and biglycan, indicating modification of the proteins with dermatan sulfate chains of increased sulfation. Immunostaining of corneal sections showed an abnormal stromal localization of biglycan in pathologic corneas.The increased dermatan sulfate associated with chronic corneal pathologic conditions results from stromal accumulation of decorin and particularly of biglycan in the affected corneas. These proteins bear dermatan sulfate chains with increased sulfation compared with normal stromal proteoglycans. |
| 1998 | A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: biochemical characterization, crystallization, myotoxic and edematogenic activity. | Toxicon | Bothtrops moojeni snake venom was fractionated on a CM-Sepharose column which was previously equilibrated with 0.05 M ammonium bicarbonate buffer at pH 8.0 and subsequently eluted with an ammonium bicarbonate concentration gradient from 0.05 to 0.5 M at constant pH (8.0) and temperature (25 degrees C). The fraction which eluted last (M-VI) showed, after direct lyophilization, a single band by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE, indicating an approximate Mr of 14000 and 27000, in the presence and absence of dithiothreitol, respectively. Its amino acid composition revealed a high level of hydrophobic and basic amino acids as well as 14 half-cystine residues. Its isoelectric point and extinction coefficient (E(1.0 mg/ml) (1.0 cm) at 278 nm and pH 7.0) were 8.2 and 1.170, respectively. M-VI was devoid of phospholipase A2 (PLA2) activity on egg yolk, as well as of hemorrhagic, anticoagulant and coagulant activities, but could induce drastic necrosis on skeletal muscle fibres as well as rapid and transient edema on the rat paw. Its N-terminal sequence: SLFELGKMILQETGKNPAKSYGVYGCNCGVGGRGKPKDATDRCCYVHKCCYK... revealed high homology with other Lys 49 PLA2-like myotoxins from other bothropic venoms. Orthorhombic crystals of M-VI, which diffracted to a maximal resolution of 1.6 A, were obtained and indicated the presence of a dimer in the asymmetrical unit. |
| 1998 | Thalassophryne nattereri fish venom: biological and biochemical characterization and serum neutralization of its toxic activities. | Toxicon | Envenomation by Thalassophryne nattereri fishes are an important medical problem in northeast of Brazil, causing in human victims considerable pain and edema followed by necrosis. Venom obtained from fresh captured specimens of this fish was tested in vitro or in animal models for a better characterization of its toxic activities. Intradermal injection of the venom in the foot pad of mice induced local edema and hemorrhage followed a few hours later by necrosis. Subcutaneous injection of the venom induced systemic effects consisting in jerking motions, paralysis of hind limbs, erection of hair, rotational movements and violent convulsions followed by death. Dead animals showed hyperemia of the small intestine and lungs. The venom showed distinct edematous, necrotizing and hemolytic activities, a low level of hemorrhagic, myotoxic and proteolytic activities and no detectable phospholipase A2 activity. SDS-PAGE analysis of the crude venom showed at least 17 components with the major band located around Mw = 19,000. Almost all proteins stained by amido black were also revealed by Western blotting with antibodies to T. nattereri venom. Fractionation of the venom by either gel filtration or cation exchange chromatography resulted in a few distinct peaks but in both situations the biological activities were located in only one of the peaks which corresponded to basic proteins with approximately Mw = 47,000. Heating of the venom at 56 degrees C for 60 min completely destroyed its biological activities. All venom toxic activities except edema were completely neutralized after in vitro incubation with anti-T. nattereri serum. |
| 1998 | Fermentation, purification, and characterization of protective antigen from a recombinant, avirulent strain of Bacillus anthracis. | Appl Environ Microbiol | Bacillus anthracis, the etiologic agent for anthrax, produces two bipartite, AB-type exotoxins, edema toxin and lethal toxin. The B subunit of both exotoxins is an M(r) 83,000 protein termed protective antigen (PA). The human anthrax vaccine currently licensed for use in the United States consists primarily of this protein adsorbed onto aluminum oxyhydroxide. This report describes the production of PA from a recombinant, asporogenic, nontoxigenic, and nonencapsulated host strain of B. anthracis and the subsequent purification and characterization of the protein product. Fermentation in a high-tryptone, high-yeast-extract medium under nonlimiting aeration produced 20 to 30 mg of secreted PA per liter. Secreted protease activity under these fermentation conditions was low and was inhibited more than 95% by the addition of EDTA. A purity of 88 to 93% was achieved for PA by diafiltration and anion-exchange chromatography, while greater than 95% final purity was achieved with an additional hydrophobic interaction chromatography step. The purity of the PA product was characterized by reversed-phase high-pressure liquid chromatography, sodium dodecyl sulfate (SDS)-capillary electrophoresis, capillary isoelectric focusing, native gel electrophoresis, and SDS-polyacrylamide gel electrophoresis. The biological activity of the PA, when combined with excess lethal factor in the macrophage cell lysis assay, was comparable to previously reported values. |
| Differential effects of ozone on lung epithelial lining fluid volume and protein content. | Exp Lung Res | Urea dilution has been used to estimate the volume of epithelial lining fluid (ELF) in the respiratory tract. However, ELF volume may be overestimated as the result of rapid net diffusion of urea from tissues into the bronchoalveolar lavage (BAL) fluid. This study established a protocol for rat BAL in a manner that minimizes this problem and then used this procedure to examine the edemagenic effects of ozone (O3) exposure on ELF volume and the concentrations of ELF protein and albumin. One passage lavage with variable dwell times up to 30 s showed no difference in recovered urea, protein, and albumin and ELF volume between 0 and 4 s, but a progressive increase of each thereafter. The calculated concentrations of protein and albumin in ELF did not vary significantly with dwell time. By increasing the number of lavage passages from one to three, the amounts of recovered urea, protein, and albumin and estimated ELF volume were increased with each passage. Again, the calculated concentrations of protein and albumin in ELF did not vary appreciably. When a single lavage passage and no added dwell time were used, it was observed that exposure of rats to 2 but not 0.5 and 1 ppm O3 increased urea, protein, and albumin in the BAL immediately after 6 h exposure. In addition, at 18 h postexposure to 1 ppm O3, ELF volume increased only 21%, but protein and albumin concentrations in ELF were 2.3- and 4.5-fold of control values, respectively. A higher O3 concentration (2 ppm) moderately increased ELF volume (+83%) and exerted even greater effects on concentrations of ELF protein (7.8-fold) and albumin (19-fold) while lower O3 dosage (0.5 ppm) had no significant effect. SDS-PAGE analysis showed that small serum proteins including albumin were greatly enriched in lung BAL fluid of 1 ppm O3-exposed rats. These results demonstrate that movement of water and protein into the airspaces after O3 exposure is not strictly coupled, and that protein recovery by BAL should cautiously be used to indicate airspace edema as a result of O3 injury. |
| 1995 | Purification and partial immunochemical characterization of proteins of fimbriae F107 from Escherichia coli isolated from edema disease of pigs. | Folia Microbiol (Praha) | The paper describes the isolation, purification and characterization of F107-fimbrial proteins, obtained by thermoelution from Escherichia coli 107/86. Isolation of the pure F107 protein was done by FPLC chromatography, employing Superose 12, Mono Q, and Phenyl-Superose columns. The highest purity of the F107 protein was achieved with Superose 12 HR 10/30. Purity checking by a HPLC system Waters 625 LC (Millipore) proved the absence of protein admixtures in a fraction from Superose 12. Analysis of the molar mass of F107 proteins by SDS PAGE revealed that F107 fimbriae consist of two proteins, one of M = 43 kDa (minor), and other of M = 18.9 kDa (major). Western blot analysis with rabbit polyclonal antiserum confirmed that the 18.9 kDa protein was the major characteristic unit of F107 fimbriae. |
| 1994 | Intratumoral PEG-interleukin-2 therapy in patients with locoregionally recurrent head and neck squamous-cell carcinoma. | Ann Oncol | An enhanced efficacy of local as compared to systemic administration of interleukin-2 (IL-2) has been demonstrated in several experimental tumors. We previously reported that guinea pigs with palpable tumors and regional micrometastases could be cured by intratumoral injections of polyethylene glycol-modified IL-2 (PEG-IL-2). In the present study this treatment schedule was applied in a clinical situation.Nineteen patients with 11 local and 11 regional recurrences of head and neck squamous cell carcinoma (HNSCC) were treated with intratumoral injections of 200,000 U of PEG-IL-2 3 times weekly in courses of 4 weeks.Treatment was given on an out-patient basis, and was well tolerated. Temporary regional swelling and redness developed in 10 patients, and in 9 of them systemic eosinophilia was documented. Median duration of treatment was 4 weeks (range 2-14 weeks). Seventeen patients were evaluable for response. One complete response (CR; 6%; duration 91 weeks), and 6 stable diseases (SDs; duration 8-57+ weeks) were recorded. The CR and the 3 best SDs (23, 40, 57+ weeks) occurred in patients with a single regional tumor recurrence of relatively small size. During treatment, all 4 developed locoregional edema and redness, and high levels of circulating eosinophils. Median survival was 23 weeks for all patients, and 45+ weeks for the patients with SD.Intratumoral injection of PEG-IL-2 in patients with HNSCC is feasible. This treatment appears beneficial for highly selected patients. The objective response rate is insufficient to justify wide clinical application. |
| 1994 | Anthrax protective antigen forms oligomers during intoxication of mammalian cells. | J Biol Chem | The protective antigen component (PA) of anthrax toxin binds to receptors on target cells and conveys the toxin's edema factor (EF) and lethal factor (LF) components into the cytoplasm. PA (83 kDa) is processed by a cellular protease, yielding a 63-kDa fragment (PA63), which binds EF and/or LF. When exposed to acidic pH, PA63 inserts into membranes and forms ion-conductive channels. By electron microscopy, a significant fraction of purified PA63 was found to be in the form of a multi-subunit ring-shaped oligomer (outer diameter, 10.4 nm). The rings are heptameric, as judged by inspection and by rotational power spectra. Purified PA63 showed a high M(r) band, apparently corresponding to the oligomer, on SDS-polyacrylamide gels, and oligomer of similar size was formed in cells in a time-dependent manner after addition of full-length PA. Inhibitors of internalization and endosome acidification blocked conversion of cell-associated PA to a high molecular weight species, and medium at pH 5.0 induced oligomer formation in the presence or absence of the inhibitors. These results correlate PA63 oligomerization with conditions required for translocation of EF and LF across lipid bilayers, implying that the PA63 oligomer may function in translocation. |
| 1994 | Purification, characterization and biological activities of phospholipase A from Russell's viper (Vipera russelli) venom. | Int J Biochem | 1. The major phospholipase A has been purified to electrophoretic homogeneity from the venom of Vipera russelli (Russell's viper). 2. The molecular weight of the purified enzyme was estimated to be 31,000 by Sephadex G-75 gel filtration chromatography and 29,000 by SDS-polyacrylamide gel electrophoresis. The enzyme exhibited an apparent Km value of 2.3 x 10(-2) M. 3. The phospholipase A showed edema forming, indirect hemolytic and myonecrotic activities but not hemorrhagic activity. |
| 1993 | Aldose reductase in human retinal pigment epithelial cells. | Exp Eye Res | Sugar alcohols have been reported to accumulate in retinal pigment epithelium (RPE) of diabetic animals. This finding has raised interest in the role of RPE in diabetes-associated retinal changes such as cystoid macular edema. To confirm the presence of aldose reductase in this tissue, the NADPH-dependent enzyme was purified to an apparent homogeneity from cultured human RPE cells, characterized, and its biochemical properties investigated. The induction of aldose reductase by hypertonic stress was also examined. The purification of aldose reductase was performed by a series of chromatographic steps which include gel filtration, affinity chromatography and chromatofocusing. Final purity achieved was monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The kinetic properties and susceptibility to inhibition of the purified aldose reductase were essentially identical to aldose reductase purified from human placenta and kidney. In addition to aldose reductase, chromatofocusing demonstrated the presence of aldehyde reductase, another NADPH-dependent reductase. However, the amounts of aldehyde reductase present were much smaller than those of aldose reductase and the levels of aldehyde reductase appeared too small to contribute to the polyol production in the RPE cells. Culture of RPE cells in hypertonic medium containing 150 mM sodium chloride (600 mosmol total) increased both reductase activity, monitored with DL-glyceraldehyde as substrate, and immunoblot staining for aldose reductase. Chromatofocusing of RPE cells cultured in hypertonic media resulted in a prominent increase in the peak corresponding to aldose reductase compared to the peak height of cells grown in control medium. No increase in aldehyde reductase from RPE cells cultured in hypertonic medium was observed.(ABSTRACT TRUNCATED AT 250 WORDS) |
| 1993 | Biological and biochemical activities of Vipera berus (European viper) venom. | Toxicon | Vipera berus is widely distributed throughout the northern part of Europe and Asia. Characterization of several toxic effects of its venom in the mouse, as well as of in vitro enzymatic activities was performed. Vipera berus venom displayed in vitro proteolytic, fibrinolytic, anticoagulant, and phospholipase A2 activities. The i.p. LD50 of the venom for Swiss mice was 0.86 micrograms/g (95% confidence limits 0.71-1.01 microgram/g). Significant local tissue-damaging effects, including edema, hemorrhage and myonecrosis, were observed. The local edema was characterized by rapid onset, reaching a maximum after 0.5-1 hr, and with dose-dependent persistence. The hemorrhagic potency was measured by a skin test, giving a minimum hemorrhagic dose value of 3.2 micrograms. The venom also induced a moderate local myonecrosis, evidenced by histological evaluation of injected tissue (gastrocnemius), and by biochemical parameters (increase of plasma creatine kinase activity, and decrease of muscle residual MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)-reducing activity). Characterization of the venom by SDS-polyacrylamide gel electrophoresis revealed 10 (reduced) or 11 (unreduced) main protein bands, which were further analyzed in relation to mol. wt and relative concentration by densitometry. A rabbit antiserum to V. berus venom recognized all main venom bands by immunoblotting. This antiserum cross-reacted to a variable extent with several crotaline venoms, as assessed by enzyme immunoassay. |
| 1992 | Proteinuria of B700, a 67 kD albumin-like melanoma-specific antigen. | Pigment Cell Res | B700 is a murine melanoma antigen that is closely related to, but distinct from, serum albumin. The present study examined the metabolic fate and anatomic distribution of radioiodinated B700 and mouse serum albumin (MSA) administered s.c. to mice. In blood, both proteins were associated with the plasma fraction where the halflife of B700, a glycoprotein, was 0.5 days, compared to 2.7 days for MSA. Of particular interest was the observation that B700, a 67 kD anionic protein, was excreted primarily in urine. The selective B700-proteinuria did not alter urinary volumes or produce hematuria or edema. SDS-polyacrylamide gel electrophoresis and western blot analysis using the H-2-3-3 B700-specific monoclonal antibody revealed that B700 proteinuria occurred in B-16 murine melanoma bearing animals but not in control mice. These studies demonstrate that the tumor-bearing host readily distinguishes between very similar normal protein (MSA) and tumor-associated antigen (B700) molecules and processes them differently. |
| 1991 | Purification and partial characterization of stonustoxin (lethal factor) from Synanceja horrida venom. | Comp Biochem Physiol B | 1. The lethal factor of the stonefish (Synanceja horrida) venom, designated as the stonustoxin, was purified to homogeneity by a two-step procedure on Sephacryl S-200 High Resolution (HR) gel permeation and DEAE Bio-Gel A anion exchange chromatography. 2. Stonustoxin has a native mol. wt of 148,000 and an isoelectric point of 6.9. 3. SDS-polyacrylamide gel electrophoresis revealed two subunits (designated alpha and beta) with mol. wts of 71,000 and 79,000, respectively. 4. The amino acid composition of both subunits and the N-terminal amino acid sequence of the beta subunit were also determined. 5. Purified stonustoxin had an LD50 of 0.017 microgram/g which is 22-fold more potent than that of the crude venom. 6. The toxin exhibited potent haemolytic activity in vitro and edema-inducing activity with a minimum edema dose (MED) of 0.15 micrograms in mouse paw. The edema effect was not antagonized by diphenhydramine. |
| 1990 | Dysfunctional C1 inhibitor Ta: deletion of Lys-251 results in acquisition of an N-glycosylation site. | Proc Natl Acad Sci U S A | Hereditary angioneurotic edema is inherited as an autosomal dominant disorder and is characterized by potentially life-threatening episodic angioedema. In type II hereditary angioneurotic edema, a dysfunctional C1 inhibitor molecule is present together with low levels of normal C1 inhibitor. About 70% of these dysfunctional proteins result from reactive center (Arg-444) mutations. We describe the deletion of nucleotides encoding Lys-251 (AAG) in C1 inhibitor Ta, the dysfunctional C1 inhibitor from a family with type II hereditary angioneurotic edema. DNA sequence analysis was derived from clones obtained through polymerase chain reaction amplification of blood monocyte C1 inhibitor mRNA. As expected, clones with both normal and abnormal sequence were isolated. The deletion was verified by protein sequence analysis. These data, together with biochemical analysis of the protein and cell-free translation studies, suggest that this deletion, by altering the normal amino acid sequence from Asn-Lys-Ile-Ser to Asn-Ile-Ser, creates a new glycosylation site. The additional carbohydrate accounts for the larger size on SDS/PAGE and very likely interferes with protein function. |
| 1990 | Purification and characterization of two acidic phospholipase A2 enzymes from king cobra (Ophiophagus hannah) snake venom. | Int J Biochem | 1. The two major phospholipase A2 enzymes (OHPLA-DE1 and OHPLA-DE2) of king cobra (Ophiophagus hannah) venom have been purified to electrophoretic homogeneity. 2. The isoelectric points of OHPLA-DE1 and OHPLA-DE2 were 3.81 and 3.89, respectively and the Mws were 14,000 and 15,000, respectively, as estimated by Sephadex G-75 gel filtration chromatography; and 14,000 as estimated by SDS-PAGE. 3. The enzymes were not lethal to mice at a dosage of 10 micrograms/g body wt by i.v. route. Both phospholipase A2 enzymes, however, exhibited moderate edema-inducing and anti-coagulant activities. 4. Bromophenacylation of the enzymes reduced the enzymatic activity drastically but did not affect the edema-inducing activity of the enzymes. |
| 1990 | Effect of fibrinogen degradation products and lung ground substance on surfactant function. | Biol Neonate | Acute lung injury syndromes have many characteristics including protein-rich alveolar edema, hyaline membranes, and abnormal surface tension at the alveolar air-liquid interface. Increased surface tension can occur because of a relative surfactant deficiency and/or dysfunction. It has been previously demonstrated that surfactant dysfunction occurs when plasma protein inhibitors leak into the alveolar space during the induction of the lung injury and edema formation. The present study investigated whether inhibitors that would be generated during the stage of repair from lung injury could impair surfactant function. We determined whether fibrinogen degradation products (FDP) which would be released during lysis of the fibrin(ogen)-containing alveolar exudate and hyaline membranes, and components of the lungs' ground substance could inhibit the in vitro function of a lipid extract surfactant preparation. FDP were prepared by incubating human fibrinogen with plasmin or neutrophil elastase for 4 min to 60 h and were characterized by SDS-PAGE. Early (fragment X and Y) and late (fragment D and E) plasmin-derived FDP (MW greater than 40,000) inhibited surfactant function as assessed by a bubble surfactometer. The early elastase-derived FDP also inhibited surfactant, but the later and much smaller fragments (MW less than 15,000) did not affect surfactant function. Laminin also inhibited surfactant in a dose-dependent manner. Neither hyaluronic acid nor heparan sulfate affected surfactant performance in vitro. We conclude that plasmin-induced lysis of intraalveolar fibrinogen and hyaline membranes will result in prolonged generation (i.e. days) of surfactant inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS) |
| 1989 | Purification and characterization of a myotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom. | Toxicon | A major phospholipase A2 (NN-XIII-PLA2) which constitutes 20% of the whole Naja naja naja venom was purified to homogeneity on CM-Sephadex C-25 column chromatography. NN-XIII-PLA2 is a basic protein with a mol. wt of 11,200 by SDS-PAGE. This enzyme has low enzymatic activity but is more toxic to mice than the whole venom. The LD50 value (i.p.) of NN-XIII-PLA2 is 2.4 mg/kg body weight (whole venoms LD50 is 2.8 mg/kg body weight). It induces neurotoxic-like signs in experimental animals. It induces myotoxicity when injected i.m. into the thigh muscle of mice and edema when injected into the foot pads of mice. This enzyme has a fluorescence maxima between 310-316 nm which is typical of tyrosine residues. |
| 1989 | A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo). | Toxicon | A new muscle damaging toxin, myotoxin II, was purified from the venom of Bothrops asper by ion-exchange chromatography on CM-Sephadex C-25. The toxin is a dimeric, basic protein with a monomer mol.wt of 13,341, according to the amino acid composition, and 16,000 on the basis of SDS-polyacrylamide gel electrophoretic mobility. It has a high number of aspartate and lysine residues, as well as of hydrophobic amino acids. Upon i.m. injection into mice, the toxin induces myonecrosis and increase in serum creatine kinase levels. In addition, myotoxin II induces edema in the mouse foot pad. Immunochemical tests, mol.wt, and amino acid composition indicate a high degree of homology between myotoxin II and a previously characterized myotoxin from this venom, myotoxin I. However, in contrast to myotoxin I, myotoxin II lacks phospholipase A2 and anticoagulant activities in vitro. |
| 1989 | Purification and characterization of a major phospholipase A2 from Russell's viper (Vipera russelli) venom. | Toxicon | A major phospholipase A2 (VRV PL-VIIIa) which constitutes 24% of the whole Vipera russelli venom was purified to homogeneity by CM-Sephadex C-25 column chromatography followed by gel filtration on Sephadex G-50. VRV PL-VIIIa is a basic protein with a molecular weight of 11,800 by SDS-PAGE. This enzyme contributes 45% of the total PLA2 activity of the venom, but it is least toxic compared to other purified basic PLA2 enzymes prepared from V. russelli venom. The LD50 value (i.p.) of VRV PL-VIIIa is 5.3 mg/kg body wt. It shows neurotoxic symptoms and damages vital organs such as lung, liver and kidney at LD50 doses. It induces myonecrosis when injected i.m. into the thigh muscle of mice and edema when injected into the foot pads. |
| 1989 | [Proteinuria in normal pregnancy and in EPH gestosis]. | Acta Med Austriaca | EPH-gestosis (pre-eclampsia-eclampsia) characterized by edema, proteinuria and hypertension occurs primarily in the nullipara, usually after the 20th gestational week. As in normal pregnancy there is striking change in both renal blood flow and glomerular filtration rate a slight increase in urinary protein secretion is not considered abnormal until it exceeds 300 mg/day. Abnormal proteinuria commonly accompanies pre-eclampsia and may be minimal, moderate or severe (even exceeding greater than 25 g/l). Proteinuria was typed mainly of nonselective glomerular origin by using the SDS-disc-electrophoresis. Additionally the clearance ratio of IgG to transferrin in all patients with abnormal proteinuria was evaluated. In none of the patients studied the ratio was less than 0.1 (highly selective). As severe proteinuria is associated with fetal growth retardation, preterm deliveries and prenatal mortality the quantitation and typing of early proteinuria is essential for considering patients who are at risk for developing EPH-gestosis. |
| 1989 | Isolation and characterization of the major phospholipase A2 from the venom of Trimeresurus purpureomaculatus (shore pit viper). | Int J Biochem | 1. The major phospholipase A2 (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity. 2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14,000 as estimated by SDS-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC greater than PE greater than PS =0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme. 4. The phospholipase A2 exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 micrograms/g by i.v. route. |
| 1988 | Lavage angiotensin-converting enzyme as a marker of lung injury. | Am Rev Respir Dis | The source of angiotensin-converting enzyme (ACE) in lavage fluid remains controversial. I reassessed the levels of ACE in lavage fluid in contrasting models of lung injury in rats. In all 3 models examined, there was at least a 3-fold elevation in both ACE activity and total protein levels. However, determination of enzyme activity relative to total protein content (enzyme specific activity) revealed contrasting patterns. High dose intratracheal bleomycin instillation (500 micrograms) induced an increase in ACE activity relative to total protein. When a lower dose of bleomycin (50 micrograms) or when a 30-h exposure to 100% oxygen were used, both ACE and protein increased but enzyme specific activity did not change. In contrast, exposure to 100% oxygen for 48 h or to alpha-naphthylthiourea, an agent that rapidly induces protein edema without cell injury, caused a decrease in enzyme specific activity. The enzyme found in lavage fluid in these models is identical to the lung tissue enzyme with respect to cofactor requirements, inhibition by captopril, and has an apparent molecular weight of 175 kDa by SDS-polyacrylamide gel electrophoresis. These studies clearly refute the null hypothesis that lavage ACE levels in the injured lung merely reflect altered bulk protein transmigration. |
| 1988 | Comparison of posttranslational protein modification by amino acid addition after crush injury to sciatic and optic nerves of rats. | Exp Neurol | Posttranslational protein modifications by the addition of amino acids are reactions which occur in intact sciatic and optic nerves of rats. The nerves differ, however, in that 2 h after crush injury these reactions are activated in sciatic but not in optic nerves. As sciatic nerves will eventually regenerate, whereas optic nerves will not, we have proposed that the activation of these reactions is correlated with the ability of a nerve to regenerate. The current experiments examined the posttranslational addition of amino acids to proteins at times greater than 2 h after nerve crush, during sciatic nerve regeneration and optic nerve degeneration. We also examined the optic nerve for morphologic correlates to changes in protein modification and partially characterized the proteins modified by [3H]Lys in the regenerating sciatic nerve using two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In a segment of sciatic nerve taken from a region just proximal to the site of crush, protein modification by covalent addition of [3H]Arg, [3H]Lys and [3H]Leu increased during both posttraumatic (2 h postcrush) and regenerative (6 days and 14 days postcrush) stages. Two-dimensional PAGE of [3H]Lys modified sciatic nerve proteins 6 days after crush injury showed labeling of proteins having molecular masses in the 18,000- to 20,000-, 30,000- to 40,000-, and 80,000- to 100,000-Da ranges, with neutral or basic isoelectric points (pI 7.1 to 8.0). In the retinal portion of the crushed optic nerve, incorporation of the same amino acids was unchanged or depressed to 21 days postcrush, except at 6 days postcrush when the incorporation of all three amino acids into proteins was increased threefold. These increases correlated with the appearance of terminal end bulbs in the portion of nerve analyzed. Histological examination of each nerve 2 h postcrush showed marked edema in the optic but not the sciatic nerve, a condition which may be related to the ability of sciatic and inability of optic nerves to activate protein modification reactions. |
| 1988 | Purification and characterization of a lethal factor in venom from the crown-of-thorns starfish (Acanthaster planci). | Toxicon | A lethal factor in venom of the crown-of-thorns starfish (Acanthaster planci) was obtained in an electrophoretically pure state by chromatography on CM-cellulose and Sephadex G-100. The purified lethal factor is a basic (pI 10.6) glycoprotein (carbohydrate content 3.5%). The mol. wt was estimated to be 20,000 by gel filtration or 25,000 by SDS-disc electrophoresis, suggesting that the lethal factor has no subunit structure. Despite its basicity, the lethal factor was richer in acidic amino acids than in basic amino acids. The lethal factor had an LD50 of 0.43 mg/kg (i.p. injection into mice). Hemolytic, edema-forming and capillary permeability-increasing activities, though very weak, were also exhibited by the lethal factor, while hemorrhagic and phospholipase A activities were not present. |
| 1987 | Dynamics of immune responses related to clinical status in Brugia pahangi-infected dogs. | Am J Trop Med Hyg | Clinical signs of lymph node enlargement, limb edema, lymph duct fibrosis, and microfilaremia were monitored in dogs with chronic Brugia pahangi infections. During the study a single rear limb of each dog was reinfected with multiple low doses of infective larvae. The changing immune responses to parasite antigens prepared from three sources--Brugia pahangi adult worm homogenate extract, adult worm excretory-secretory products, and microfilaria excretory-secretory products--were monitored by Western blot ELISA of antigens fractionated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by microtiter plate ELISA. Assays were used to detect antibodies in both the the IgG and IgE classes. A wide range of clinical manifestations was demonstrated in response to reinfection: asymptomatic, amicrofilaremic; asymptomatic, microfilaremic; acute short duration node enlargement and/or limb edema with microfilaremia; and chronic limb edema, amicrofilaremic. On microtiter plate ELISA, the dogs demonstrating the highest anti-adult worm homogenate titers were amicrofilaremic and were asymptomatic or developed chronic limb edema, dogs with high anti-mf ES titers were persistently amicrofilaremic, and the most marked increases against all three antigen sources upon reinfection occurred in low or amicrofilaremic dogs. Quantitative changes in antibody levels against the three crude antigen sources following reinfection were often paralleled by distinct changes in recognition of specific bands of antigens fractionated by SDS-PAGE. |
| 1987 | Interactions of plasma kallikrein and C1-s with normal and dysfunctional C1(-)-inhibitor proteins from patients with hereditary angioneurotic edema: analytic gel studies. | Blood | Purified preparations of normal C1(-)-inhibitor (C1(-)-INH) formed high mol wt complexes with plasma kallikrein that were stable during sodium dodecyl sulfate (SDS)-gel electrophoresis, but most of the dysfunctional C1(-)-INH proteins isolated from plasma of patients with type II hereditary angioneurotic edema (HANE) did not. Two of eight dysfunctional C1(-)-INH proteins were cleaved to lower mol wt forms that were not seen following the reaction of normal C1(-)-INH with equimolar amounts, or less, of plasma kallikrein. Only the higher mol wt component of normal C1(-)-INH (106,000 mol wt) appeared to form a stable complex with the plasma kallikrein, whereas both the 106,000 and 96,000 mol wt forms made stable complexes with C1-s. When a preparation of normal C1(-)-INH containing a homogeneous single band of C1(-)-INH was exposed to C1-s or kallikrein, a "doublet" form evolved in which the heaviest band was in the original position of native C1(-)-INH; C1-s cleavage provided a second band of 96,000; and cleavage by kallikrein, a second band of 94,000 mol wt. We conclude that dysfunctional C1(-)-INH proteins from plasma of persons with type II hereditary angioneurotic edema have impaired interactions with plasma kallikrein and are heterogeneous with respect to these interactions. Moreover, the requirements for the formation of stable complexes between normal C1(-)-INH and plasma kallikrein differed from those for stable complex formation with C1-s. The doublet form of C1(-)-INH, which purified preparations frequently demonstrate, may be due to prior cleavage by C1-s or kallikrein. |
| 1987 | Purification of the 65 kD protein from Mycobacterium gordonae and use in skin test response to Mycobacterium leprae. | Int J Lepr Other Mycobact Dis | The cell wall-associated protein of Mycobacterium gordonae (Mr = 65,000) was purified by affinity chromatography using a murine monoclonal antibody produced in response to the crossreactive 65 kD protein of M. leprae. The affinity-purified material was analyzed for purity by protein and carbohydrate analyses, SDS-PAGE, and immunoblotting. The final preparation contained a major protein band on SDS-PAGE analysis (Mr = 65,000) with no detectable carbohydrates. The affinity fraction was prepared at 250 micrograms/ml (protein) in sterile saline and 0.1 ml injected intradermally into guinea pigs immunized 30 days earlier. Gross changes at 48 hr were consistent with the characteristics of a delayed hypersensitivity skin reaction measuring 2.5 mm, 3.4 mm, and 2.7 mm in animals which had been immunized with M. leprae, M. gordonae, or M. bovis (BCG), respectively. Histologically, all 65 kD protein skin-test sites showed marked edema and infiltration by numerous lymphocytes, macrophages, and scattered neutrophils. Animals injected with Freund's incomplete adjuvant showed a minimal or no reaction (1.8 mm) to the purified protein. These results further define the immunogenicity of the 65 kD protein of M. gordonae and by inference M. leprae, and demonstrate the ability of crossreactive epitopes of the 65 kD protein to sensitize lymphocytes involved in delayed-type hypersensitivity reaction to M. leprae, M. gordonae, and M. bovis (BCG). |
| 1986 | Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummifer. | Toxicon | A myotoxin from the venom of the snake Bothrops nummifer was purified to homogeneity by ion-exchange chromatography on CM-Sephadex. The toxin is a basic dimer with a subunit molecular weight of 16,000, as estimated by SDS-polyacrylamide gel electrophoresis. The toxin lacks phospholipase A2 activity when tested on egg yolk lecithin and skeletal muscle homogenates. It induces skeletal muscle damage both in vivo and in vitro. When injected i.m. it promotes a drastic increase in serum creatine kinase levels; the isozyme CK-MM is responsible for this increment. A rapid release of creatine kinase was observed when mouse gastrocnemius muscle was incubated with the toxin, suggesting that it induces the formation of relatively large 'lesions' in the plasma membrane of muscle cells. Moreover, analysis of the dose-response data indicated that the myotoxin affects muscle sarcolemma by a 'one hit' mechanism. Skeletal muscle cells are affected by the toxin when calcium is eliminated from the medium. The myotoxin has an i.v. LD50 of 3.9 mg/kg body weight in mice, and induces edema when injected in the foot pad. On the other hand, it is not directly hemolytic, anticoagulant, hemorrhagic nor cytotoxic for lymphocytes. The myotoxin shows partial immunologic identity with a myotoxic phospholipase A2 isolated from Bothrops asper venom. The polyvalent antivenom produced in Costa Rica forms a precipitation arc against B. nummifer myotoxin on immunoelectrophoresis. |
| 1985 | Relationship between SH group reactivity and concentration of bovine serum albumin and rat plasma. | Pharmacol Res Commun | This study revealed that SH group reactivity (R) and concentration (C) of bovine serum albumin (BSA) and rat plasma are proportional in the sulphydryl-disulfide (SH-SS) exchange reaction with 5-5' dithiobis (2nitrobenzoic acid) (DTNB). The existence of the R/C proportionality suggests the use of Kr ratio and C as parameters for characterizing the biological properties of plasma SH groups. Moreover it was found that the electrophilic agents, diethylmaleate (DEM) and ethacrynic acid (ETHAC) that react with SH groups, determine an in vitro decrease in plasma SH group concentration and a Kr increase. The Kr increase seemed to be independent of SH group blocking as results obtained with sodium dodecyl sulfate (SDS) and SDS plus DEM indicated. The Kr biological significance might be related to a conformational change of albumin. In vivo treatment with 0.6 and 1.2 ml/kg of DEM confirmed the plasmatic linear relationship between R and C and showed a Kr increase in accordance with in vitro results. In carrageenan paw edema, decreased SH group plasma levels and an increased Kr were obtained. |
| 1985 | Variability in purified dysfunctional C1(-)-inhibitor proteins from patients with hereditary angioneurotic edema. Functional and analytical gel studies. | J Clin Invest | C1(-)-inhibitor (C1(-)-INH) proteins from normal persons and members of eight different kindred with dysfunctional C1(-)-INH proteins associated with hereditary angioneurotic edema (HANE) were compared with respect to their inhibitory activity against purified preparations of C1s-, plasma kallikrein, activated forms of Hageman factor, and plasmin. Each dysfunctional C1(-)-INH protein showed a unique spectrum of inhibitory activity against these enzymes. Although none of the dysfunctional C1(-)-INH proteins significantly impaired amidolysis by plasmin, all but one inhibited activated Hageman factor. One purified dysfunctional C1(-)-INH (Ta) inhibited purified C1s- to a normal degree. Another C1(-)-INH (Za) had almost seven times as much inhibitory activity as normal C1(-)-INH against activated Hageman factor, but had decreased activity against C1s- and no activity against plasmin. Analyses of mixtures of plasmin and C1(-)-INH proteins in SDS gel electrophoresis revealed variability in the patterns of complex formation and cleavage of dysfunctional proteins after exposure to C1s- and plasmin. Some bound to plasmin and were cleaved, even though none significantly impaired the amidolytic activity of plasmin. Two were cleaved by C1s-, whereas neither normal or other dysfunctional C1(-)-INH were cleaved. Dysfunctional C1(-)-INH proteins from patients with HANE are thus heterogeneous in their inhibitory properties and there must be different structural requirements for the inhibition of the various plasma enzymes that can be regulated by normal C1(-)-INH. The data suggest that in addition to common sites of interactions between these proteases and C1(-)-INH, there are also points of contact that are specific for each protease. Genetic mutations leading to structural changes at some of these sites may have differing effects on the interaction between individual proteases and abnormal C1(-)-INH proteins. These alterations may allow these proteins to serve as probes for structural requirements for inhibitory actions of normal C1(-)-INH. |
| 1984 | Intravitreal ceftriaxone in a rabbit model. Dose- and time-dependent toxic effects and pharmacokinetic analysis. | Arch Ophthalmol | Ceftriaxone's toxic effects were assessed in albino rabbits after intravitreal injection. Doses up to 5 mg did not alter the B-wave amplitude. Following 7.5-and 20-mg doses, B-wave amplitude ratios were depressed at 24 hours and normal at seven and 14 days. A 50-mg dose caused a temporarily flat B-wave 24 hours after injection. At two weeks this ratio exhibited a moderate increase but remained 2 SDs below the mean preinjection ratio. Eyes receiving up to 20 mg were normal histologically at 14 days. A 50-mg dose induced generalized retinal edema and disruption of the retinal layers 24 hours after injection; at two weeks there were no histologic changes in the retina. Immediately after a 2-mg intravitreal injection, vitreous ceftriaxone levels were 1,345 +/- 4.9 mg/L; by 72 hours they had decreased to 17.6 +/- 1.4 mg/L. The mean peak aqueous level was 80.2 +/- 12.2 mg/L at 72 hours. |
| 1984 | Fulminating haemophilus influenzae b meningitis. | Can J Neurol Sci | Haemophilus influenzae type b (HIb) is the most common cause of bacterial meningitis in children with a mortality rate ranging from 1.6% to 14%. Most patients have a 2-3 day history of symptoms prior to admission. A few have fulminating disease with rapid neurological deterioration. Review of 191 cases of HIb meningitis revealed a mortality rate of 2.1% but all who died had fulminating meningitis (FM). Four of six patients with FM died. FM patients had symptoms for less than 24 hours before rapid neurological deterioration with increased ICP, seizures, coma and/or respiratory arrest. Review of 10 FM cases revealed that on admission, 5 had hypotension, 3 had thrombocytopenia, and 8 had coma. Typical CSF changes were seen in only 7. All fatal cases died within 24 hours. Brain swelling and tonsillar herniation were found at autopsy. SDS-PAGE outer membrane protein subtyping did not show one "killer strain". Animal and autopsy data suggest that diminished CSF outflow and cerebral edema contribute to increased ICP. To improve survival of FM patients, initial treatment must (1) decrease ICP below levels impairing cerebral perfusion, (2) maintain adequate ventilation and blood pressure, and include (3) LP when stable, (4) antibiotics, and (5) close monitoring. Utilizing these principles, two FM patients survived without major sequelae. |
| 1983 | Immunocytochemical staining for glial fibrillary acidic protein and the metabolism of cytoskeletal proteins in experimental allergic encephalomyelitis. | Brain Res | Spinal cord sections from Lewis rats with acute experimental allergic encephalomyelitis (EAE) showed greatly increased staining of astrocytes when stained immunocytochemically for glial fibrillary acidic protein (GFAP). Fibrous processes in white matter were heavily stained early in the course of the disease when paralysis was first evident (10-12 days after injection of guinea pig spinal cord myelin), then protoplasmic astrocytes were stained in the gray matter and became more heavily stained at 20 days post-injection. The stained astrocytes were evenly distributed throughout the tissue, and did not correspond to the sites of the lesions. Spinal cord slices of control and EAE rats were incubated with [3H]amino acids, then cytoskeletal proteins were prepared in an enriched fraction, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein bands counted for radioactivity. In the EAE rat all cytoskeletal proteins, including the neurofilaments, vimentin, microtubules, GFAP and actin, showed increased uptake of radioactive amino acids. Immunoprecipitation of GFAP with specific antiserum showed increased radioactivity in the complex beginning at day 10 when cellular infiltration was beginning in the EAE animals. As the disease became acute, the radioactivity in the immunoprecipitated GFAP increased, in some cases to very high levels, then by day 18 when recovery was underway, the radioactivity had fallen to normal levels. Possible agents causing metabolic activation of protein synthesis in EAE animals include stimulating substances elaborated by infiltrating lymphoid cells, and the generalized edema accompanying the demyelinative condition. The activation of GFAP protein staining and metabolism in EAE might serve as a model for the activated growth of astrocyte processes which cause the severe gliosis seen in multiple sclerosis. |
| 1983 | Characterization of canine distemper viruses adapted to neural cells and their neurovirulence in mice. | Microbiol Immunol | Interaction of the Onderstepoort strain of canine distemper virus (CDV) with three established human neural cells, i.e. IMR-32 neuroblastoma, 118-MGC glioma and KG-1 oligodendroglioma, was examined, and adaptation of CDV to these cells was also attempted. The unadapted virus was found to grow at relatively low titers in the three neural cells inducing moderate to minimal cytopathic effects (CPE). The virus was successfully grown at high titers in these cells after 8 to 10 passages. Biological characteristics such as growth rate, morphology of CPE and plaque size changed after adaptation. Analysis by SDS-polyacrylamide gel electrophoresis, however, failed to show any difference in the molecular weight of component proteins among the unadapted and three adapted viruses. Inbred DDD strain of mice developed clinical signs after intracerebral inoculation with the unadapted virus but most of them survived with histological lesions of encephalitis. Neuroblastoma-adapted virus induced only transient clinical signs in some animals with mild encephalitic lesions in the gray matter. Increases in neurovirulence were found for viruses adapted to glioma and oligodendroglioma cells. Almost all mice inoculated with these two viruses at 3 weeks of age died within 8 days with histological lesions consisting of hyperemia, edema, severe degeneration of nerve cells and a few giant cells. Demyelinating lesions in the absence of inflammatory changes were observed in the cerebellum, pons and medulla oblongata of animals inoculated with oligodendroglioma-adapted virus. |
| 1981 | Effect of different doses of D-penicillamine and combined administration of D-penicillamine and methylprednisolone on collagen, glycosaminoglycans, DNA and RNA of granulation tissue, skin, bone and aorta in rats. | Acta Pharmacol Toxicol (Copenh) | D-penicillamine (D-pen) in doses of 20, 100 and 500 mg/kg/day or D-pen 100 mg/kg/day plus methylprednisolone (MP) 2.0 mg/kg/day was administered daily for 42 days to rats implanted with viscose-cellulose sponges. Operated, pairfed rats served as controls. D-pen increased the DNA content of granulation tissue, but had no effect on the amount of tissue produced. In contrast, high dose D-pen reduced the content of DNA and collagen in skin. A dose related inhibition of collagen crosslink formation occurred in all tissues, particularly in skin, as indicated by increased proportions of extractable collagen with increased alpha/beta chain ratio and aldehyde content. Moreover, low doses of D-pen increased the hydroxyproline/proline ratio of acid soluble skin collagen, presumably due to solubilization of type III collagen as demonstrated by SDS-polyacrylamide gel electrophoresis in the presence of 3.6 M urea. These changes were associated with increased skin fragility and edema plus excess elastin deposition in the aorta after high dose D-pen treatment. Low dose D-pen stimulated the 35S-sulphate uptake into the sulphated glycosaminoglycans (GAGs) of granulation tissue without altering their relative amounts, whereas high dose D-pen reduced the concentration of chondroitin-4/6-sulphate in skin. MP antagonized the solubilizing effect of D-pen on collagen, probably by inhibition of the collagen synthesis. In addition, MP inhibited the cell proliferation and GAG metabolism. Food restriction reduced the DNA content of granulation tissue. The inhibitory effect of D-pen on the formation of granuloma collagen crosslinks in the presence of unaltered rate of collagen biosynthesis may diminish the amount of fibrotic tissue due to increased degradability of crosslink deficient collagen. Simultaneous administration of MP may facilitate this effect by inhibiting the biosynthesis of collagen. However, long-term D-pen treatment seems to increase the susceptibility of normal tissues to mechanical injury. |
| 1975 | Studies on human plasma C1 inactivator-enzyme interactions. II. Structural features of an abnormal C1 inactivator from a kindred with hereditary angioneurotic edema. | J Clin Invest | The function and several of the structural features of the C1 inactivator protein isolated from the plasma of a mother and daughter with the variant form of hereditary angioneurotic edema have been examined. These abnormal inhibitors shared immunologic identity with the normal C1 inactivator protein; however, they were inactive in inhibiting the functional activity of C1s. Analysis of the abnormal inhibitors by sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis suggested that each consisted of a single polypeptide chain, the mobility of which was slower than that of the normal C1 inactivator. The apparent molecular weight of the patients' inhibitors was 109,000 daltons as contrasted to 105,000 daltons, that of the normal C1 inactivator. The abnormal inhibitors failed to form a complex with C1s or plasmin as analyzed by SDS-acrylamide gels. The large proteolytic derivatives resulting from the plasmin- and trypsin-induced degradation of the abnormal inhibitors were approximately 3,000 daltons heavier than the corresponding products derived from normal C1 inactivator. Thus, the structural abnormality identified appeared to be a property of the core molecule. Treatment of the inhibitors with neuraminidase failed to demonstrate a difference between the normal and patient-derived C1 inactivator molecule. Neither were major differences found between the amino acid composition of the defective and normal inhibitors; however, the acidic amino acids tended to be higher in the patients' inhibitors, and the phenylalanine content lower. Thus, these studies have identified both structural and functional abnormalities in the C1 inactivator protein isolated from two related patients with hereditary angioneurotic edema. Examination of the interaction between endopeptidases and the inhibitors has further delineated the abnormal structural features. |