| 2018 | Hsp72 protects against liver injury via attenuation of hepatocellular death, oxidative stress, and JNK signaling. | J Hepatol | Heat shock protein (Hsp) 72 is a molecular chaperone that has broad cytoprotective functions and is upregulated in response to stress. To determine its hepatic functions, we studied its expression in human liver disorders and its biological significance in newly generated transgenic animals.Double transgenic mice overexpressing Hsp72 (gene Hspa1a) under the control of a tissue-specific tetracycline-inducible system (Hsp72-LAP mice) were produced. Acute liver injury was induced by a single injection of acetaminophen (APAP). Feeding with either a methionine choline-deficient (MCD; 8 weeks) or a 3,5-diethoxycarbonyl-1,4-dihydrocollidine-supplemented diet (DDC; 12 weeks) was used to induce lipotoxic injury and Mallory-Denk body (MDB) formation, respectively. Primary hepatocytes were treated with palmitic acid.Patients with non-alcoholic steatohepatitis and chronic hepatitis C infection displayed elevated HSP72 levels. These levels increased with the extent of hepatic inflammation and HSP72 expression was induced after treatment with either interleukin (IL)-1β or IL-6. Hsp72-LAP mice exhibited robust, hepatocyte-specific Hsp72 overexpression. Primary hepatocytes from these animals were more resistant to isolation-induced stress and Hsp72-LAP mice displayed lower levels of hepatic injury in vivo. Mice overexpressing Hsp72 had fewer APAP protein adducts and were protected from oxidative stress and APAP-/MCD-induced cell death. Hsp72-LAP mice and/or hepatocytes displayed significantly attenuated Jnk activation. Overexpression of Hsp72 did not affect steatosis or the extent of MDB formation.Our results demonstrate that HSP72 induction occurs in human liver disease, thus, HSP72 represents an attractive therapeutic target owing to its broad hepatoprotective functions.HSP72 constitutes a stress-inducible, protective protein. Our data demonstrate that it is upregulated in patients with chronic hepatitis C and non-alcoholic steatohepatitis. Moreover, Hsp72-overexpressing mice are protected from various forms of liver stress. |
| 2013 | Transition between acute and chronic hepatotoxicity in mice is associated with impaired energy metabolism and induction of mitochondrial heme oxygenase-1. | PLoS One | The formation of protein inclusions is frequently associated with chronic metabolic diseases. In mice, short-term intoxication with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to hepatocellular damage indicated by elevated serum liver enzyme activities, whereas only minor morphological changes are observed. Conversely, chronic administration of DDC for several weeks results in severe morphological damage, characterized by hepatocellular ballooning, disruption of the intermediate filament cytoskeleton, and formation of Mallory-Denk bodies consisting predominantly of misfolded keratins, Sqstm1/p62, and heat shock proteins. To evaluate the mechanistic underpinnings for this dichotomy we dissected the time-course of DDC intoxication for up to 10 weeks. We determined body weight change, serum liver enzyme activities, morphologic alterations, induction of antioxidant response (heme oxygenase-1, HO-1), oxidative damage and ATP content in livers as well as respiration, oxidative damage and the presence and activity of HO-1 in endoplasmic reticulum and mitochondria (mtHO-1). Elevated serum liver enzyme activity and oxidative liver damage were already present at early intoxication stages without further subsequent increase. After 2 weeks of intoxication, mice had transiently lost 9% of their body weight, liver ATP-content was reduced to 58% of controls, succinate-driven respiration was uncoupled from ATP-production and antioxidant response was associated with the appearance of catalytically active mtHO-1. Oxidative damage was associated with both acute and chronic DDC toxicity whereas the onset of chronic intoxication was specifically associated with mitochondrial dysfunction which was maximal after 2 weeks of intoxication. At this transition stage, adaptive responses involving mtHO-1 were induced, indirectly leading to improved respiration and preventing further drop of ATP levels. Our observations clearly demonstrate principally different mechanisms for acute and chronic toxic damage. |
| 2013 | Superoxide dismutase (SOD) in boar spermatozoa: purification, biochemical properties and changes in activity during semen storage (16°C) in different extenders. | Reprod Biol | The antioxidant system in semen is composed of enzymes, low-molecular weight antioxidants and seminal plasma proteins. Loss of enzymatic activity of superoxide dismutase (SOD) during semen preservation may cause insufficient antioxidant defense of boar spermatozoa. The aim of this study was to isolate and characterize SOD molecular forms from spermatozoa and to describe changes in SOD activity in boar sperm during preservation at 16°C. Sperm extracts were prepared from fresh or diluted semen and used for SOD purification or activity measurement. Ion-exchange chromatography and gel filtration was used to purify SOD molecular forms. BTS, Dilu Cell, M III and Vitasem were used as diluents for 5-day storage of semen at +16°C. The molecular form of SOD released from spermatozoa after cold shock and homogenization had a molecular weight of approximately 67kDa. The activity of the SOD form was the highest at pH 10 within the temperature range between 20 and 45°C. The enzymatic activity of form released after cold shock was inhibited by H2O2 and diethyldithiocarbamate (DDC; by 65 and 40%, respectively). The SOD form released by homogenization was inhibited by H2O2 and DDC (40%). The molecular form released after urea treatment was a 30kDa protein with maximum activity at 20°C and pH 10. Enzymatic activity of this form was inhibited by H2O2 by 35%, DDC by 80% and 2-mercaptoethanol by 15%. The antigenic determinants of SOD isolated from boar seminal plasma and spermatozoa were similar to each other. Susceptibility of spermatozoa to cold shock increased during storage, but the differences between extenders were statistically non-significant. |
| 2010 | A Neurogenetic Dissociation between Punishment-, Reward-, and Relief-Learning in Drosophila. | Front Behav Neurosci | What is particularly worth remembering about a traumatic experience is what brought it about, and what made it cease. For example, fruit flies avoid an odor which during training had preceded electric shock punishment; on the other hand, if the odor had followed shock during training, it is later on approached as a signal for the relieving end of shock. We provide a neurogenetic analysis of such relief learning. Blocking, using UAS-shibire(ts1), the output from a particular set of dopaminergic neurons defined by the TH-Gal4 driver partially impaired punishment learning, but left relief learning intact. Thus, with respect to these particular neurons, relief learning differs from punishment learning. Targeting another set of dopaminergic/serotonergic neurons defined by the DDC-Gal4 driver on the other hand affected neither punishment nor relief learning. As for the octopaminergic system, the tbh(M18) mutation, compromising octopamine biosynthesis, partially impaired sugar-reward learning, but not relief learning. Thus, with respect to this particular mutation, relief learning, and reward learning are dissociated. Finally, blocking output from the set of octopaminergic/tyraminergic neurons defined by the TDC2-Gal4 driver affected neither reward, nor relief learning. We conclude that regarding the used genetic tools, relief learning is neurogenetically dissociated from both punishment and reward learning. This may be a message relevant also for analyses of relief learning in other experimental systems including man. |
| 2007 | The orphan nuclear receptor DHR38 influences transcription of the DOPA decarboxylase gene in epidermal and neural tissues of Drosophila melanogaster. | Genome | The DOPA decarboxylase gene (Ddc) belongs to the "early-late" class of ecdysone-inducible genes in Drosophila melanogaster. Its expression is up-regulated in epidermal tissues by the ecdysone receptor acting through a response element, EcRE. In this paper, we show that another member of the nuclear receptor superfamily, DHR38, may act as a repressor of epidermal Ddc while inducing Ddc expression in neuronal cells. DHR38 does not behave as a classical co-repressor of the ecdysone receptor though, since the site through which DHR38 acts is distinct from the EcRE. Ectopic expression of a Dhr38 cDNA from a heat-shock promoter completely repressed transcription from the endogenous Ddc promoter and from an intact reporter construct in the hypoderm and in imaginal discs. Ectopic DHR38 had no effect on the transcription of a reporter driven by a Ddc fragment missing the DHR38 binding site. Neither reporter expression nor endogenous Ddc transcript levels were affected in a Dhr38 mutant background. Because most mutant organisms pupariate apparently normally and many of these survive to eclose, we believe that some functional redundancy exists within the Dhr38 regulatory network operating in epidermal tissues. In contrast to its apparent repressor function in epidermal tissues, DHR38 may act as a positive regulator of neural Ddc expression. Ectopic expression of DHR38 throughout the CNS induced as much as a 20-fold increase in Ddc transcripts in the set of neurons in which DDC normally appears. |
| 2005 | Interaction of stress proteins with misfolded keratins. | Eur J Cell Biol | Misfolded and aggregated proteins are a characteristic feature of a variety of chronic diseases. Examples include neurofibrillary tangles in Alzheimer disease, Lewy bodies in Parkinson disease and Mallory bodies (MBs) in chronic liver diseases, particularly alcoholic and non-alcoholic steatohepatitis (ASH and NASH). MB formation is at least in part the result of chronic oxidative cell stress in hepatocytes and can be induced in mice by long-term intoxication with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Proteomic analysis revealed that MBs consist of ubiquitinated keratins and the stress proteins Hsp70, Hsp25, and p62. Furthermore, marked overexpression of clusterin, which shares functional properties with small heat shock proteins, was identified by gene expression profiling of DDC-treated mice livers. To investigate whether clusterin has a function in the stress response to misfolded keratins, we performed transfection studies utilizing expression constructs encoding ubiquitin, p62, Hsp27, clusterin, keratin 8, and keratin 18. Ubiquitin was found in a strong and constant association with keratin aggregates, whereas binding of p62 to keratin was variable. Hsp27 did not colocalize with keratin aggregates under these experimental conditions. In contrast, clusterin associated with misfolded keratin only if its signal peptide was deleted and its secretion inhibited. This suggests that clusterin has ability to bind misfolded proteins, including keratins but its physiological function is restricted to the extracellular space. The extracellular localization of clusterin was underlined by immunohistochemical studies in Alzheimer disease brains, where clusterin was constantly found in association with amyloid plaques; in contrast, cytoplasmic inclusions such as neurofibrillary tangles as well as MBs in ASH were negative. Furthermore, we found clusterin in association with elastic fibers in the extracellular matrix in several chronic liver diseases, including ASH and alpha1-antitrypsin deficiency, implying a possible role of clusterin in liver fibrosis. |
| 2003 | Decreased survival of prostate cancer cells in vitro by combined treatment of heat and an antioxidant inhibitor diethyldithiocarbamate (DDC). | Exp Toxicol Pathol | The aim of this study was to examine a modulation of thermotolerance by treatment with combination of heat and the antioxidant inhibitor diethyldithiocarbamate (DDC) of the PC-3 prostate cancer cells. To determine thermotolerance, cells were heated once or twice. Two 1 h exposures at 43 degrees C, with a recovery period in between, revealed better survival/recovery of cells after the second exposure than after the first (fig. 1A + 1B). Additional experiments were performed, heating cells twice (fig. 1B + 1C). First, cells were heated at 43 degrees C for 1 h and, after various recovery times (intervals) at 37 degree C, subsequently reheated at 44 degrees C for 1 h. To ensure effective cell killing, efficiency of the combined treatments of 1 mM DDC and heating at 43 or 44 degrees C for 1 h was estimated by measuring cell survival, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity and heat shock protein 70 (hsp 70) expression. To obtain a more effective method for subsequent heat exposure, cells were heated twice after a 24 h interval in the presence or absence of 1 mM DDC. ROS generation and SOD activity immediately increased correlating with duration of heating, but their levels gently decreased with time after discontinuation of heating. On the other hand, hsp 70 levels slowly increased, also correlating with duration of heating but continued to increase with time after discontinuation of heating for a certain period. DDC administration coupled with heating at 43 or 44 degrees C significantly decreased cell survival compared to heating alone (p < 0.05). Furthermore, significant decreases in numbers of viable cells were observed for cells after the first heat exposure when combined with DDC as compared to heat alone at 43 and 44 degrees C (p < 0.05). These findings suggest that heat combined with DDC could have potential benefits in the treatment of prostate cancer. |
| 2002 | Modulation of heat-induced cell death in PC-3 prostate cancer cells by the antioxidant inhibitor diethyldithiocarbamate. | BJU Int | To examine the relationships between the form of cell death (apoptosis or necrosis), reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity and the level of heat-shock protein 70 (hsp 70) expression after thermotherapy of PC-3 prostate cancer cells; also assessed were the tumoricidal effects of combined treatment with both heat and the antioxidant inhibitor diethyldithiocarbamate (DDC).PC-3 cells were treated with thermotherapy at 42, 43 or 44 degrees C for 30, 60, 90 or 120 min. Cell proliferation, ROS generation, SOD activity and cellular hsp 70 level were determined using tetrazolium-based cytotoxicity, fluorescent dichlorofluorescein (DCF) and nitroblue tetrazolium assays, Western blot analysis and flow cytometry, respectively. The apoptotic and necrotic cells were determined by staining with propidium iodide and fluorescein isothiocyanate-labelled annexin V. These variable were also measured after combined treatment of PC-3 cells with 1 mmol/L DDC and thermotherapy at 43 or 44 degrees C for 60 min.Cell survival was significantly lower after heating cells at 43 degrees C for 60, 90 and 120 min and at 44 degrees C for all periods tested (P<0.05). At 43 degrees C apoptosis increased with the duration of heating and was similarly enhanced after heating at 44 degrees C for 30 min. Necrosis was not increased by heating at 42 or 43 degrees C, but was markedly enhanced after heating at 44 degrees C with both the duration of heating and with time after heating. Significant increases in DCF production were induced by heating at 43 degrees C for 60, 90 and 120 min (P<0.05) and at 44 degrees C at all times (P<0.010-0.005). There was a significant correlation between the level of ROS generation and necrosis (P<0.001) but no correlation between the ROS level and apoptosis. SOD activity increased in cells after heating at 43 degrees C, with significant differences among cells heated for 60, 90 and 120 min (P<0.05). After heating at 44 degrees C, SOD activity was maximal in cells heated for 30 min (P<0.005), by 30 min and then decreased with time after heating. There were significant increases in hsp 70 level in cells heated at 43 degrees C for 90 and 120 min (P<0.05) and at 44 degrees C for 30 and 60 min (P<0.05 and <0.025, respectively). Hsp 70 levels decreased after heating at 44 degrees C for 90 and 120 min. The combination of DDC and heating significantly increased ROS generation and the percentage of cell death, and decreased SOD activity (P<0.05).These findings show a qualitative change in the form of cell death induced by thermotherapy of PC-3 cells, which changed from apoptosis to necrosis according to the degree and duration of heating. Mild thermotherapy induced marginally low occurrence of apoptosis of PC-3 cells and DDC may represent a useful future strategy for the treatment of prostate carcinoma. |
| 2002 | Mallory body--a disease-associated type of sequestosome. | Hepatology | Mallory bodies (MBs) consist of abnormal keratins, ubiquitin, heat shock proteins, and the protein p62. p62 is encoded by an immediate-early response gene that rapidly responds to a variety of extracellular signals involved in cell proliferation, differentiation, and particularly oxidative stress. It acts as an adapter in signal transduction and binds noncovalently to ubiquitin, possibly being involved in the regulation of the fate of ubiquitinated proteins by segregation (i.e., sequestosome or aggresome formation). The presence of p62 together with ubiquitinated abnormal keratins in the MB characterizes MBs as a disease-associated type of sequestosome. A detailed study on the expression of p62 and its relationship to MB formation in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-treated mouse liver is reported based on immunohistochemical, immunoblot, and Northern blot analyses. The results indicate that p62 is rapidly induced in hepatocytes of intoxicated animals preceding MB formation. As suggested by experiments with short-term DDC-treated naive mice and mice refed DDC after recovery from long-term DDC treatment (primed mice), p62 does not exert an initiating effect on MB formation but the appearance of MBs requires the presence of abnormal keratins, which associate with p62 after ubiquitination. The rapid induction of p62 and its association with MBs further support the role of oxidative stress in MB formation. In conclusion, the constant presence of p62 in MBs suggests that binding of p62 to abnormal keratins may allow hepatocytes to dispose potentially harmful proteins in a biologically inert manner. |
| 2001 | Sequence of events in the assembly of Mallory body components in mouse liver: clues to the pathogenesis and significance of Mallory body formation. | J Hepatol | Chronic intoxication of mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or griseofulvin (GF) results in appearance of Mallory bodies (MBs) and alterations of the keratin cytoskeleton, which are reversible upon drug withdrawal but recur after readministration within 2-3 days.DDC- or GF-treated and recovered mice were reintoxicated with the original drugs but also colchicine and lumicolchicine. Cytoskeletal alterations of hepatocytes and MB formation were monitored by immunofluorescence microscopy using keratin, MB-specific antibodies, antibodies to phosphoepitopes and to HSP70. Keratin 8/18 mRNA expression and protein levels were determined by competitive reverse transcription-polymerase chain reaction, in situ-hybridization and western blotting.Duration of pretreatment was important for the efficiency of MB triggering. Rapid increase of keratin 8/18 mRNA and proteins was found in all reintoxicated mice concomitant with MB formation, whereby keratin 8 prevailed over keratin 18. Keratins and a protein with heat shock characteristics (M(M) 120-1 antigen) were the earliest detectable MB components, whereas ubiquitination and phosphorylation followed later.Overproduction of keratins is a major but not the only step responsible for MB formation. Additional components (e.g. M(M) 120-1 antigen) and excess of keratin 8 over keratin 18 are essential. |
| 1999 | Molecular mechanism of the short-term cardiotoxicity caused by 2',3'-dideoxycytidine (ddC): modulation of reactive oxygen species levels and ADP-ribosylation reactions. | Biochem Pharmacol | The short-term cardiac side effects of 2',3'-dideoxycytidine (ddC, zalcitabine) were studied in rats in order to understand the biochemical events contributing to the development of ddC-induced cardiomyopathy. In developing animals, ddC treatment provoked a surprisingly rapid appearance of cardiac malfunctions characterized by prolonged RR, PR, and QT intervals and J point depression. The energy metabolism in the heart was compromised, characterized by a decreased creatine phosphate/creatine ratio (from 2.05 normal value to 0.75) and a decreased free ATP/ADP ratio (from 332 normal value to 121). The activity of respiratory complexes (NADH: cytochrome c oxidoreductase and cytochrome oxidase) also decreased significantly. Southern blot and polymerase chain reaction analysis did not show deletions or a decrease in the quantity of mitochondrial DNA (mtDNA) deriving from ddC-treated rat hearts, indicating that under our experimental conditions, ddC-induced heart abnormalities were not the direct consequence of mtDNA-related damage. The ddC treatment of rats significantly increased the formation of reactive oxygen species (ROS) in heart and skeletal muscle as determined by the oxidation of non-fluorescent dihydrorhodamine123 to fluorescent rhodamine123 and the oxidation of cellular proteins determined from protein carbonyl content. An activation of the nuclear poly-(ADP-ribose) polymerase (EC 2.4.2.30) and an increase in the mono-ADP-ribosylation of glucose-regulated protein and desmin were observed in the cardiac tissue from ddC-treated animals. A decrease in the quantity of heat shock protein (HSP)70s was also detected, while the level of HSP25 and HSP60 remained unchanged. Surprisingly, ddC treatment induced a skeletal muscle-specific decrease in the quantity of three proteins, one of which was identified by N-terminal sequencing as myoglobin, and another by tandem mass spectrometer sequencing as triosephosphate isomerase (EC 5.3.1.1). These data show that the short term cardiotoxicity of ddC is partially based on ROS-mediated signalling through poly- and mono-ADP-ribosylation reactions and depression of HSP70 levels, whose processes represent a new mtDNA independent mechanism for ddC-induced cell damage. |
| 1999 | Superoxide: a major factor for stress protein induction in reoxygenation injury in the intestinal cell line Caco-2. | Digestion | Acute intestinal ischemia is followed by cellular destruction and loss of mucosal barrier function. Posthypoxic injury of cellular proteins leads to the synthesis of heat shock proteins. The role of oxygen radicals in this process, however, is not fully established.In the present study, using the intestinal cell line Caco-2, we investigated the relationship between the synthesis of the heat shock protein HSP70, detected by Western blot and oxygen radicals as well as lactate dehydrogenase (LDH) release, as measured in photometrical tests.Various periods of hypoxia and 30 min of reoxygenation resulted in an increased generation of superoxide as measured by the tetrazolium base 3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide. The inhibitor of superoxide dismutase (SOD), diethyldithiocarbamate (DDC) increased and addition of SOD decreased intracellular superoxide levels. HSP70 synthesis was detectable after 2 h of hypoxia. Similar to superoxide production, DDC increased and SOD reduced the HSP70 synthesis. In contrast, the increased LDH release from the cells observed after hypoxia was not significantly altered by DDC and SOD.The production of superoxide correlates with HSP70 induction, but not with LDH release. We conclude that hypoxia/reoxygenation induces heat shock protein production, a result of protein damage, by increased superoxide generation, whereas superoxide does not correlate with membrane damage in Caco-2 cells. |
| 1997 | Effects of oxidative stress on the expression of limbic-specific protease neuropsin and avoidance learning in mice. | Brain Res | We evaluated the effects of oxidative stress in mouse brain induced by the intraperitoneal injection of diethyldithiocarbamate (DDC) on gene expression of the novel serine protease, neuropsin, and on shock-avoidance learning. The level of neuropsin mRNA in the hippocampal pyramidal neurons increased at 2 h after DDC treatment and decreased thereafter. At 7 days neuropsin mRNA significantly decreased to 60% of the pretreated control level and then returned to the control level at 30 days. Genes for tissue plasminogen activator, manganese superoxide dismutase, and heat shock protein did not differ in DDC-treated mice vs. the control group at 7 and 30 days. The shuttle-box avoidance learning was retarded at 7 days after DDC administration. However, it recovered to the control level at 30 days after DDC administration. The results suggest that generation of reactive oxygen species has an important role in neuropsin transcript in the limbic areas which might be related to the disturbance in avoidance learning. |
| 1995 | Heat shock in vivo induces Mallory body formation in drug primed mouse liver. | Exp Mol Pathol | Perturbations in keratin intermediate filament organization and Mallory body (MB) formation are associated with alcoholic hepatitis. Inducible heat shock proteins (HSPs) are expressed in a variety of liver diseases including alcoholic liver disease. Therefore, we investigated whether heat shock protein induction can lead to MB formation. Mice were primed by a 5-month feeding of griseofulvin (GF) or diethyl 1,4-dehydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) followed by drug withdrawal for 1 month. The animals were then subjected to an in vivo heat shock treatment or sham heat treatment. Liver morphology, HSP expression, liver regeneration (PCNA-labeled nuclei), and MB formation were monitored during a 7-day posttreatment period. Numerous MBs developed in the livers of mice exposed to GF or DDC for 5 months, but very few small MBs remained after 1-month withdrawal of either drug. No MBs were found at Day 1 post heat shock, whereas numerous MBs were observed at Day 7. The frequency of PCNA-labeled nuclei increased during the same period. At Day 1 posttreatment, a variable liver centrilobular necrosis was observed accompanied by a prominent increase in HSP-25 and HSP70 expression, but HSP-90 expression was not increased. In drug-primed mouse liver, a heat shock treatment induces the expression of specific HSPs prior to the formation of MBs, indicating that HSP expression may play a role in the pathogenesis of MB formation. We speculate that this role is through the protein unfolding function of HSP, which leads to the aggregation of the cytokeratins to form MBs as well as to polyubiquitin binding to these proteins in a manner analogous to amyloid formation. |
| 1993 | Tissue-specific alternative splicing of the Drosophila dopa decarboxylase gene is affected by heat shock. | Mol Cell Biol | The Drosophila dopa decarboxylase gene, Ddc, is expressed in the hypoderm and in a small number of cells in the central nervous system (CNS). The unique Ddc primary transcript is alternatively spliced in these two tissues. We investigated whether Ddc splicing in the CNS is a general property of the CNS or a unique property of the cells that normally express Ddc by expressing the Ddc primary transcript ubiquitously under the control of an Hsp70 heat shock promoter. Under basal expression conditions, Ddc splicing shows normal tissue specificity, indicating that the regulation of Ddc splicing in the CNS is tissue specific rather than cell specific. Previous studies have shown that severe heat shock blocks mRNA splicing in cultured Drosophila melanogaster cells. Our results show that splicing of the heat shock-inducible Hsp83 transcript is very resistant to heat shock. In contrast, under either mild or severe heat shock, the splicing specificity of the heat shock-induced Ddc primary transcript is affected, leading to the accumulation of inappropriately high levels of the CNS splice form in non-CNS tissues. The chromosomal Ddc transcript is similarly affected. These results show unexpected heterogeneity in the splicing of individual mRNAs as a response to heat shock and suggest that the Ddc CNS-specific splicing pathway is the default. |
| 1991 | Effects of 2-(E-2-decenoylamino)ethyl 2-(cyclohexylethyl) sulfide on various ulcer models in rats. | Chem Pharm Bull (Tokyo) | The effects of 2-(E-2-decenoylamino)ethyl 2-(cyclohexylethyl) sulfide (compd. III-1a) on various experimental ulcers were investigated. The oral administration of compd. III-1a at doses ranging from 30 to 300 mg/kg inhibited the acute gastric ulcerations induced by ethanol, HCl.aspirin and indomethacin in rats. Compound III-1a significantly inhibited the water immersion stress-induced gastric ulcer at doses of 3 mg/kg, p.o. The anti-ulcer activity of plaunotol as a reference drug was equivalent on an ethanol-induced ulcer to that of compd. III-1a, but weaker on HCl.aspirin, indomethacin and stress-induced ulcers than that of compd. III-1a. On indomethacin-produced gastric antral ulcer, compd. III-1a showed the same significant inhibitory activity as spizofurone did at a dose of 100 mg/kg, p.o. Compound III-1a also inhibited hemorrhagic shock-, diethyldithiocarbamic acid (DDC)-and platelet activating factor (PAF)-induced ulcers dose-dependently. Plaunotol only showed significant inhibitory activity on PAF-induced ulcer in these three ucler models. The consecutive administration of compd. III-1a (100 mg/kg, p.o.) twice a day significantly accelerated the healing of an acetic acid-induced ulcer and that of plaunotol (200 mg/kg, p.o.) showed the same activity. Moreover, orally administered compd. III-1a at a dose of 100 mg/kg decreased the gastric acid secretion in pylorus-ligated rats. The results in the present study suggest that compd. III-1a has the dual action on ulcer formation. |
| 1990 | Closely related DNA sequences specify distinct patterns of developmental expression in Drosophila melanogaster. | Mol Cell Biol | Three short synthetic DNA sequences, which are closely related to one another, confer three distinct patterns of developmental expression on the heat shock hsp70 gene in transgenic Drosophila melanogaster lines. These results show that small variations or even single base pair changes in a repeated element of a regulatory sequence can create promoters that display new specificities of tissue and developmental regulation. Interestingly, the three patterns of developmental expression conferred by the synthetic DNAs resemble in part those of the known developmental genes: glucose dehydrogenase (Gld), Dopa decarboxylase (Ddc), and salivary gland secretory proteins (Sgs), respectively. In each case, the defined regulatory region of the known developmental gene contains multiple sequences that are similar or identical to the synthetic sequence that confers a similar pattern of developmental expression on the hsp70 gene. Thus, these results are congruent with the view that short sequence elements in multiple copies can confer either simple or relatively complex patterns of developmental expression on a receptive promoter like that of hsp70. Furthermore, the fact that the three variants tested produced three distinct patterns of expression in transgenic animals suggests that the number of different elements is large. |
| 1989 | Effects of diethyldithiocarbamate and endogenous polyamine content on cellular responses to hydrogen peroxide cytotoxicity. | Biochem J | In exponential-phase Chinese-hamster cells, 0.1 mM-diethyldithiocarbamate (DDC) afforded greater than 1 log survival protection to cultures treated before and during exposure to 1 mM-H2O2. Both DDC and H2O2 treatment stimulated the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, within 4 h of exposure. DDC, and to a lesser degree H2O2, also stimulated the activity of spermidine N1-acetyltransferase (SAT), the rate-limiting enzyme in polyamine catabolism. The increase in SAT activity, after exposure to DDC or another stress (heat shock), was inhibited in cells depleted of putrescine and spermidine by alpha-difluoromethylornithine (DFMO), the enzyme-activated suicide inhibitor of ODC. Pretreatment with DFMO or heat shock also induced resistance to H2O2 cytotoxicity. Since SAT activity is low in resting cells, yet stimulation of enzyme activity depends on endogenous spermidine pools, these results suggest that the expression of SAT activity occurs by a mechanism involving a stress-dependent displacement of spermidine into a new intracellular compartment. The stimulation of ODC and SAT activities does not appear to be a necessary component of the mechanism by which DDC protects cells from H2O2 cytotoxicity, although spermidine displacement may be a common facet of the cellular response to stress. |
| 1986 | Neurochemical and behavioral effects of catecholamine and protein synthesis inhibitors in mice. | Pharmacol Biochem Behav | A series of biochemical and behavioral experiments tested the hypothesis that anisomycin (ANI), a protein synthesis inhibitor, produced decrements in long-term memory by raising free tyrosine levels and by the accumulation of catecholamines (CAs) rather than by its primary effect on protein synthesis. We compared the effects of ANI and three catecholamine synthesis inhibitors (CAIs)--diethyldithiocarbamic acid, alpha-methyl-p-tyrosine, and tetrabenazine--on cerebral concentrations of tyrosine and CAs and on the rate of accumulation of CAs. ANI had a relatively small effect, whereas the CAIs resulted in large reductions. When ANI and a CAI were used in combination, effects on CA levels were determined mainly by the CAI. The amnestic effects of ANI and the CAIs were also compared across seven experimental paradigms. Pretraining administration of any of the four drugs could result in amnesia for passive avoidance training, but only when training was weak. With an increase in training strength, a series of three injections of ANI (one pre- and two post-training) caused amnesia, but a similar series of CAI injections did not. Substituting one CAI injection for the second of three successive ANI injections did not cause amnesia, but substituting cycloheximide, another protein synthesis inhibitor, resulted in amnesia. With an active avoidance test, ANI caused amnesia while AMPT did not; d-amphetamine blocked the amnestic effect of ANI but caused amnesia in AMPT injected mice. Whereas ANI lengthened the temporal gradient over which electroconvulsive shock produced amnesia, AMPT or DDC did not. DDC caused only transient amnesia for passive avoidance training, while the amnestic effect of ANI remained constant at 24-hr and 1-week retention tests. We conclude that ANI and CAIs have distinctly different abilities to produce amnesia. These experiments provide additional support for the hypothesis that protein synthesis is required for formation of long-term memory. |
| 1980 | On the evaluation of photoreceptor properties by micro-fluorimetric measurements of fluorochrome diffusion. | Biophys Struct Mech | By use of the microfluorimetric technique it is possible to study the diffusion of the fluorochrome di-dansylcystine (DDC) within isolated frog rod outer segments (ros) which are immobilysed in agarose gel. For this purpose, by a short hypotonic shock a leak is applied to one end of the ros. By this open end the DDC enters the rod and migrates through the whole outer segment. Following the propagation of the fluorescence boundary with time the cytoplasmatic diffusion constant can be determined if a chromatographic model is used to allow for the considerable binding of DDC to the inner membrane surface. With a binding constant K = 5 . 10(-4) cm the cytoplasmatic diffusion constant was found to be D = 1.3 . 10(-6) cm2/s whereas Dg = 2 . 10(-6 cm2/s and Dr = 3.5 . 10(-6) cm2/s were found in agarose gel or ringer solution, respectively. Using the mobility reduction factor given by D/Dr approximately equal to 0.4 to calculate the cytoplasmatic conductivity an inner resistance per length of 1.7 M omega/mu could be calculated for a frog rod which is in good agreement with corresponding data obtained from electrophysiological measurements. |