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Biomedical subjects

R Bhatnagar

Publications and source records attributed to R Bhatnagar.

At least 37 records · Page 2Linked to original sources
PubMed1997

A long-term dose-response study of mitomycin in glaucoma filtration surgery.

OBJECTIVE: To establish the long-term, dose-response relationship between the concentration of and duration of exposure to mitomycin to a decrease in intraocular pressure (IOP) and fewer complications. METHODS: We performed a prospective double-masked, placebo-controlled, 1-year study evaluating the decrease in IOP and fewer complications of fornix-based trabeculectomy surgery in 300 eyes equally divided among therapy with placebo; mitomycin, 0.2 mg/ mL, applied for 2 minutes; mitomycin, 0.4 mg/mL, applied for 4 minutes; or mitomycin, 0.4 mg/mL, applied for 2 minutes. All of the eyes had vertical and horizontal cup-disc ratios greater than 0.7. RESULTS: We observed significant treatment-related differences in IOP, with a decrease in IOP in all 3 mitomycin-treated groups for all of the times beyond 1 month. The number of eyes achieving strict IOP control and the development of cataract suggest a possible dose-response effect for concentration and time of exposure. Progressive lens opacification was the most frequent complication in 54 eyes (18.1%). The incidence of progressive lens changes markedly increased in subjects receiving 4 minutes of mitomycin therapy. Cataract formation was unrelated to IOP. Other complications were rare. Macular folds developed in 6 patients, with visual acuity returning to better than 20/40 in all but 1 patient. CONCLUSIONS: A possible dose-response relationship seemed to exist between the concentration of and duration of exposure to mitomycin. Length of exposure seems to be more important than concentration. The benefits of additional decreases in IOP must be weighed against the potential for increases in the risk of complications.

Antibiotics, Antineoplastic↗
PubMed1997

Modulation of macrophage heat shock proteins (HSPs) expression in response to intracellular infection by virulent and avirulent strains of Leishmania donovani.

We have examined the differential expression of heat shock proteins of murine macrophage-like cell line J774 G8 following infection with Leishmania donovani. In response to infection with virulent promastigotes, the up-regulation of HSP70 and 90 and a selective down-regulation of HSP60 was observed using monoclonal antibodies specific for host HSPs. However, infection with avirulent strain failed to alter the expression of host HSPs. The maximum alterations in HSPs expression were noted at 18 h post infection, a time period which coincided with the transformation of parasite from promastigote to the amastigote form. Data indicates that host HSPs may play a role in parasite differentiation/survival during infection with L. donovani.

Animals↗
PubMed1997

Up-regulation of reactive astrogliosis in the rat glioma 9L cell line by combined mechanical and chemical injuries.

The 9L rat glioma cells grown in culture, when subjected to a mechanical injury (scratch wound) and/or a chemical injury (CdCl2) manifest changes which are characteristic of an astrocyte reaction (astrogliosis) in the central nervous system. Such changes include cell hypertrophy and an increase in immunostaining for the astrocytic marker proteins, glial fibrillary acidic protein and J1-31 antigen. Mitochondria also increase in size and number, and the endoplasmic reticulum expands in area. These mechanical and chemical injuries are coordinated, and act synergistically to induce a considerably more intense astroglial reaction by 9L cells than can be elicited with either injurious agent alone, and this occurs without any interactions with microglia, neurons or oligodendroglia. The phenomenon suggests that more than one transcriptional mechanism is involved in the activation of astrocytes, and that mechanical and CdCl2-induced injuries, respectively, probably affect different receptors and second- and third-messenger pathways. There are a number of questions concerning the molecular biology of reactive astrocytes which can be addressed through the use of the 9L rat glioma cell model. This model offers certain advantages over primary cultures of astrocytes, namely a low basal level of reactivity (because the cells are not subjected to mechanical injury prior to experimentation), an absence of contaminating microglial cells, greater ease of reproducibility of results, lower costs and avoidance of the use of animals.

Animals↗
PubMed1997

Gap junctions in 9L and C6 glioma cells: correlation with growth characteristics.

The relationship between growth properties and gap junctional communication properties of two glioma cell lines, 9L and C6, was compared. Using microinjection of a gap junction permeable fluorescent dye, it was demonstrated that 9L cells were extensively dye coupled through gap junctions, whereas C6 cells had low levels of dye coupling between the cells. More gap junctions were observed between 9L cells than between C6 cells both by electron microscopy and immunofluorescent labelling with specific antibodies. Immunoblotting and immunofluorescence labelling of cellular proteins from 9L and C6 cells also showed differences in the Cx43 connexin contents of these cells, reflecting the differences in their gap junctional communication. Northern blot analyses confirmed that the level of Cx43 mRNA was higher in 9L than in C6 cells. In spite of these differences in gap junctions, the two cell lines did not differ in their growth rates in cultures. These results suggest a lack of direct correlation between gap junctional communication and cell growth regulation in these two glioma cell lines.

Animals↗
PubMed1997

Inhibition of connexin 43 synthesis by antisense RNA in rat glioma cells.

The 9L rat glioma cells communicate via gap junctions which are formed of connexin 43. The gap junctional communication was inhibited in these cells by transfecting them with an antisense Cx43 DNA construct, and its effect on their growth rate was investigated. This construct was induced by a Zn(2+)-inducible metallothionein promoter. Results showed that the induction of antisense RNA expression in rat 9L glioma cells produced the loss of gap junctions which was reflected in the loss of gap junctional communication. By inducing the antisense construct with zinc acetate, and using specific antibodies to connexin 43, the synthesis of this connexin was inhibited in the transfected cells and gap junctions were lost on the cell-cell appositions. There was no such effect on the untransfected cells. The loss of gap junctions at cell-cell appositions also correlated with the loss of Lucifer yellow fluorescent dye transfer between the cells. The effect of loss of gap junctions on the growth rate of the cells was assessed. In spite of the drastic decrease in the number of gap junctions between cells, their growth rate was only approximately 20% less than that of the transfected but non-induced cells. Therefore, gap junction communication is not negatively related to the rate of growth of these cells.

Animals↗
PubMed1996

Thermostabilization of protective antigen--the binding component of anthrax lethal toxin.

Protective antigen (PA) is the binding component of anthrax lethal toxin produced by Bacillus anthracis, and constitutes a major ingredient of the vaccine against anthrax. PA and lethal factor when added together are cytolytic to mouse macrophages and J774G8 macrophage cell line. This in vitro lethal toxicity assay is very useful in understanding the molecular mechanism of action of lethal toxin. Effective utilization of PA is, however, hampered due to its thermolability. On prolonged storage at 37 degrees C, PA was found to lose its activity almost completely. The effect of solvent additives like trehalose, sorbitol, xylitol, sodium citrate and magnesium sulphate on the thermal stabilization of PA was examined. The results indicated an increase in the stability of PA when the incubation at 37 degrees C was carried out in the presence of solvent additives used in the 1-3 M range. Magnesium sulphate helped retain the activity up to 82.7% against the control in which no additive was used, as judged by cytolytic assay using J774G8 macrophage cell line. Trehalose or sodium citrate also showed an appreciable protection of PA activity, while sorbitol or xylitol were not very effective. Competitive binding assay using radiolabeled PA showed that PA had lost capacity of binding to macrophage cells on prolonged incubation at 37 degrees C. Circular dichroism results at 4, 18, and 37 degrees C indicated an increase in secondary structure at 37 degrees C relative to that at 4 or 18 degrees C, supporting the activity data.

Animals↗
PubMed1995

Expression of DnaK and GroEL homologs in Leuconostoc esenteroides in response to heat shock, cold shock or chemical stress.

The mechanism of adaptation of bacteria to survive at elevated temperature in the human host and the expression of heat-shock proteins in response to stress was examined by labelling with [35S]methionine. An increase in culture temperature from 26 degrees C to 37 degrees C induced expression of certain bacterial proteins (70 and 60 kDa). Heat shock at 40 degrees C, cold shock (10 degrees C), ethanol treatment or arsenite treatment also led to an increased expression of heat shock proteins of 70 and 60 kDa. Actinomycin D completely blocked the induction, indicating that transcription is required for the overexpression of stress proteins in Leuconostoc mesenteroides. N-terminal sequence analysis showed that these proteins were homologous to the highly conserved chaperone proteins DnaK and GroEL of Escherichia coli, respectively.

Arsenites↗
PubMed1995

Tumour necrosis factor-alpha induces preferential expression of stress proteins in virulent promastigotes of Leishmania donovani.

Intracellular replication of Leishmania donovani inside macrophages is essential for production of disease and development of the parasite. Tumour necrosis factor-alpha (TNF) plays an integral role in host response to Leishmania. The effect of TNF on expression of heat-shock proteins (Hsp) was examined in promastigotes of L. donovani. TNF treatment led to an increased expression of Hsp83, Hsp70 and Hsp65 in virulent, but not avirulent, parasites. In response to stress by H2O2 or sodium arsenite, an increased expression of Hsp60 was observed in the virulent, but not avirulent, parasites. The virulent promastigotes were found to be more resistant to the toxic effects of TNF and other stresses. The data indicated that Hsp expressed in response to stress encountered in macrophages may confer protection to parasites and play a crucial role in their survival in the mammalian host.

Animals↗
PubMed1995

L-alanine: 4,5-dioxovalerate transaminase in Leishmania donovani that differs from mammalian enzyme.

Leishmania protozoans are the causative agents of leishmaniasis, a major parasitic disease in humans. The parasites manifest a nutritional requirement for heme compounds since they are deficient in heme biosynthesis. In this study we have demonstrated for the first time the presence of the enzyme L-alanine: 4,5-dioxovalerate transaminase in Leishmania donovani. This enzyme catalyzes the synthesis of 5-aminolevulinic acid (ALA), the first committed step in heme synthesis. Thus the defect in heme biosynthesis pathway in Leishmania must lie at some enzymatic level subsequent to synthesis of ALA. The enzyme was found to be present in both virulent and avirulent strains of L. donovani. The virulent promastigotes showed a 41% higher specific activity as compared to the avirulent strain. The enzyme activity was found to be inhibited in the presence of heme and methylglyoxal. Immunoblot analysis revealed that L-alanine: 4,5-dioxovalerate transaminase in L. donovani was immunologically different from that in mammals.

Alanine↗
PubMed1995

Rat glioma cell line as a model for astrogliosis.

In this investigation 9L rat glioma cells were grown on coverslips in culture, and when subjected to mechanical trauma, they were capable of mounting an astroglial response, without interaction with neurons, as evidenced by cell hypertrophy and increased immunostaining for glial fibrillary acidic protein and J1-31 antigen. It is of particular interest that this neoplastically transformed cell line is capable of mounting an astroglial response when subjected to mechanical trauma in vitro. The advantages of this model over in vivo models of central nervous system injury include the ease of reproducibility of results, and the ability to investigate molecular mechanisms under controlled conditions.

Animals↗
PubMed1995

Optimal preservation of the J1-31 antigen in astroglia examined by immunogold labelling.

Several methods for the detection of immunogold labelling for the monoclonal antibody (Mab) J1-31 in astroglial cells are described. The optimal preservation of the gold labelling is achieved in sections after cryoultramicrotomy or LR white embedding. The results obtained are consistent with the previous findings that the Mab J1-31 antigen is an intermediate filament-associated protein in the astroglial cells.

Animals↗
PubMed1994

The dynamic progression of evolved character states for aromatic amino acid biosynthesis in gram-negative bacteria.

A systematic analysis of the evolution of aromatic amino acid biosynthesis in the Proteobacteria, previously focussed mainly upon the gamma subdivision, has now been extended to the beta subdivision. Five lineages were studied, represented by Neisseria gonorrhoeae, Nitrosomonas europaea, Alcaligenes faecalis, rRNA Group-III pseudomonads/Rubrivivax gelatinosus, and rRNA Group-II pseudomonads/Rhodocyclus tenuis. Within the phenylalanine pathway, the bifunctional P-protein (chorismate mutase/prephenate dehydratase) was present in each lineage and must have evolved in a common ancestor of the beta and gamma subdivisions. Each P-protein was found to be subject to activation by L-tyrosine, and to feedback inhibition by L-phenylalanine. Phenylalanine-inhibited (DS-phe) and tyrosine-inhibited (DS-tyr) isoenzymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase probably existed in the common beta-subdivision ancestor, with DS-tyr being lost in N. gonorrhoeae and A. faecalis. The participation of DS-phe in a dissociable multienzyme complex with one or more other common-pathway enzymes is known to exist in N. gonorrhoeae. The same complex is indicated by two peaks of DS-phe seen in chromatographic profiles of Group-III pseudomonads and A. faecalis. It is concluded that the contemporary DS-phe species present in subdivision gamma and beta must have had independent origins. Tyrosine biosynthesis was found to be quite diverse within the beta subdivision. Nit. europaea possessed an arogenate dehydrogenase which was specific for NADP+. In all other lineages, a broad-specificity cyclohexadienyl dehydrogenase (CHD) was present. In N. gonorrhoeae the CDH was specific for NAD+ while the remaining CDH species could utilize either NAD+ or NADP+. Only the CDH species within the rRNA Group-II pseudomonad/R. tenuis lineage was feedback-inhibited by L-tyrosine, and this correlated with an allosteric pattern where activation of the prephenate dehydratase component of the P-protein by L-tyrosine was relatively poor. However, the CDH enzyme present in N. gonorrhoeae and A. faecalis was subject to inhibition by 4-hydroxyphenylpyruvate, this being competitive with respect to the cyclohexadienyl substrate. The monofunctional species of chorismate mutase (CM-F) and cyclohexadienyl dehydratase, widely distributed among the gamma-subdivision assemblage and recently shown to be periplasmic enzymes, were demonstrated in Pseudomonas pickettii, a member of rRNA homology Group-II.

Alcaligenes↗
PubMed1994

Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity.

Anthrax lethal toxin, which is composed of two proteins, i.e., protective antigen and lethal factor, is cytolytic to mouse peritoneal macrophages and the macrophage-like cell line J774A.1. After exposure of cells to lethal toxin, inhibition of protein synthesis occurred only slightly before the onset of cytolysis. Thus, cell death did not appear to be due to inhibition of protein synthesis. However, prior treatment of J774A.1 cells with cycloheximide or puromycin, which inhibited protein synthesis, protected them completely against lethal toxin-induced cytolysis, which suggested that continuous protein synthesis is required for the expression of lethal toxin activity. Inhibition of protein synthesis had no appreciable effect on the binding of protective antigen to the cell surface receptor or on proteolytic cleavage of surface-bound protective antigen. Furthermore, inhibition of protein synthesis did not alter the uptake of toxin, which suggested that protein synthesis is required at a later stage of the intoxication process. The protection provided by inhibition of protein synthesis was effective, even up to 1 h after exposure to anthrax lethal toxin. The increased uptake of calcium observed in cells exposed to lethal toxin did not occur when they were protected by blocking protein synthesis. Identifying the protein(s) synthesized during the intoxication process may help to understand the mechanism of cell death produced by anthrax lethal toxin.

Animals↗
PubMed1994

Differential expression of stress proteins in virulent and attenuated promastigotes of Leishmania donovani.

Induction of stress proteins in response to heat shock and/or pH shock was compared in virulent and attenuated promastigotes of Leishmania donovani. The virulent strain showed increased expression of heat shock proteins (HSPs) of 110, 83, 70, 65, 40 and 12 kDa on heat shock (37 degrees C), while the attenuated form showed increased expression of HSPs 83, 70 and 65 only. In response to a low pH (4.5), the virulent promastigotes showed a significantly greater increase in the relative synthesis of HSP83 and HSP 70 as compared to the attenuated promastigotes. A combination of heat shock and pH shock led to an increase in the synthesis of HSP 83 and HSP 70 in the virulent parasites, while the attenuated strain showed marked increase in HSP 70 only. These results point to an important role of HSPs in the differentiation and/or virulence of L. donovani.

Animals↗
PubMed1993

Diversity among reactive astrocytes: proximal reactive astrocytes in lacerated spinal cord preferentially react with monoclonal antibody J1-31.

An Astrocyte-specific antigen recognized by monoclonal antibody J1-31 is a more intense marker for proximal reactive astrocytes in lacerated rat spinal cord than is glial fibrillary acidic protein (GFAP). Thus, MAb J1-31 recognizes reactive astrocytes in the immediate vicinity of the lesion, whereas reactive astrocytes that are located at a distance from the lesion are not detected by immunofluorescent staining. These findings are relevant to the biochemical heterogeneity manifested respectively by reactive astrocytes located proximal and distal to a laceration-type injury of the spinal cord, and those that develop following axotomy with retrograde degeneration. Reactive astrocytes in the axotomy model are not stained with MAb J1-31, but are positive for GFAP.

Animals↗
PubMed1993

Characterization of macrophage sensitivity and resistance to anthrax lethal toxin.

Anthrax lethal toxin, which consists of two proteins, protective antigen and lethal factor, is cytolytic for macrophages. Macrophages from different mouse strains were found to vary in their sensitivities to toxin. C3H mouse macrophages lysed by lethal factor concentrations of 0.001 micrograms/ml were 100,000 times more sensitive than those from resistant A/J mice. We analyzed various stages of the intoxication process to determine the basis for this resistance. Direct binding studies with radioiodinated protective antigen revealed that the affinity (Kd, approximately 0.5 nM) and number of receptors per cell (25,000 to 33,000) were the same in sensitive and resistant cells. Proteolytic activation of protective antigen by a cell surface protease and subsequent binding of lethal factor were also the same in both sensitive and resistant macrophages. Resistant A/J macrophages were not cross-resistant to other toxins and a virus which, like lethal toxin, require vesicular acidification for activity, implying that resistance is not due to a defect in vesicular acidification. When introduced into the cytosol by osmotic lysis of pinosomes, lethal factor in the absence of protective antigen was cytolytic for the sensitive macrophages while resistant cells were unaffected. Thus, lethal factor by itself possesses the toxic activity of lethal toxin. These results suggest that macrophage resistance is due to a defect at a stage occurring after toxin internalization. A/J macrophages may lack the putative lethal factor target in the cytosol or be defective in the further processing or activation of lethal factor in the cytosol or in endocytic vesicles.

Animals↗
PubMed1992

Rat glioma cell lines C6 and 9L synthesize type 1 collagen in vitro.

Glial cell lines (C6, a glioma and 9L, a gliosarcoma) grown in vitro produce type 1 collagen which is detectable in the extracellular matrix by immunocytochemistry. Northern blot analysis using a cDNA specific for the proalpha2 (I) chain of procollagen indicates the presence of a single transcript with an apparent size of 4.8 kb in the C6 cell line, whereas two transcripts with apparent sizes of 5.8 and 4.8 kb are visualized in the 9L cells. The stimulatory effect of ascorbic acid on collagen production is detectable by a 20-27% increase in the concentration of hydroxyproline in the culture medium from the two glioma cell lines. Therefore these glioma cell lines provide a valuable model system for comparative investigations on the regulation of type 1 collagen synthesis by nonmesenchymal cells of neuroepithelial origin.

Animals↗