Biomedical subjects
Publications and source records attributed to S Roy.
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PURPOSE: We examined the metal-catalyzed oxidation of brain-derived neurotrophic factor (BDNF) using the Cu(II)/ascorbate/O2 model oxidative system. METHODS: Electrospray ionization mass spectrometry, peptide mapping and amino acid analysis were utilized to determine the nature of the covalent modification induced by the metal-catalyzed oxidative system. Additionally, analytical ultracentrifugation, the Bradford assay, circular dichroism and ANSA dye-binding were used to determine the nature of any conformational changes induced by the oxidation. RESULTS: Exposure of BDNF to the Cu(II)/ascorbate/O2 system led to the modification of ca. 35% of Met92 to its sulfoxide, and to subsequent conformational changes. The proteolytic digestion procedure was sensitive to this conformational change, and was unable to detect the modification. Chemical digestion with CNBr, however, was not sensitive to this change, and allowed for the identification of the site of modification. CONCLUSIONS: The modification of Met92 to its sulfoxide rendered the oxidized BDNF inaccessible to proteolytic digestion, due to conformational changes associated with the oxidation.
Sepsis induces lymphocyte apoptosis and prevention of lymphocyte death may improve the chances of surviving this disorder. We compared the efficacy of a selective caspase-3 inhibitor to a polycaspase inhibitor and to caspase-3-/- mice. Both inhibitors prevented lymphocyte apoptosis and improved survival. Caspase-3-/- mice shared a decreased, but not total, block of apoptosis. The polycaspase inhibitor caused a very substantial decrease in bacteremia. Caspase inhibitors did not benefit RAG-1-/- mice, which had a > tenfold increase in bacteremia compared to controls. Adoptive transfer of T cells that overexpressed the anti-apoptotic protein Bcl-2 increased survival. T cells stimulated with anti-CD3 and anti-CD28 produced increased interleukin 2 and interferon gamma by 6 h. Thus, caspase inhibitors enhance immunity by preventing lymphocyte apoptosis and lymphocytes act rapidly, within 24 h, to control infection.
The 20 cysteine residues of tubulin are heterogeneously distributed throughout its three-dimensional structure. In the present work, we have used the reactivity of these cysteine residues with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe to detect the global conformational changes of tubulin under different experimental conditions. The 20 sulfhydryl groups can be classified into two categories: fast and slow reacting. Colchicine binding causes a dramatic decrease in the reactivity of the cysteine residues and causes complete protection of 1.4 cysteine residues. Similarly, other colchicine analogs that bind reversibly initially decrease the rate of reaction; but unlike colchicine they do not cause complete protection of any sulfhydryl groups. Interestingly, in all cases we find that all the slow reacting sulfhydryl groups are affected to the same extent, that is, have a single rate constant. Glycerol has a major inhibitory effect on all these slow reacting sulfhydryls, suggesting that the reaction of slow reacting cysteines takes place from an open state at equilibrium with the native. Ageing of tubulin at 37 degrees C leads to loss of self-assembly and colchicine binding activity. Using DTNB kinetics, we have shown that ageing leads to complete protection of some of the sulfhydryl groups and increased reaction rate for other slow reacting sulfhydryl groups. Ageing at 37 degrees C also causes aggregation of tubulin as indicated by HPLC analysis. The protection of some sulfhydryl groups may be a consequence of aggregation, whereas the increased rate of reaction of other slow reacting sulfhydryls may be a result of changes in global dynamics. CD spectra and acrylamide quenching support such a notion. Binding of 8-anilino-1-naphthalenesulfonate (ANS) and bis-ANS by tubulin cause complete protection of some cysteine residues as indicated by the DTNB reaction, but has little effect on the other slow reacting cysteines, suggesting local effects.
In this report, we examined the possible functions of the cell death protease, caspase-3, in the axotomy-induced apoptosis of facial motoneurons in newborn rodents. Using in situ hybridization and Western blot, we found higher levels of caspase-3 mRNA and pro-caspase-3 protein expression in motoneurons of neonatal and 2-week-old rats than adult rats. Following facial motoneuron axotomy, caspase-3 mRNA and protein expression increased in motoneurons of both neonatal and adult rats. However, using an antibody directed to the activated form of the caspase-3 protease, we found that catalytically active caspase-3 was present only in axotomized neonatal motoneurons. As motoneurons in neonatal but not adult rodents are susceptible to axotomy-induced apoptosis, we hypothesized that caspase-3 may play a role in their demise. To determine the necessity of caspase-3 activation in axotomy-induced apoptosis, we counted the number of surviving motoneurons at 4 and 7 days following axotomy in wild type mice and caspase-3 gene-deleted mice. There were nearly three times more surviving motoneurons in caspase-3 gene-deleted mice than in wild type mice at both 4 days (mean 1074 vs. 464, P<0.005) and 7 days (mean 469 vs. 190, P<0.005) following injury, indicating a slower rate of death. Examination of the dying motoneurons using TUNEL staining (for fragmented DNA) and bisbenzimide staining (for nuclear morphology) revealed incomplete nuclear condensation in caspase-3-deficient motoneurons. These results demonstrate that caspase-3 activation plays important roles in the rapid demise of axotomized neonatal motoneurons.
PURPOSE: The purpose of this study was to visualise the rabbit aqueous outflow pathway using a numeric imaging system with ICG and fluorescein injection in the anterior chamber. MATERIAL AND METHODS: We performed a simultaneous injection of Indocyanin Green (ICG) and Fluorescein into the anterior chamber of rabbit eyes. Using a digital camera, we took several sequenced pictures to visualize the distribution of the dyes within the outflow pathway. We observed the dynamics of the outflow over time. RESULTS: In the early phases, the shape of the outflow canal around the limbus was clearly seen. Several collecting veins close to the recti muscles were also identified. There was only slight fluorescein leakage during the early phases, allowing adequate visualization of the morphology of the outflow system. In the late phases, the sclera was stained with the fluorescein, and no details were thus visible. The ICG dye allowed better recognition of the fine details of the outflow structure. CONCLUSION: This method was relatively simple, safe and precise, and allowed us to visualise the details of the outflow pathways in the rabbit eyes. These results could be of great value in further evaluating the outcome of filtering surgeries in animal models.
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BACKGROUND: Interleukin-4 (IL-4) treatment after lipopolysaccharide (LPS) induction inhibits macrophage (Mphi) IL-12 synthesis; however, IL-4 pretreatment (PreTx) primes the Mphi for increased LPS-induced IL-12 production. In this study we study the role of c-fos in the IL-4 priming of Mphi IL-12 synthesis. METHODS: With a murine in vitro peritoneal M phi model, we studied the effect of either c-fos deficiency (wild type, WT; homozygous c-fos knockout, Homo KO) or c-fos overexpression to study the role of c-fos in IL-4 priming of LPS-induced M phi IL-12 synthesis. RESULTS: (1) We first show that IL-4 PreTx results in a 72% decrease in Mphi c-fos mRNA compared with vehicle PreTx. (2) With respect to IL-12 p70 protein, IL-4 PreTx in the WT group increased LPS-induced Mphi IL-12 p70 2.2-fold compared with vehicle PreTx. Compared with vehicle PreTx in the WT group, vehicle PreTx in the Homo KO group followed by LPS stimulation resulted in a 2.8-fold increase in IL-12 p70 in the Homo KO group. IL-4 PreTx did not significantly increase IL-12 p70 over vehicle PreTx in the Homo KO group. (3) We studied the effect of c-fos overexpression on LPS-induced Mphi IL-12 production when primed with IL-4. Overexpression of c-fos completely inhibited IL-4 primed LPS-induced IL-12 p70 protein synthesis. CONCLUSIONS: These data demonstrated that down-regulation of c-fos is an integral part of the IL-4 priming process for Mphi IL-12 production.
The objective of this study was to assess the effects of gender on the secondary mechanisms by which dietary soluble fiber lowers plasma LDL cholesterol. For that purpose, male, female and ovariectomized (to mimic menopause) guinea pigs (8-10 per group) were allocated to two dietary treatments. Diets were identical in composition except for the fiber source: the control diet contained 10 g/100 of cellulose and 2.5 g/100 g of guar gum, while the soluble fiber (SF) diet contained 5 g/100 of psyllium, 5 g/100 of pectin and 2.5 g/100 g of guar gum. SF intake resulted in 44% lower plasma LDL cholesterol, 64% lower apo B and 22% lower plasma triacylglycerol (TAG) concentrations (P < 0.01) compared to guinea pigs fed the control diet. However, ovariectomized guinea pigs had higher plasma cholesterol, apo B and TAG concentrations (P < 0.01) compared to males and females, even those fed SF. Plasma HDL-cholesterol was higher in females than in males (P < 0.05). LDL size, as measured by LDL composition and fast protein liquid chromatography, was larger in females than males. Guinea pigs fed SF had smaller LDL than controls. LDL susceptibility to oxidation was 80% lower in male and females fed the SF diet (P < 0.001) than in controls, while there was no effect of diet in ovariectomized guinea pigs. Hepatic free cholesterol and TAG were lower, and activities of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase and cholesterol 7alpha-hydroxylase were higher in guinea pigs fed SF (P < 0.05) than in controls. These results indicate that gender plays an important role in the metabolic responses to dietary soluble fiber and that estrogen deprivation leads to a detrimental lipoprotein profile.
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A lambda-repressor mutant, S228N, which is defective in tetramer formation in the free state but retains full cooperativity, was studied in detail. Isolated single operator-bound S228N repressor shows association properties similar to those of the wild-type repressor. Fluorescence anisotropy studies with dansyl chloride-labeled repressor show a dimer-monomer dissociation constant of around 10(-5) M. The structure of the mutant repressor was studied by circular dichroism, acrylamide quenching and sulfhydryl reactivity at protein concentrations of < or =10(-6) M, where it is predominantly monomeric. The results suggest no significant perturbations in the structure of the S228N mutant repressor from that of the wild-type repressor. Urea denaturation studies also indicate no significant change in the stability of the repressor. The results were used to calculate energetics of loop formation in the cooperative binding process.
The ability of the leishmanial parasite UR6 to act as an immunoprophylactic and immunotherapeutic agent against Leishmania donovani infection in BALB/c mice was investigated. Unlike the virulent L. donovani AG83 (MOHOM/IN/1983/AG83), UR6 given through intracardiac route failed to induce visceral infection, but when it was injected subcutaneously, UR6 induced a short-lived and localized self-healing skin lesion. Priming of peritoneal macrophages with UR6 in vitro induced superoxide (O(2)(-)) generation, whereas similar experiments with virulent AG83 inhibited O(2)(-) generation. It was observed that priming of mice with either live or sonicated UR6 in the absence of any adjuvant provided strong protection against subsequent virulent challenge. Further, UR6-primed infected mice not only displayed a strong antileishmanial delayed-type hypersensitivity (DTH) response but also showed an elevated level of the serum antileishmanial immunoglobulin G2a (IgG2a) isotype, whereas infected mice failed to mount any antileishmanial DTH response and showed an elevated level of IgG1. This indicates that UR6 priming and subsequent L. donovani infection allowed the expansion of Th1 cells. Our studies indicate that UR6 has potential to be used as an immunoprophylactic and immunotherapeutic agent against experimental visceral leishmaniasis.
The efficacy of sodium lauryl sulfate (SLS), a sulfated anionic chaotropic surfactant, and dextran sulfate (DS), a polysulfated carbohydrate, against herpes simplex virus (HSV) and human immunodeficiency virus (HIV) infections was evaluated in cultured cells and in different murine models of HSV infection. Results showed that both SLS and DS were potent inhibitors of the infectivities of various HSV-1 and HSV-2 strains. Pretreatment of HIV-1 (strain NL4-3) with SLS also reduced its infectivity to 1G5 cells. DS prevented the binding of HSV to cell surface receptors and therefore its entry into cells. Pretreatment of HSV-1 (strain F) with 50 microM SLS resulted in a complete loss of virus infectivity to Vero cells. However, viruses were able to enter into cells and to produce in the nuclei capsid shells devoid of a DNA core. The amount of the glycoprotein D gene produced in these cells remained unchanged compared to controls, suggesting that SLS could interfere with the maturation of the virus. At a higher SLS concentration (100 microM), HSV was highly damaged by SLS pretreatment and only a few viral particles could enter into cells to produce abnormal capsids. Although DS was a more potent inhibitor of HSV infectivity in vitro, it was unable to provide any protection in murine models of HSV infection. However, SLS conferred a complete protection of animals infected cutaneously with pretreated viruses. In addition, skin pretreatment of mice with a polymer formulation containing SLS completely prevented the development of cutaneous lesions. More interestingly, intravaginal pretreatment of mice with SLS in a buffered solution also completely protected against lethal HSV-2 infection. Taken together, our results suggest that SLS could thus represent a candidate of choice as a microbicide to prevent the sexual transmission of HIV, HSV, and possibly other pathogens that cause sexually transmitted diseases.
Orphan nuclear receptor fetoprotein transcription factor (FTF) was previously identified as a specific regulator of the alpha(1)-fetoprotein gene during early liver development and in response to hormonal signals (L. Galarneau, J.-F. Paré, D. Allard, D. Hamel, L. Lévesque, J. D. Tugwood, S. Green, and L. Bélanger, Mol. Cell. Biol. 16:3853-3865, 1996). Here we report a functional analysis of FTF interactions with the hepatitis B virus (HBV) nucleocapsid promoter. DNA-protein-binding assays show that the HBV core promoter contains two high-affinity FTF-binding sites and a third, lower-affinity site shared with other receptors. Transfections in HepG2, Hep3B, and PLC/PRF/5 hepatoma cells using chloramphenicol acetyltransferase reporter genes with the nucleocapsid promoter linked or not linked to enhancer I indicate that FTF is a potent activator of the HBV core promoter, more efficient than HNF4alpha, HNF3alpha, HNF3beta, or C/EBPalpha. Steroidogenic factor 1, a close FTF homolog which binds to the same DNA motif and is expressed ectopically in HepG2 cells, seems to be an even stronger inducer than FTF. Point mutations of the FTF-binding sites indicate direct FTF activatory effects on the core promoter and the use of both high-affinity sites for productive interaction between the core promoter and enhancer I. Coexpression assays further indicate that FTF and HNF4alpha are the most efficient partners for coactivation of the pregenomic core promoter, which may largely account for the hepatic tropism and the early amplification of HBV infection. Carboxy terminus-truncated FTF behaves as a dominant negative mutant to compete all three FTF sites and strongly deactivate core promoter interactions with enhancer I; this suggests possible new ways to interfere with HBV infection.
Reactive oxygen species (ROS) generated by activated leukocytes play an important role in the disruption of endothelial cell (EC) integrity, leading to barrier dysfunction and pulmonary edema. Although ROS modulate cell signaling, information remains limited regarding the mechanism(s) of ROS-induced EC barrier dysfunction. We utilized diperoxovanadate (DPV) as a model agent to explore the role of tyrosine phosphorylation in the regulation of EC barrier function. DPV disrupted EC barrier function in a dose-dependent manner. Tyrosine kinase inhibitors, genistein, and PP-2, a specific inhibitor of Src, reduced the DPV-mediated barrier dysfunction. Consistent with these results, DPV-induced Src activation was attenuated by PP-2. Furthermore, DPV increased the association of Src with cortactin and myosin light chain kinase, indicating their potential role as cytoskeletal targets for Src. Transient overexpression of either wild-type Src or a constitutively active Src mutant potentiated the DPV-mediated decline in barrier dysfunction, whereas a dominant negative Src mutant attenuated the response. These studies provide the first direct evidence for Src involvement in DPV-induced EC barrier dysfunction.
Nine districts in West Bengal, India, and 42 districts in Bangladesh have arsenic levels in groundwater above the World Health Organization maximum permissible limit of 50 microg/L. The area and population of the 42 districts in Bangladesh and the 9 districts in West Bengal are 92,106 km(2) and 79.9 million and 38,865 km(2) and 42.7 million, respectively. In our preliminary study, we have identified 985 arsenic-affected villages in 69 police stations/blocks of nine arsenic-affected districts in West Bengal. In Bangladesh, we have identified 492 affected villages in 141 police stations/blocks of 42 affected districts. To date, we have collected 10,991 water samples from 42 arsenic-affected districts in Bangladesh for analysis, 58,166 water samples from nine arsenic-affected districts in West Bengal. Of the water samples that we analyzed, 59 and 34%, respectively, contained arsenic levels above 50 microg/L. Thousands of hair, nail, and urine samples from people living in arsenic-affected villages have been analyzed to date; Bangladesh and West Bengal, 93 and 77% samples, on an average, contained arsenic above the normal/toxic level. We surveyed 27 of 42 districts in Bangladesh for arsenic patients; we identified patients with arsenical skin lesions in 25 districts. In West Bengal, we identified patients with lesions in seven of nine districts. We examined people from the affected villages at random for arsenical dermatologic features (11,180 and 29,035 from Bangladesh and West Bengal, respectively); 24.47 and 15.02% of those examined, respectively, had skin lesions. After 10 years of study in West Bengal and 5 in Bangladesh, we feel that we have seen only the tip of iceberg.
The complications of diabetic microangiopathy have been well documented clinically for many years; however, the specific cellular mechanisms that lead to the dysfunction of small vessels in diabetes are unknown. Histopathological studies on diabetic microangiopathy have established thickened vascular basement membrane as one of the first and foremost structural abnormalities. Evidence suggests that this fundamental structural lesion develops, at least in part, from excess accumulation of basement membrane material in the vessel walls. Several vascular basement membrane components have been identified whose expression is up-regulated in diabetes. Enzymes responsible for the degradation of basement membrane components have also been reported to exhibit reduced activity. Strategies are being developed to regulate the abnormal accumulation of extracellular matrix (ECM) components in diabetic microangiopathy. In experimental diabetes, aldose reductase inhibitors have been found to be effective in preventing vascular basement membrane thickening when applied soon after the onset of hyperhexosemia. Clinical trials have been initiated with inhibitors of protein kinase C, an enzyme that up-regulates the synthesis of ECM components in diabetes. Currently, gene therapy strategies are also being developed to inhibit specific gene expression responsible for excess ECM synthesis in diabetes. Recent studies in the authors' laboratory have shown the applicability and efficacy of antisense oligonucleotides toward reducing vascular basement membrane component synthesis. Several gene regulatory approaches are currently being developed to modulate and prevent vascular basement membrane component synthesis. Such strategies may help identify key events that underlie the complex developmental stages of diabetic microangiopathy.