| 2020 | Efficiency and safety of one-year anti-TNF-α treatment in Crohn's disease: a Polish single-centre experience. | Prz Gastroenterol | Anti-TNF-α therapy of Crohn's disease (CD) represents considerable progress in inflammatory bowel disease (IBD) treatment; however, many patients still require surgical intervention. The Polish National Insurance Fund currently only covers up to 2 years of infliximab (IFX) therapy in CD patients and 1 year of adalimumab (ADA).To estimate the effectiveness and side effects of the anti-TNF-α Polish therapeutic program in CD patients.In this retrospective study, medical documentation of 80 CD patients treated with anti-TNF-α (IFX or ADA) was analysed. Fifty-two patients finished 1 year of therapy, and 28 individuals did not complete it due to lack of response to treatment or severe side effects.After treatment, 27 (67.50%) patients achieved a semi-annual remission and 14 (35%) achieved yearly remission. Twenty percent of patients experienced severe side effects such as anaphylactic shock, pneumonia, shingles, or upper respiratory tract infections. A strong negative correlation between the number of patients in remission and the period since therapy termination ( = -0.996, < 0.001) was found. During the 1-year follow-up, 20 patients were re-enrolled in the biological therapy program (the median time to next therapy was 231 days IQR: 126.5-300.5).Anti-TNF-α treatment in CD is relatively safe. The restricted time period of the therapy affects the clinical course of the disease and entails the need to resume biological therapy. |
| 2019 | A Fit-for-Purpose Method for the Detection of Human Antibodies to Surface-Exposed Components of BMS-986263, a Lipid Nanoparticle-Based Drug Product Containing a siRNA Drug Substance. | AAPS J | ND-L02-s0201/BMS-986263 is a lipid nanoparticle (LNP) drug product containing a heat shock protein 47 (HSP47)-specific small interfering ribonucleic acid (siRNA) and being developed for the treatment of liver and idiopathic pulmonary fibrosis. To address immunogenicity-related issues, we developed a robust, fit-for-purpose (FFP) three-tier electrochemiluminescent (ECL) anti-drug antibody (ADA) assay for the detection of antibodies (Abs) generated to surface-exposed components of BMS-986263. The drug was coated directly on plates, and several Abs specific for polyethylene glycol (PEG) and other surface components were tested for use as positive quality controls (QCs). Following selection of a rabbit monoclonal anti-PEG Ab, the assay was optimized, and various method development challenges specific to the modality and pseudo surrogate rabbit control were addressed. Screening, confirmatory, and titer cut points were validated following a statistical evaluation of 41 individual KEDTA human plasma samples at a minimum required dilution (MRD) of 100. Assay precision, sensitivity, selectivity, drug tolerance, and hook effect were determined for the rabbit Ab prepared in human KEDTA plasma matrix. The assay was used to interrogate anti-drug Ab (ADA) responses in normal human subjects who were administered 90 mg of the drug intravenously (IV) once every week for 3 weeks in phase I clinical trials. All pre- and post-dose samples were found to be negative for ADA. Based on these results, we concluded that BMS-986263 is not immunogenic. To the best of our knowledge, this work represents the first ADA method developed and reported for an LNP-based drug product. |
| 2019 | Clinical and Laboratory Predictors associated with Complicated Scrub Typhus. | Infect Chemother | Scrub typhus, a mite-borne disease caused by bites of -infected chiggers, is endemic in Asia-Pacific countries. In Korea, it is a seasonal disease prevalent in autumn and one of the important causes of acute undifferentiated febrile illness. The purpose of this study was to identify the risk factors for the prediction of the severe clinical course of scrub typhus and to investigate the differences in the clinical and laboratory findings of hospitalized elderly and non-elderly patients with scrub typhus.This study retrospectively reviewed the medical records of patients diagnosed with scrub typhus.A total of 710 patients were enrolled and 43.9% of them were elderly patients. The number of patients with complicated scrub typhus was 168 (23.7%) and the most common complication of severe scrub typhus was hepatic dysfunction (10.7%) followed by pneumonia (7.2%), acute kidney injury (4.9%) and shock (2.4%). Blood urea nitrogen ≥20 mg/dL, adenosine deaminase (ADA) ≥100 IU/L, pulmonary edema or pleural effusion, lactate dehydrogenase ≥500 U/L, alkaline phosphatase ≥400 IU/L, ferritin ≥500 ng/mL and absence of skin rash were independently associated with severe scrub typhus. There was no significant difference in the incidence of complicated scrub typhus between elderly and non-elderly patients. Absence of skin rash, pulmonary edema, pleural effusion, serum creatinine ≥1.5 mg/dL, total bilirubin ≥1.5 mg/dL, ADA ≥100 IU/L and ferritin ≥500 ng/mL were significantly associated with a longer hospitalization (≥10 days).The several independent predictors of complicated scrub typhus were identified in this study. Absence of skin rash, the increased levels of serum ADA and ferritin were identified as the predictors of complicated scrub typhus, which were also associated with a prolonged hospitalization. |
| 2018 | Glucocorticoids use in kidney transplant setting. | Expert Opin Drug Metab Toxicol | Despite major advances in kidney transplant, glucocorticoids (GCs) or steroids remain one of the mainstay treatments. They possess adverse events (AEs) that are related to cumulative dosage, as documented in experimental and clinical studies. Therefore, it is important to comprehend and interpret experimental data and equally important to critically review clinical studies. Areas covered: This article provides a broad overview of the structure, pharmacokinetics, and pharmacodynamics of systemically administered GCs in transplant setting. It further discusses at length the results of in vitro and pre-clinical studies, as well as steroid avoidance (SA) or withdrawal (SW)-based clinical studies. We summarized the main AEs and discussed the alternatives to minimize these events. Some clinically relevant drug-drug interactions are also highlighted. Expert opinion: Although SA/SW in kidney transplant is a desirable strategy due to its AEs, there is no evidence to support that strategy based on the available data, despite some encouraging reports. Furthermore, early diagnosis and treatment of GC-induced AEs seem to be the most efficacious strategies. Likewise, some risks factors predate transplant and could be used to risk-stratify patients to determine appropriate risk-reduction strategies. Additional randomized-controlled studies are required to assess the impact of SA/SW during short and long follow-ups.ACTH: Adrenocorticotropic hormone (also adrenocorticotropin and corticotropin); ADA: American Diabetes Association; AEs: Adverse events; ADX: Adrenalectomized; AR: Acute rejection; AUC: Area under the curve; BMI: Body mass index; BMD: Bone mineral density; BPAR: Biopsy-proven acute rejection; cAMP: cyclic adenosine monophospahte; CBG: Corticosteroid-binding globulin; CBP: CREB binding protein; C: Mean maximal serum concentration; CNIs: Calcineurin inhibitors; COX-2: cyclo-oxygenase-2; cPLA: cytosolic phospholipase A CSA: Cyclosporine; CYP: Cytochrome; DEX: Dexamethasone; DM: Diabetes mellitus; ESW: Early steroid withdrawal; GCs: Glucocorticoids; GRs: Glucocorticoid receptors; GREs: Glucocorticoid receptor elements; CSA: Cyclosporine; DEX: Dexamethasone; Glycated hemoglobin: HbA; HDL: High-density lipoproteins; HLA: Human leukocyte anrigen; HPA axis: Hypothalamic-pituitary-adrenal axis; HR: Hazard ratio; HSP: Heat shock proteins; 11β-HSD: 11β-Hydroxysteroid dehydrogenase; IC: Half of maximal inhibitory concentration; IFG: Impaired fasting glucose; IGT: Impaired glucose tolerance; imTOR: Inhibitors of mammalian target of rapamycin; iNOS: Inducible oxide nitric synthase; K: Dissociation constant; KDIGO: Kidney Disease: Improving Global Outcomes; LSW: Late steroid withdrawal; LDL: Low-density lipoproteins; MCP-1: Monocyte chemoattractant protein-1; MMF: Mycophenolate mofetil; MPS: Mycophenolate sodium; NSAIDS: non-steroidal anti- inflammatory drugs; NF-κB: Nnuclear factor-κB TNF; OPTN/UNOS: Organ Procurement and Transplant Network/United Network of Organ Sharing database; PK/PD: Pharmacokinetic/pharmacodynamics; PRA: Panel reactive antibodies; PTDM: Post-transplantation diabetes mellitus; PTH: Parathyroid hormone; RCT: Randomized controlled trial; RR: Risk ratio; SA: Steroid avoidanceSGLT-2: Sodium-glucose co-transporter 2; SW: Steroid withdrawal; TAC: Tacrolimus or FK506; TG/HDL: Triglyceride to high-density lipoprotein ratio; TNF-α: Tumor necrosis factor-α; TGF-β: Transforming growth factor-β. |
| 2018 | Anticancer activity of methylene blue via inhibition of heat shock protein 70. | Biomed Pharmacother | Heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) chaperones are indispensable to lung cancer cells for their survival and proliferation. In this study we evaluated and compared anticancer potential of methylene blue (MB) as an Hsp70 inhibitor, novobiocin (NB) a well-known Hsp90 inhibitor and their combination.In vitro evaluation was done by cell viability assays, fluorescent staining, and flow cytometry analysis using A549 non-small cell lung cancer cells. In vivo anticancer activity was investigated by evaluating oxidative stress, tumor biomarkers, weight, lung microarchitecture, and Hsp70 and Hsp90 inhibitions via immunoblotting in benzo[a]pyrene induced lung carcinogenesis mice model.Using A549 NSCLC cells, we found MB demonstrated lower cell viability versus NB. Together, MB + NB resulted in further decrease in cell viability. SRB assay revealed significantly superior and similar potency for MB versus NB and MB + NB (1:1) versus MB, respectively. Fluorescent staining and flow cytometry analysis displayed early apoptosis by MB (11.4%); early and late apoptosis by MB + NB (13.8%). In vivo, MB significantly inhibited Hsp70. Furthermore, MB significantly alleviated tumor biomarkers (ADA and LDH) and improved lung histopathological features more than NB. Additionally, MB significantly improved SOD, not more than MB + NB or NB and improved LPO.MB demonstrated potent anticancer activity in vitro and in vivo via inhibition of Hsp70 in benzo[a]pyrene induced lung carcinogenesis in mice. |
| 2018 | The SAGA/TREX-2 subunit Sus1 binds widely to transcribed genes and affects mRNA turnover globally. | Epigenetics Chromatin | Eukaryotic transcription is regulated through two complexes, the general transcription factor IID (TFIID) and the coactivator Spt-Ada-Gcn5 acetyltransferase (SAGA). Recent findings confirm that both TFIID and SAGA contribute to the synthesis of nearly all transcripts and are recruited genome-wide in yeast. However, how this broad recruitment confers selectivity under specific conditions remains an open question.Here we find that the SAGA/TREX-2 subunit Sus1 associates with upstream regulatory regions of many yeast genes and that heat shock drastically changes Sus1 binding. While Sus1 binding to TFIID-dominated genes is not affected by temperature, its recruitment to SAGA-dominated genes and RP genes is significantly disturbed under heat shock, with Sus1 relocated to environmental stress-responsive genes in these conditions. Moreover, in contrast to recent results showing that SAGA deubiquitinating enzyme Ubp8 is dispensable for RNA synthesis, genomic run-on experiments demonstrate that Sus1 contributes to synthesis and stability of a wide range of transcripts.Our study provides support for a model in which SAGA/TREX-2 factor Sus1 acts as a global transcriptional regulator in yeast but has differential activity at yeast genes as a function of their transcription rate or during stress conditions. |
| 2017 | Coat Protein Regulation by CK2, CPIP, HSP70, and CHIP Is Required for Potato Virus A Replication and Coat Protein Accumulation. | J Virol | We demonstrate here that both coat protein (CP) phosphorylation by protein kinase CK2 and a chaperone system formed by two heat shock proteins, CP-interacting protein (CPIP) and heat shock protein 70 (HSP70), are essential for potato virus A (PVA; genus Potyvirus) replication and that all these host proteins have the capacity to contribute to the level of PVA CP accumulation. An E3 ubiquitin ligase called carboxyl terminus Hsc70-interacting protein (CHIP), which may participate in the CPIP-HSP70-mediated CP degradation, is also needed for robust PVA gene expression. Residue Thr within the CK2 consensus sequence of PVA CP was found to be essential for viral replication and to regulate CP protein stability. Substitution of Thr either with a phosphorylation-mimicking Asp (CP) or with a phosphorylation-deficient Ala (CP) residue in CP expressed from viral RNA limited PVA gene expression to the level of nonreplicating PVA. We found that both the CP mutant and CK2 silencing inhibited, whereas CP mutant and overexpression of CK2 increased, PVA translation. From our previous studies, we know that phosphorylation reduces the RNA binding capacity of PVA CP and an excess of CP fully blocks viral RNA translation. Together, these findings suggest that binding by nonphosphorylated PVA CP represses viral RNA translation, involving further CP phosphorylation and CPIP-HSP70 chaperone activities as prerequisites for PVA replication. We propose that this mechanism contributes to shifting potyvirus RNA from translation to replication.Host protein kinase CK2, two host chaperones, CPIP and HSP70, and viral coat protein (CP) phosphorylation at Thr are needed for potato virus A (PVA) replication. Our results show that nonphosphorylated CP blocks viral translation, likely via binding to viral RNA. We propose that this translational block is needed to allow time and space for the formation of potyviral replication complex around the 3' end of viral RNA. Progression into replication involves CP regulation by both CK2 phosphorylation and chaperones CPIP and HSP70. |
| 2016 | Breaking the Chain of Infection: Dental Unit Water Quality Control. | J Clin Diagn Res | The air-water syringes, ultrasonic scalers, high speed air turbine handpieces are connected to dental units by a network of small-bore plastic tubes through which water and air travel to activate or cool the instruments and it had been shown that this system is extensively contaminated with microbial biofilms and pose a potential risk of infection for patients as well as dental professionals.To evaluate and compare the efficacy of various disinfectants in reducing the microbial colony count in water derived from Dental Unit Waterlines.Five random dental units were selected and samples were collected before and after intervention with 5 disinfectants (0.02% H2O2 continuously, 0.02% H2O2 continuously with shock treatment with 0.25% H2O2 weekly, 0.12% Chlorohexidine and 12% Ethanol overnight, 1:50 Original Listerine overnight, 2% Sodium Perborate and 2% EDTA 5 minutes in morning) using different disinfection methods for 4 weeks. Samples were cultured on Reasoner's 2A (R2A) agar for microbial counting.Results were recorded as Colony forming units/ml (cfu/ml) and were evaluated statistically. Results showed that all the dental unit waterlines were heavily contaminated with microbes before any intervention. After 1 day of disinfection regime the counts reduced significantly and showed progressive reduction in consecutive weeks. Goals set by ADA & CDC were ultimately achieved at the end of 4 weeks.All the disinfectants were equally effective in reducing the microbial colony count of DUWLs, irrespective of their concentration and method of disinfection. |
| 2016 | Transcriptomics analysis and hormonal changes of male and female neonatal rats treated chronically with a low dose of acrylamide in their drinking water. | Toxicol Rep | Acrylamide is known to produce follicular cell tumors of the thyroid in rats. RccHan Wistar rats were exposed to a carcinogenic dose of acrylamide (3 mg/Kg bw/day) from gestation day 6 to delivery and then through their drinking water to postnatal day 35. In order to identify potential mechanisms of carcinogenesis in the thyroid glands, we used a transcriptomics approach. Thyroid glands were collected from male pups at 10 PM and female pups at 10 AM or 10 PM in order to establish whether active exposure to acrylamide influenced gene expression patterns or pathways that could be related to carcinogenesis. While all animals exposed to acrylamide showed changes in expected target pathways related to carcinogenesis such as DNA repair, DNA replication, chromosome segregation, among others; animals that were sacrificed while actively drinking acrylamide-laced water during their active period at night showed increased changes in pathways related to oxidative stress, detoxification pathways, metabolism, and activation of checkpoint pathways, among others. In addition, thyroid hormones, triiodothyronine (T3) and thyroxine (T4), were increased in acrylamide-treated rats sampled at night, but not in quiescent animals when compared to controls. The data clearly indicate that time of day for sample collection is critical to identifying molecular pathways that are altered by the exposures. These results suggest that carcinogenesis in the thyroids of acrylamide treated rats may ensue from several different mechanisms such as hormonal changes and oxidative stress and not only from direct genotoxicity, as has been assumed to date. |
| 2015 | Novel Blood Purification System for Regulating Excessive Immune Reactions in Severe Sepsis and Septic Shock: An Ex Vivo Pilot Study. | Ther Apher Dial | Promising results have been reported with blood purification as adjuvant treatment; however, the immunological mechanisms remain unclear. We have been developing a new blood purification system for regulating excessive immune reactions in severe sepsis and septic shock using a granulocyte adsorbing column (Adacolumn [Ada]), and a cytokine-adsorbing hemofilter (AN69ST hemofilter [AN69]). Fresh porcine blood was circulated for 6 h in five experimental groups including Ada and AN69 to assess the effects of leukocyte adsorption, phagocytic activity and adhesiveness of granulocytes. In the present study, we found that Ada mainly adsorbed granulocytes and monocytes, but not lymphocytes. The phagocytic activity level of granulocytes decreased, and adhesiveness increased, but the number of CD11b-positive cells markedly decreased in the current system. Elevated cytokine levels (IL-1β, IL-8 and IL-10) at the outlet of Ada were significantly lower than at the outlet of AN69 due to cytokine adsorption. Further studies are needed to better understand cellular interactions. |
| 2012 | Insulin/IGF-1 and hypoxia signaling act in concert to regulate iron homeostasis in Caenorhabditis elegans. | PLoS Genet | Iron plays an essential role in many biological processes, but also catalyzes the formation of reactive oxygen species (ROS), which can cause molecular damage. Iron homeostasis is therefore a critical determinant of fitness. In Caenorhabditis elegans, insulin/IGF-1 signaling (IIS) promotes growth and reproduction but limits stress resistance and lifespan through inactivation of the DAF-16/FoxO transcription factor (TF). We report that long-lived daf-2 insulin/IGF-1 receptor mutants show a daf-16-dependent increase in expression of ftn-1, which encodes the iron storage protein H-ferritin. To better understand the regulation of iron homeostasis, we performed a TF-limited genetic screen for factors influencing ftn-1 gene expression. The screen identified the heat-shock TF hsf-1, the MAD bHLH TF mdl-1, and the putative histone acetyl transferase ada-2 as activators of ftn-1 expression. It also revealed that the HIFα homolog hif-1 and its binding partner aha-1 (HIFβ) are potent repressors of ftn-1 expression. ftn-1 expression is induced by exposure to iron, and we found that hif-1 was required for this induction. In addition, we found that the prolyl hydroxylase EGL-9, which represses HIF-1 via the von Hippel-Lindau tumor suppressor VHL-1, can also act antagonistically to VHL-1 in regulating ftn-1. This suggests a novel mechanism for HIF target gene regulation by these evolutionarily conserved and clinically important hydroxylases. Our findings imply that the IIS and HIF pathways act together to regulate iron homeostasis in C. elegans. We suggest that IIS/DAF-16 regulation of ftn-1 modulates a trade-off between growth and stress resistance, as elevated iron availability supports growth but also increases ROS production. |
| 2004 | Defibrillators. | J Am Dent Assoc | Sudden cardiac arrest can strike anyone, anywhere and at any time, often without warning. Reported survival rates for cardiac arrest with ventricular fibrillation are low: from 3 to 10 percent. Studies have shown that rapid defibrillation after out-of-hospital cardiac arrest with ventricular fibrillation is the most important determinant of survival. That is why training people to use automated external defibrillators, or AEDs, in public places can double the odds that a person in cardiac arrest will survive. The ADA recently awarded the Seal of Acceptance to HeartStart OnSite and FR2+ defibrillators. HeartStart defibrillators are easy to use. HeartStart defibrillators are designed specifically for the lay responder. They provide clear, easy-to-follow voice instructions and a simple user interface to guide the responder through an emergency reliable. HeartStart defibrillators perform comprehensive daily, weekly and monthly self-tests to help ensure readiness. A highly visible status indicator shows at a glance that the device is ready for use safe. HeartStart defibrillators have an innovative design that makes it virtually impossible to shock someone who is not in cardiac arrest. The heart rhythm first is analyzed to ensure that a shock is needed. The device will not allow the user to deliver a shock that the analysis determines is unnecessary. Philips offers two defibrillator models for the dental office: the HeartStart OnSite Defibrillator and the HeartStart FR2+ Defibrillator. The HeartStart OnSite Defibrillator is intended to be easy to use for responders in an office setting. It provides step-by-step verbal instructions on how to perform cardiopulmonary resuscitation, or CPR. The HeartStart FR2+ Defibrillator is designed for first responders, such as flight attendants, nurses, police officers and designated workplace responders who participate in regular training. Each HeartStart defibrillator comes with a nonrechargeable long-life battery, two sets of adult pads and complete instructions for use. |
| 2004 | Impaired fasting glucose and cardiogenic shock in patients with acute myocardial infarction. | Eur Heart J | In-hospital outcome after acute myocardial infarction (MI) has not yet been evaluated with regard to the new category of Impaired Fasting Glucose level (IFG) patients defined by the American Diabetes Association (ADA).Nine hundred and ninety-nine patients with acute MI from the RICO survey were included in the study. Fasting blood glucose was measured after admission. Patients were grouped according to ADA definitions: Diabetes Mellitus (DM) (FG >/=7mmol/l or personal history of DM); IFG (FG 6.1 to 7mmol/l); NFG (normal FG <6.1mmol/l).Three hundred and eighty-one patients (38%) had DM, 145 (15%) IFG and 473 (47%) NFG. Mortality in the IFG group was twice that of the NFG group (8% vs 4%, P=0.049). A significant increase in cardiogenic shock (12% vs 6%, P=0.011) and ventricular arrhythmia (15% vs 9%, P=0.035) was observed in the IFG vs NFG group. IFG, after adjustment for confounding factors (age, sex, anterior location, and LVEF), was a strong independent predictive factor for cardiogenic shock (P=0.005).MI patients with IFG had an overall worse outcome, characterized by a higher risk of developing cardiogenic shock during their hospital stay. |
| 2001 | Altered transcription in yeast expressing expanded polyglutamine. | Proc Natl Acad Sci U S A | Expanded polyglutamine tracts are responsible for at least eight fatal neurodegenerative diseases. In mouse models, proteins with expanded polyglutamine cause transcriptional dysregulation before onset of symptoms, suggesting that this dysregulation may be an early event in polyglutamine pathogenesis. Transcriptional dysregulation and cellular toxicity may be due to interaction between expanded polyglutamine and the histone acetyltransferase CREB-binding protein. To determine whether polyglutamine-mediated transcriptional dysregulation occurs in yeast, we expressed polyglutamine tracts in Saccharomyces cerevisiae. Gene expression profiles were determined for strains expressing either a cytoplasmic or nuclear protein with 23 or 75 glutamines, and these profiles were compared to existing profiles of mutant yeast strains. Transcriptional induction of genes encoding chaperones and heat-shock factors was caused by expression of expanded polyglutamine in either the nucleus or cytoplasm. Transcriptional repression was most prominent in yeast expressing nuclear expanded polyglutamine and was similar to profiles of yeast strains deleted for components of the histone acetyltransferase complex Spt/Ada/Gcn5 acetyltransferase (SAGA). The promoter from one affected gene (PHO84) was repressed by expanded polyglutamine in a reporter gene assay, and this effect was mitigated by the histone deacetylase inhibitor, Trichostatin A. Consistent with an effect on SAGA, nuclear expanded polyglutamine enhanced the toxicity of a deletion in the SAGA component SPT3. Thus, an early component of polyglutamine toxicity, transcriptional dysregulation, is conserved in yeast and is pharmacologically antagonized by a histone deacetylase inhibitor. These results suggest a therapeutic approach for treatment of polyglutamine diseases and provide the potential for yeast-based screens for agents that reverse polyglutamine toxicity. |
| 1999 | Altered association of transcriptionally active DNA with the nuclear-matrix after heat shock. | Int J Radiat Biol | Exposure of human cells to heat leads to denaturation and aggregation of proteins. Within the nucleus, it has been suggested that protein aggregation is linked to the selective inhibition by hyperthermia of nucleotide excision repair in transcriptionally active genes. In this study it was investigated in detail whether and how the inhibition of repair of transcriptionally active genes might be related to alterations in their association with the nuclear-matrix.Different protocols for nuclear-matrix isolation (high salt and lithium 3',5'-diiodosalycilate [LIS] extraction of nuclei) were used to compare DNA loop organization and positioning of transcriptionally active genes in both heated and non-heated cells.DNaseI digestion of total genomic DNA in Cu2+ -stabilized LIS-extracted nuclei revealed that heat shock perturbed the formation of nuclear-matrix attachment sites. Specific labelling of active genes indicated that the number of nuclear-matrix attachment sites in transcriptionally active DNA was increased due to the heat shock. At the level of individual genes, heat treatment led to stabilization of the 5' matrix attachment site (MAR) in the transcriptionally active adenosine deaminase (ADA) housekeeping gene. Moreover, heat shock resulted in the formation of an additional MAR at the 3' end of the ADA gene. The inactive 754 locus was unassociated, irrespective of a heat shock.The reported changes in chromatin structure might underlie the selective inhibition of repair in transcriptionally active genes and consequently may be mechanistically linked to the sensitization of heated cells to ionizing radiation. |
| 2000 | The heat shock cognate protein hsc73 assembles with A(1) adenosine receptors to form functional modules in the cell membrane. | Mol Cell Biol | A(1) adenosine receptors (A(1)Rs) are G protein-coupled heptaspanning receptors that interact at the outer face of the plasma membrane with cell surface ecto-adenosine deaminase (ecto-ADA). By affinity chromatography the heat shock cognate protein hsc73 was identified as a cytosolic component able to interact with the third intracellular loop of the receptor. As demonstrated by surface plasmon resonance, purified A(1)Rs interact specifically with hsc73 with a dissociation constant in the nanomolar range (0.5 +/- 0.1 nM). The interaction between hsc73 and A(1)R led to a marked reduction in the binding of the ligands and prevented activation of G proteins, as deduced from (35)S-labeled guanosine-5'-O-(3-thio)triphosphate binding assays. Interestingly this effect was stronger than that exerted by guanine nucleotide analogs, which uncouple receptors from G proteins, and was completely prevented by ADA. As assessed by immunoprecipitation a high percentage of A(1)Rs in cell lysates are coupled to hsc73. A relatively high level of colocalization between A(1)R and hsc73 was detected in DDT(1)MF-2 cells by means of confocal microscopy, and no similar results were obtained for other G protein-coupled receptors. Colocalization between hsc73 and A(1)R was detected in specific regions of rat cerebellum and in the body of cortical neurons but not in dendrites or synapses. Remarkably, agonist-induced receptor internalization leads to the endocytosis of A(1)Rs by two qualitatively different vesicle types, one in which A(1)R and hsc73 colocalize and another in which hsc73 is absent. These results open the interesting possibility that signaling via G protein-coupled receptors may be regulated by heat shock proteins. |
| 1999 | Inducible acid tolerance mechanisms in enteric bacteria. | Novartis Found Symp | Enteric micro-organisms have developed several inducible mechanisms for surviving transient periods of extreme acid stress. Salmonella typhimurium possesses an acid tolerance response (ATR) induced in minimal medium by short exposures to mild acid stress. More than 50 acid shock proteins (ASPs) are induced during adaptation. Eight ASPs are regulated by the major iron regulatory protein, Fur, in an unusual iron-independent manner. The two-component regulator, PhoP, is an autoinduced ASP that controls the induction of three additional ASPs. The stress sigma factor sigma S is an ASP that regulates induction of eight ASPs. Acid induction of sigma S is due to its decreased proteolytic turnover via the ClpXP protease in conjunction with the two-component-type response regulator MviA (RssB in Escherichia coli). Mutations in any of these three regulators leads to a defective ATR. Repair of pH stress-induced DNA damage appears to require the Ada protein (O6-methylguanine methyltransferase) since an ada mutant is both acid and alkaline sensitive. In contrast to S. typhimurium, E. coli and Shigella have acid resistance systems induced in complex media that include a glucose-repressed system protective at pH 2.5 without amino acid supplementation, a glutamate decarboxylase system that requires glutamate and an arginine decarboxylase system unique to E. coli. |
| 1998 | A low pH-inducible, PhoPQ-dependent acid tolerance response protects Salmonella typhimurium against inorganic acid stress. | J Bacteriol | The acid tolerance response enables Salmonella typhimurium to survive exposures to potentially lethal acidic environments. The acid stress imposed in a typical assay for acid tolerance (log-phase cells in minimal glucose medium) was shown to comprise both inorganic (i.e., low pH) and organic acid components. A gene previously determined to affect acid tolerance, atbR, was identified as pgi, the gene encoding phosphoglucoisomerase. Mutations in pgi were shown to increase acid tolerance by preventing the synthesis of organic acids. Protocols designed to separate the stresses of inorganic from organic acids revealed that the regulators sigma38 (RpoS), Fur, and Ada have major effects on tolerance to organic acid stress but only minor effects on inorganic acid stress. In contrast, the two-component regulatory system PhoP (identified as acid shock protein ASP29) and PhoQ proved to be important for tolerance to inorganic [corrected] acid stress but had little effect against organic acid stress. PhoP mutants also failed to induce four ASPs, confirming a role for this regulator in acid tolerance. Acid shock induction of PhoP appears to occur at the transcriptional level and requires the PhoPQ system. Furthermore, induction by acid occurs even in the presence of high concentrations of magnesium, the ion known to be sensed by PhoQ. These results suggest that PhoQ can sense both Mg2+ and pH. Since phoP mutants are avirulent, the low pH activation of this system has important implications concerning the pathogenesis of S. typhimurium. The involvement of four regulators, two of which are implicated in virulence, underscores the complexity of the acid tolerance stress response and further suggests that features of acid tolerance and virulence are interwoven. |
| 1997 | An alkB gene homolog is differentially transcribed during the Caulobacter crescentus cell cycle. | J Bacteriol | A Caulobacter crescentus alkB gene homolog was identified in a clone previously shown to contain the heat shock genes dnaK and dnaJ; the homolog is located upstream of dnaK and is transcribed in the opposite orientation. An analysis of the alkB gene has shown that the deduced amino acid sequence is that of a 21-kDa protein, which is 42% identical and 78% similar to Escherichia coli AlkB. Furthermore, an alkB-null mutant was constructed by gene disruption and was shown to be highly sensitive to the alkylating agent methyl methanesulfonate (MMS). However, the alkB gene of C. crescentus, unlike its E. coli counterpart, is not located downstream of the ada gene, and its transcription is not induced by alkylating agents. In addition, no acquired enhanced resistance to MMS toxicity by treatment with low MMS doses was observed, suggesting that no adaptive response occurs in C. crescentus. Nevertheless, transcription of the alkB gene is cell cycle controlled, with a pattern of expression similar to that of several Caulobacter genes involved in DNA replication. |
| 1997 | Gene induction in response to unfolded protein in the endoplasmic reticulum is mediated through Ire1p kinase interaction with a transcriptional coactivator complex containing Ada5p. | Proc Natl Acad Sci U S A | In eukaryotic cells, accumulation of unfolded protein in the endoplasmic reticulum induces transcription of a family of genes encoding endoplasmic reticulum protein chaperones through a conserved unfolded protein response element. In Saccharomyces cerevisiae, activation of a transmembrane receptor kinase, Ire1p (Ern1p), initiates signaling, although the mediators immediately downstream of Ire1 kinase are unknown. Here we demonstrate interaction of Ire1p with the transcriptional coactivator, Gcn5p (for general control nonrepressed; also known as Ada4p). Gcn5p associates with other Ada (for alteration/deficiency in activation) gene products in a heteromeric complex and has histone acetyltransferase activity. We show that the Gcn5/Ada complex is selectively required for the unfolded protein response but not for the heat shock response. A novel mechanism is proposed in which activation of a receptor kinase recruits a transcription coactivator complex to a specific chromosomal locus to mediate localized histone acetylation, thus making specific gene sequences accessible for transcription. |
| 1995 | Inhibition of erythro-myeloid differentiation by constitutive expression of a DNA binding-deficient c-myb mutant: implication for c-myb function. | Blood | The c-myb proto-oncogene encodes a nuclear protein involved in the regulation of cell proliferation, differentiation, and development. Myb protein contains a DNA binding and a transactivating domain thought to mediate its biologic properties. The DNA binding domain consists of three repeats (R1, R2, and R3), each containing a highly conserved motif of tryptophan residues. A c-myb mutant (DR1-myb) lacking the last 46 amino acids of R1 and 23 amino terminal residues of R2, a region homologous to the ADA-2 yeast transcriptional adaptor, lost DNA binding ability, but remained able to transactivate the human heat-shock promoter. Transfection of murine 32D and murine erythroleukemia (MEL) cell lines with DR1-myb caused inhibition of cellular differentiation induced by granulocyte colony-stimulating factor (G-CSF) and dimethyl sulfoxide (DMSO), respectively. A second c-myb mutant (D-ADA2-myb) lacking the first 23 amino acids of R2, also lost DNA binding and transactivation activity, but did not inhibit DMSO-induced differentiation of MEL transfected cells. These findings suggest that deletion of R1 activates a DNA binding-independent mechanism of c-myb function, which may involve interaction of Myb with cellular factors. |
| 1995 | Repair of UV-induced pyrimidine(6-4)pyrimidone photoproducts is selectively inhibited in transcriptionally active genes after heat treatment of human fibroblasts. | Int J Radiat Biol | In normal human fibroblasts, repair of (6-4)PP in the active adenosine deaminase (ADA) gene occurs with similar rate in the transcribed and non-transcribed strand of the ADA gene, and removal of (6-4)PP from the active ADA gene is faster than from the inactive X-chromosomal 754 locus. Heat shock decreased the rate of repair of the active ADA gene down to the level of inactive genes, whereas the rate of repair of the inactive 754 locus was not affected. |
| 1993 | Heat-shock treatment selectively affects induction and repair of cyclobutane pyrimidine dimers in transcriptionally active genes in ultraviolet-irradiated human fibroblasts. | Radiat Res | The effect of hyperthermia on induction and repair of UV-radiation-induced cyclobutane pyrimidine dimers was investigated in the genome overall and in transcriptionally active and inactive genes in confluent human fibroblasts. Hyperthermia treatment (30 min, 45 degrees C) of human fibroblasts resulted in an increase in the protein content of isolated nuclei (protein aggregation) similar to that observed for HeLa S3 cells. The faster rate of disaggregation of nuclear proteins and the higher survival rate of heated fibroblasts in comparison with those for HeLa cells provide further evidence for a possible role of protein aggregation in heat-induced cell killing. Determination of the frequencies of cyclobutane pyrimidine dimers in the genome overall and in restriction fragments of the active adenosine deaminase (ADA) gene and inactive 754 locus revealed that hyperthermia selectively inhibits the induction of cyclobutane pyrimidine dimers in transcriptionally active DNA. Removal of cyclobutane pyrimidine dimers from the ADA gene was strongly delayed during the first 8 h in 10 J/m2 UV-irradiated fibroblasts. Such inhibition of repair of cyclobutane pyrimidine dimers was not observed for the 754 gene, indicating that inhibition of repair by hyperthermia is generally not mediated by inactivation of repair enzymes. It is proposed that the inhibition of induction and repair of cyclobutane pyrimidine dimers in active genes by hyperthermia is related to the heat-induced aggregation of proteins with the nuclear matrix, proximal to which active genes are located. Our results are consistent with a functional compartmentalization of DNA repair at the nuclear matrix. |
| 1992 | Effect of heat shock on expression of proteins not involved in the heat-shock regulon. | Eur J Biochem | The effect of heat shock on the expression of some genes of Escherichia coli was tested. To avoid side effects, promoters of the genes were fused to lacZ and their expression measured by the level of beta-galactosidase. The results show that expression of umuC, recA and polB, after induction of the SOS response, was somewhat higher in the heat-shocked than in the non-shocked cells, whereas expression of ada, alkB and alkA genes, after induction of the adaptive response, was about the same. Unexpectedly, it was found that expression of lacZ from its own promoter was drastically lowered in the heat-shocked cells. This effect, however, seems not to be dependent on the induction of heat-shock proteins. |
| 1987 | [Dissecting aortic aneurysm]. | Vutr Boles | Twenty cases with aortic dissecting aneurysm (ADA) are presented, at an average age of 52.5 (from 14 to 75), 15 of them males and 5--females. Nineteen had acute form of the disease and 1--chronic. Etiology, clinical picture, diagnostic approach and treatment were analyzed. Pain as leading symptom was present in all patients with various intensity and predominantly antero-thoracal localization. Seventeen of the patients (70%) were in shock at admittance. Pulse asymmetry was established in 8 (40%) and in 4, out of 12 cases (33%) with ADA, complicated by cardiac tamponade, a freshly appeared diastolic murmur was present. Enlarged aortic shadow at X-ray investigation had 14 (70%) of the patients with a dynamics of the enlargement in 5 patients. The disease has been clinically diagnosed in 19 (95) of the patients. Five out of 20 patients underwent operation, two of them followed for two years after the surgical treatment. Only one patient out of the non-operated 15 patients, survived. The modes of therapeutic behaviour are discussed on the base of the authors' experience and literature data. |