Biomedical subjects

R S Gupta

Publications and source records attributed to R S Gupta.

At least 109 records · Page 6Linked to original sources

PubMed1994

Heat shock protein immunoreactivity in CSF: correlation with oligoclonal banding and demyelinating disease.

The detection of raised immunoglobulin and the presence of oligoclonal bands (OCBs) on electrophoresis of multiple sclerosis (MS) CSF has been a useful diagnostic test, but a universal antigen to which these MS antibodies are directed has yet to be found. Potentially immunogenic heat shock proteins (HSPs) are preferential expressed in vitro in human oligodendrocytes compared with other glia, and in situ in oligodendrocytes found within the plaques of MS. Immunoreactivity directed against HSPs might therefore contribute to the immune-mediated demyelinating process found in MS. We examined this possibility by quantitatively (ELISA) measuring antibodies directed against a recombinant human HSP (HSP60) in CSF from 18 MS patients, and compared them with eight patients with acute disseminated encephalomyelitis, 12 with demyelinating peripheral neuropathies, and 59 with other neurologic diseases. Immunoblotting was used to confirm the specificity of the antibodies for binding to HSP60. We found a statistically significant correlation between antibody titers to HSP60 and the presence of OCBs in CSF. These results support the notion that HSP expression in the CNS, such as that observed in MS, may be immunogenic, leading to localized HSP antibody secretion. Such HSP-directed immunoreactivity could play a role in the pathogenesis of MS and other immune-mediated disorders of the nervous system.

Antibodies↗

PubMed1994

Presence and cellular distribution of a 60-kDa protein related to mitochondrial hsp60 in Giardia lamblia.

Giardia lamblia trophozoites contain a 60-kDa protein recognized in immunoblots by antibody to mammalian hsp60, a protein localized in mitochondria in eukaryotic cells. The cellular distribution of this protein is evaluated by immunofluorescence microscopy using monoclonal antibody to human hsp60, polyclonal antibody to rodent hsp60, and 2 monoclonal antibodies to mycobacterial 65-kDa antigen, a prokaryotic hsp60 homolog. All of these antibodies, except 1, which is specific for prokaryotic hsp60, give a punctate labeling pattern throughout the cytoplasm, indicating that the 60-kDa protein is concentrated at discrete sites in the cytoplasm. The polyclonal hsp60 antiserum reveals additional punctate labeling colocalized on axonemes of the anterior flagella. Postembedment immunogold labeling and electron microscopy confirm that the antigen is clustered at foci in the cytoplasm but show no evidence of association with a membranous organelle. The hsp60 reactivity is also observed on anterior axonemes within the cytoplasm and on the adhesive disc. Hoechst 33258 DNA staining as well as electron microscopy give no evidence of endosymbionts, so a false positive due to a prokaryotic hsp60 homolog is unlikely. The presence of an hsp60-related protein in G. lamblia raises interesting questions concerning its origin.

Animals↗

PubMed1994

Identification of a GroES (CPN10)-related sequence motif in the GroEL (CPN60) chaperonins.

Significant sequence similarity has been noted between a segment of the 60 kDa heat shock family of proteins (designated as GroEL, cpn60 or hsp60) and its functional partner, the 10-12 kDa GroES (or cpn10) family of proteins. Upon introduction of a few gaps, 31 identical and 19 conserved residues were observed in a 93 amino acid overlap between the region comprising of a.a. 314 to 431 in E. coli GroEL and the entire GroES sequence from Bacillus subtilis. The functional forms of both GroEL and GroES proteins, which function as a team in the protein folding process, are toroidal rings exhibiting a highly unusual seven-fold rotational axis of symmetry. It is suggested that the information for assuming this structural form is contained within the shared sequence region between these proteins.

Amino Acid Sequence↗

PubMed1993

Mitochondrial HSP60 (P1 protein) and a HSP70-like protein (mortalin) are major targets for modification during S-(1,1,2,2-tetrafluoroethyl)-L-cysteine-induced nephrotoxicity.

The potent and site-selective nephrotoxicity of S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) in vivo has been associated with difluorothioamidyl-L-lysine formation on critical mitochondrial target proteins. Dose-response studies in the Fischer 344 rat indicate that five proteins with apparent molecular masses of 99, 84, 66, 52, and 48 kDa are predominantly adducted in vivo after nephrotoxic doses of TFEC (> 10 mg/kg, intraperitoneally). Microsequence analysis of the major difluorothioamidyl-L-lysine proteins indicated that P66 is identical, over 14 NH2-terminal residues, to mitochondrial P1 protein (HSP60, a chaperonin) and that P84 is identical, over 14 residues, to a recently isolated novel member of the HSP70 family known as mortalin. These studies indicate that mitochondrial heat shock proteins are major targets for modification by reactive metabolites of TFEC. The implications of these data in relation to the nephrotoxicity of cysteine conjugates are discussed.

Amino Acid Sequence↗

PubMed1993

A fatal, systemic mitochondrial disease with decreased mitochondrial enzyme activities, abnormal ultrastructure of the mitochondria and deficiency of heat shock protein 60.

We report on a girl presenting with facial dysmorphic features and breathing difficulties upon birth. She was hypotonic, developed a metabolic acidosis, and died two days old of heart failure. Post-mortem examination revealed abnormalities of brain, lungs, heart and liver. In cultured skin fibroblasts activities of enzymes of oxidative phosphorylation, pyruvate metabolism, beta-oxidation and other mitochondrial (mt) metabolic pathways were markedly decreased. Activities of enzymes localized in the mt outer membrane or in other cell organelles were found to be normal. The mitochondria appeared swollen and were located mainly around the nucleus. Electron micrographs showed locally disintegrated mt inner membranes and large mt vacuoles. The amount of mt heat shock protein 60 (hsp60) was about one fifth of that in controls. We conclude that this mt disorder is most likely caused by defective synthesis and maintenance of mitochondria, possibly due to a defect in mt protein import or enzyme assembly resulting from deficiency of hsp60.

Cells, Cultured↗

PubMed1993

Cloning and characterization of multiple groEL chaperonin-encoding genes in Rhizobium meliloti.

Heat-shock treatment of Rhizobium meliloti cells causes major enhancement in the synthesis of several proteins with apparent molecular weights in the range of 58-60 kDa. Using the polymerase chain reaction and degenerate oligodeoxyribonucleotide primers for conserved regions of the 60-kDa heat-shock protein (HSP60) or GroEL protein family, a 0.6-kb probe for the R. meliloti hsp60 gene was prepared. Southern blot analysis of R. meliloti DNA digested with different restriction enzymes and hybridized to R. meliloti hsp60 probes indicated the presence of between four and five hsp60 or groEL in this species. From the cloning and sequencing of several of these fragments, we have been able to deduce the complete nucleotide sequences of three groEL in R. meliloti. The deduced amino acid (aa) sequences of these proteins show extensive similarity to each other (78-85% aa identity) and to other GroEL homologues. In the upstream regions of two of the groEL, but not the third, open reading frames corresponding to GroES proteins were also identified. Analysis of various prokaryotic GroEL sequences suggests that the multiple groEL of R. meliloti have evolved by means of gene duplication events within this or a related group of organisms. Results presented in this paper also show that some of the groEL in R. meliloti are located on the two megaplasmids present in these cells. The presence of multiple GroEL homologues in R. meliloti suggests a possible role of the GroEL or HSP60 chaperonins in the nodulation (symbiosis) and nitrogen fixation processes.

Amino Acid Sequence↗

PubMed1993

Human peripheral gamma delta T cells recognize hsp60 molecules on Daudi Burkitt's lymphoma cells.

Studies with the use of polyclonal rabbit antiserum reactive with 60-kDa heat shock proteins (hsp60) suggested that hsp60-related molecules could be found on the surface of Daudi cells and were involved in their recognition by certain human gamma delta T cells. The present study confirms this finding by using a mAb specifically recognizing hsp60. This mAb can block outgrowth of human gamma delta T cells in response to stimulation with Daudi and in response to an extract of the mycobacteria H37Ra. This anti-hsp60 mAb stains the surface of Daudi cells, but does not stain either Raji or EBV-transformed B cells, cells which do not stimulate gamma delta T cell outgrowth. Anti-hsp60 mAb could immunoprecipitate a 60-kDa molecule from the H37Ra extract but was unable to precipitate this 60-kDa molecule if the mAb was first absorbed on Daudi cells. This mAb also precipitated a 60-kDa molecule from the surface of Daudi cells which shows an electrophoretic mobility pattern consistent with hsp60. These experiments demonstrate that human gamma delta T cells recognize hsp60-related epitopes on the surface of Daudi cells and within mycobacterial extracts.

Animals↗

PubMed1993

Evolution of HSP70 gene and its implications regarding relationships between archaebacteria, eubacteria, and eukaryotes.

The 70-kDa heat-shock protein (HSP70) constitutes the most conserved protein present in all organisms that is known to date. Based on global alignment of HSP70 sequences from organisms representing all three domains, numerous sequence signatures that are specific for prokaryotic and eukaryotic homologs have been identified. HSP70s from the two archaebacterial species examined (viz., Halobacterium marismortui and Methanosarcina mazei) have been found to contain all eubacterial but no eukaryotic signature sequences. Based on several novel features of the HSP70 family of proteins (viz., presence of tandem repeats of a 9-amino-acid [a.a.] polypeptide sequence and structural similarity between the first and second quadrants of HSP70, homology of the N-terminal half of HSP70 to the bacterial MreB protein, presence of a conserved insert of 23-27 a.a. in all HSP70s except those from archaebacteria and gram-positive eubacteria) a model for the evolution of HSP70 gene from an early stage is proposed. The HSP70 homologs from archaebacteria and gram-positive bacteria lacking the insert in the N-terminal quadrants are indicated to be the ancestral form of the protein. Detailed phylogenetic analyses of HSP70 sequence data (viz., by bootstrap analyses, maximum parsimony, and maximum likelihood methods) provide evidence that archaebacteria are not monophyletic and show a close evolutionary linkage with the gram-positive eubacteria. These results do not support the traditional archaebacterial tree, where a close relationship between archaebacterial and eukaryotic homologs is observed. To explain the phylogenies based on HSP70 and other gene sequences, a model for the origin of eukaryotic cells involving fusion between archaebacteria and gram-negative eubacteria is proposed.

Amino Acid Sequence↗

PubMed1993

Sarcocystis fusiformis: some protein metabolic enzymes in various fractions of sarcocysts of buffalo (Bubalus bubalis).

An investigation on the relative presence of some protein metabolic enzymes, namely aspartate aminotransferase (AST), alanine aminotransferase (ALT), NAD+ and NADP+ dependent glutamate dehydrogenase (GLDH) and arginase in cyst wall (CW), cyst fluid (CF) and zoite (ZT) fractions of the sarcocysts of Sarcocystis fusiformis in the oesophageal muscles of Indian water buffalo was carried out. Both the transaminases were present in all the fractions of the cyst, although in variable amounts. There was a higher level of AST activity than of ALT activity. AST activity was the highest in ZT, whereas ALT activity was at a maximum in the CF fraction. The levels of activity of NAD+ and NADP+ dependent GLDH and arginase remained beyond detectable limits. The study revealed that the intermediates of carbohydrate metabolism are linked to protein metabolism by transaminases. The possibility of concomitant removal of ammonia and its subsequent incorporation into the urea cycle is ruled out in this parasitic protozoan.

Alanine Transaminase↗

PubMed1993

Modulation of adjuvant arthritis in Lewis rats by recombinant vaccinia virus expressing the human 60-kilodalton heat shock protein.

The immune response to the mycobacterial 65-kDa heat shock protein (hsp65) is considered an important event in the induction of adjuvant arthritis (AA) in rats; this induction probably occurs through a molecular mimicry mechanism involving cross-reactivity against the rat homolog hsp60. To analyze the role of mammalian molecule hsp60 in arthritis, we generated a recombinant vaccinia virus (hsp60-VV) carrying the human hsp60 gene inserted into the thymidine kinase locus under the control of the 7.5k vaccinia virus promoter. Human hsp60 is almost identical to its rat homolog (97.4% linear amino acid homology) and shares about 50% of amino acid positions with Mycobacterium tuberculosis hsp65. The latter supposedly carries a critical epitope for AA induction that is not present in human hsp60. Infections with hsp60-VV of monkey cell cultures led to the expression of the human hsp60 molecule, as evidenced by immunoblotting analysis with specific monoclonal antibodies. Also, Lewis rats infected with hsp60-VV produced specific antibodies, demonstrating the in vivo expression of human hsp60 in the infected animals. Therefore, we used hsp60-VV to analyze whether the delivery of hsp60 could affect the induction of AA in Lewis rats. hsp60-VV clearly reduced and retarded arthritic symptoms when administered to rats at day 7 after AA induction. In contrast, inoculation of rats with a control recombinant vaccinia virus did not affect the course of the disease. The improvement in AA with hsp60-VV administration was associated with a specific immune response, as determined by the presence of antibodies to hsp60 in the sera and the proliferation induced by hsp60 of T cells from popliteal lymph nodes. These results support a critical role for immunity to heat shock proteins in AA. Since the protective construct is virtually identical to rat homolog hsp60, we conclude that immunity directed to conserved areas of this family of proteins is directly involved in the pathogenesis of AA.

Animals↗

PubMed1993

Staining of endothelial cells and macrophages in atherosclerotic lesions with human heat-shock protein-reactive antisera.

Our previous epidemiological studies have shown that levels of serum antibodies against mycobacterial heat-shock protein (hsp) 65 correlate positively with carotid atherosclerosis in subjects aged 40 to 79 years. To determine whether these high-titer sera also react with homologous human hsp60 and/or cell components of atherosclerotic lesions, we selected 15 human sera samples, each with high or low titers to recombinant mycobacterial hsp65, and investigated their reactivity with human arterial lesion components by immunoblotting and immunofluorescence techniques. All five higher-titer sera against hsp65 reacted with a 60-kDa band of atherosclerotic lesion proteins and human recombinant hsp60 on Western blots. Pooled sera with low antibody titers to hsp65 diluted similarly as high-titer sera did not show reactivity with atherosclerotic lesion and media proteins. By immunohistochemistry and immunofluorescence with human immunoglobulin G isolated from different sera, labeled with biotin, and visualized with a streptavidin conjugate, positive staining was observed in sections of fatty streaks and atherosclerotic plaques of carotid arteries, and weak staining was observed in the normal intima. Double immunofluorescence identified the majority of positively stained cells as macrophages, endothelial cells, and a few smooth muscle cells. In summary, serum antibodies against hsp65 cross-react with the human 60-kDa homologue present in high levels in atherosclerotic lesions and are mainly reacting with macrophages and endothelial cells, supporting our concept of a possible involvement of humoral-mediated immune reaction against hsp60 in atherogenesis.

Aged↗

PubMed1993

Synthesis and biological activity of novel thymidine derivatives of podophyllotoxin and 4'-demethylepipodophyllotoxin.

We have synthesized a number of novel derivatives of podophyllotoxin (POD) and 4'-demethylepipodophyllotoxin (DMEP) in which the nucleoside thymidine has been conjugated at the C4 position. To investigate the structure-activity relationship among these compounds, the cross-resistance patterns of these derivatives towards a set of either POD-resistant (PodR) or VP16/VM26-resistant (VpmR) mutants of Chinese hamster ovary (CHO) cells were determined. These mutants exhibit highly specific cross-resistance patterns toward compounds that show either POD- or VP16/VM26-like activity. The observed cross-resistance patterns of the thymidine derivatives suggests that these compounds display POD-like activity in vivo and show no VP16/VM26-like activity. Further, treatment of Chinese hamster cells with these compounds caused a dose-dependent increase in the mitotic index similar to the patterns observed with POD and DMEP, supporting the data from the cross-resistance assay. Most thymidine derivatives exhibited much lower activity in comparison to POD or DMEP, suggesting that the thymidine moiety interferes with the interaction of these compounds with the receptor site on the tubulin molecule. One of these derivatives which was most active in the aforementioned assays was also found to be a competitive inhibitor of radiolabelled POD binding to purified bovine brain tubulin. All other compounds were insoluble at concentrations required to perform the competition assay. Molecular modelling studies provide valuable insight regarding the three-dimensional structural requirements that distinguish POD-like compounds from their VP16/VM26-like counterparts. There appears to be a very limited spatial and electrostatic requirement for the bulky glycosidic moiety at C4 which is essential for VP16/VM26-like activity.

Animals↗

PubMed1992

Cloning of HSP70 (dnaK) gene from Clostridium perfringens using a general polymerase chain reaction based approach.

A general method to clone the HSP70 gene from any species employing polymerase chain reaction and degenerate oligonucleotide primers for conserved regions of this protein family is described. Using this method, a clone containing the entire coding sequence for the HSP70 gene from Clostridium perfringens, has been isolated and sequenced. The HSP70s from C. perfringens as well as other Gram-positive groups of bacteria contain a large deletion of 25 amino acids, near the N-terminal end, which is not seen in HSP70 from other sources.

Amino Acid Sequence↗

PubMed1992

Cloning of HSP60 (GroEL) operon from Clostridium perfringens using a polymerase chain reaction based approach.

Using degenerate oligonucleotide primers for conserved regions of HSP60, a 0.6 kilobase fragment of Clostridium perfringens DNA was amplified by the polymerase chain reaction. The amplified fragment was used as a probe to isolate a genomic clone containing the C. perfringens HSP60 operon. The clone contained two open reading frames homologous to the GroES and GroEL (or HSP60) family of bacterial and eukaryotic proteins as well as other upstream and downstream sequences. The approach described here, employing this set of degenerate oligonucleotide primers, could be used to clone HSP60 gene/cDNA from any species.

Amino Acid Sequence↗

PubMed1992

Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring.

Chaperonins are thought to participate in the process of protein folding in bacteria and in eukaryotic mitochondria and chloroplasts. While some chaperonins are relatively well characterized, the structures of the mammalian chaperonins are unknown. We have expressed a mammalian mitochondrial chaperonin 60 in Escherichia coli and purified the recombinant protein to homogeneity. Structural and biochemical analyses of this protein establish a single toroidal structure of seven subunits, in contrast to the homologous bacterial, fungal, and plant chaperonin 60s, which have double toroidal structures comprising two layers of seven identical subjects each. The recombinant mammalian chaperonin 60, together with the mammalian chaperonin 10 (but not with bacterial chaperonin 10), facilitates the formation of catalytically active ribulose-bisphosphate carboxylase from an unfolded state in the presence of K+ and MgATP. Analysis of the partial reactions involved in this in vitro reconstitution reveals that the single toroid of chaperonin 60 can form stable complexes with both unfolded or partially folded [35S]ribulose-bisphosphate carboxylase and mitochondrial (but not bacterial) chaperonin 10 in the presence of MgATP. We conclude that the minimal functional unit of chaperonin 60 is a single hepatmeric toroid.

Adenosine Triphosphate↗

PubMed1992

Activation of synovial fluid T lymphocytes by 60-kd heat-shock proteins in patients with inflammatory synovitis.

OBJECTIVE: Synovial fluid lymphocytes from patients with rheumatoid arthritis and with other forms of inflammatory synovitis demonstrate enhanced proliferative responses to Mycobacterium tuberculosis antigens, in particular, the 65-kd heat-shock protein. There is a high degree of homology between the human and the mycobacterial 60-kd family of heat-shock proteins. These studies were performed to determine if the enhanced response to the mycobacterial 65-kd heat-shock protein was due to cross-reactivity of an immune response generated against the human homolog. METHODS: These studies were performed by in vitro culture of isolated synovial fluid mononuclear cells with crude and purified antigens. RESULTS: The synovial fluid lymphocytes of a majority of patients with rheumatoid arthritis recognized the mycobacterial 65-kd heat-shock protein, as evidenced by T cell proliferation. In contrast, only 18% of all samples tested responded to a highly purified recombinant human 60-kd heat-shock protein. With only one exception, proliferative responses to the mycobacterial antigen were stronger than those to the human homolog. The proliferative responses generated against mycobacterial 65-kd heat-shock proteins from different sources were highly correlated. CONCLUSION: The findings suggest that the enhanced proliferative response to the mycobacterial 65-kd heat-shock protein noted in most patients with rheumatoid arthritis and other forms of inflammatory synovitis is not due to cross-reactivity of an immune response directed against the human heat-shock protein.

Antigens, Bacterial↗

PubMed1992

Mycobacteria and human heat shock protein-specific cytotoxic T lymphocytes in rheumatoid synovial inflammation.

OBJECTIVE: To study the cytotoxic capacity of mycobacteria-specific T lymphocyte lines and clones from sites of inflammation in patients with rheumatoid arthritis (RA). We also studied antigen specificity, surface phenotype, expression of T cell receptors (TCR), and HLA restriction. METHODS: Autologous macrophages (M phi) from the synovial membrane (SM), synovial fluid (SF), or peripheral blood (PB) were used as target cells in cytotoxicity assays. RESULTS: All SM and SF cell lines tested thus far have shown specific lysis of the autologous M phi from SM or PB that had been pulsed with BCG (bacillus Calmette-Guerin), but no cytotoxicity when the targets were pulsed with irrelevant antigens such as tetanus toxoid and Chlamydia. Both CD4+ and CD8+ cells were shown to be involved in the specific cytolysis. The majority of the cytotoxic T lymphocyte (CTL) lines were TCR alpha/beta + cells. However, both TCR alpha/beta + and TCR gamma/delta + clones (TCR delta 1+) from one RA patient showed antigen-specific lysis. Antigen-specific recognition by a number of CTL lines and clones generated from SF and SM was restricted by HLA-DR molecules. Two Mycobacterium bovis 65-kd heat shock protein (HSP)-specific TCR alpha/beta + SF T cell clones also lysed M phi that had been pulsed with a recombinant human 65-kd HSP. CONCLUSION: Joint inflammation and destruction might be partly attributable to a cross-reaction of mycobacteria-induced cytotoxic T cells with self HSP.

Antibodies, Monoclonal↗

PubMed1992

The development of monoclonal antibodies to the human mitochondrial 60-kd heat-shock protein, and their use in studying the expression of the protein in rheumatoid arthritis.

OBJECTIVE: To assess the claim that the human 60-kd heat-shock protein (HSP) is highly expressed in the joints of patients with rheumatoid arthritis (RA), but is not readily detected in normal tissues. METHODS: Monoclonal antibodies were raised against the human 60-kd mitochondrial heat-shock protein (P1 protein; hsp60), and their specificity was established. They were then applied to synovial tissue. RESULTS: HSP was expressed similarly in normal, osteoarthritic, and RA synovium. Low levels of hsp60 were detected in synovial fluid by immunoprecipitation. CONCLUSION: Minor differences in the distribution of hsp60 in synovium from RA joints were attributable to increased cellularity and to the disorganization of the tissue architecture.

Animals↗