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R S Gupta

Publications and source records attributed to R S Gupta.

At least 73 records · Page 4Linked to original sources
PubMed1997

Cell surface localization of the 60 kDa heat shock chaperonin protein (hsp60) in mammalian cells.

To investigate whether the 60-kDa heat shock chaperonin protein (hsp60) is present on the surface of mammalian cells, we used immunogold labeling of intact cells and backscattered electron imaging to image gold particles. Chinese hamster ovary cells and the human leukemic CD4-positive T-cell line CEM-SS on glass coverslips were labeled using affinity-purified monoclonal and polyclonal antibodies specific for hsp60 and 30 nm gold markers. Cells were imaged using the scanning mode of the conventional transmission electron microscope. Backscattered electron imaging provided definitive identification of the gold markers while secondary electron imaging gave information on surface architecture. Labeling intensity was 250-800 gold particles per cell in Chinese hamster ovary cells and 600-2000 in CEM-SS human lymphoblasts. The finding of hsp60 on the cell surface of mammalian cells may signify chaperone involvement in surface functions.

Animals↗
PubMed1997

The sequences of heat shock protein 40 (DnaJ) homologs provide evidence for a close evolutionary relationship between the Deinococcus-thermus group and cyanobacteria.

The genes encoding for heat shock protein 40 (Hsp40 or DnaJ) homologs were cloned and sequenced from the archaebacterium Halobacterium cutirubrum and the eubacterium Deinococcus proteolyticus to add to sequences from the gene banks. These genes were identified downstream of the Hsp70 (or DnaK) genes in genomic fragments spanning this region and, as in other prokaryotic species, Hsp70-Hsp40 genes are likely part of the same operon. The Hsp40 homolog from D. proteolyticus was found to be lacking a central 204 base pair region present in H. cutirubrum that encodes for the four cysteine-rich domains of the repeat consensus sequence CxxCxGxG (where x is any amino acid), present in most Hsp40 homologs. The available sequences from various archaebacteria, eubacteria, and eukaryotes show that the same deletion is also present in the homologs from Thermus aquaticus and two cyanobacteria, but in no other species tested. This unique deletion and the clustering of homologs from the Deinococcus-Thermus group and cyanobacterial species in the Hsp40 phylogenetic trees suggest a close evolutionary relationship between these groups as was also shown recently for Hsp70 sequences (R.S. Gupta et al., J Bacteriol 179:345-357, 1997). Sequence comparisons indicate that the Hsp40 homologs are not as conserved as the Hsp70 sequences. Phylogenetic analysis provides no reliable information concerning evolutionary relationship between prokaryotes and eukaryotes and their usefulness in this regard is limited. However, in phylogenetic trees based on Hsp40 sequences, the two archaebacterial homologs showed a polyphyletic branching within Gram-positive bacteria, similar to that seen with Hsp70 sequences.

Amino Acid Sequence↗
PubMed1997

Protein phylogenies and signature sequences: evolutionary relationships within prokaryotes and between prokaryotes and eukaryotes.

The evolutionary relationships within prokaryotes and between prokaryotes and eukaryotes is examined based on protein sequence data. Phylogenies and common signature sequences in some of the most conserved proteins point to a close evolutionary relationship between Archaebacteria and Gram-positive bacteria. The monophyletic nature and distinctness of the Archaebacterial domain is not supported by many of the phylogenies. Within Gram-negative bacteria, cyanobacteria are indicated as the deepest branching lineage, and a clade consisting of Archaebacteria, Gram-positive bacteria and cyanobacteria is supported by signature sequences in many proteins. However, the division within the prokaryotic species, viz. Archaebacteria<-->Gram-positive bacteria-->Cyanobacteria-->other groups of Gram-negative bacteria, is indicated to be not very rigid but, instead is an evolutionary continuum. It is expected that certain species will be found which represent intermediates in the above transitions. By contrast to the evolutionary relationships within prokaryotes, the eukaryotic species, which are structurally very different, appear to have originated by a very different mechanism. Protein phylogenies and signature sequences provide evidence that the eukaryotic nuclear genome is a chimera which has received major contributions from both an Archaebacterium and a Gram-negative bacterium. To explain these observations, it is suggested that the ancestral eukaryotic cell arose by a symbiotic fusion event between the above parents and that this fusion event led to the origin of both nucleus and endoplasmic reticulum. The monophyletic nature of all extant eukaryotic species further suggests that a 'successful primary fusion' between the prokaryotic species that gave rise to the ancestral eukaryotic cell took place only once in the history of this planet.

Amino Acid Sequence↗
PubMed1997

Phylogenetic analysis of mycoplasmas based on Hsp70 sequences: cloning of the dnaK (hsp70) gene region of Mycoplasma capricolum.

A 5.0-kb region containing the hsp70 (dnaK) gene was cloned from Mycoplasma capricolum and sequenced. In addition to the hsp70 gene, this sequence region also contained the complete sequences for the grpE and orfA genes and partial sequences for the clpB and dnaJ genes. The order of the above gene sequences in the cloned fragment was found to be clpB-orfA-grpE-hsp70-dnaJ, which is similar to the order seen in various other gram-positive groups of bacteria. The Hsp70 homologs from two mycoplasma species, Mycoplasma capricolum and Mycoplasma genitalium, contain a number of sequence signatures, including the absence of a large insert in the N-terminal quadrant, that are characteristics of the homologs from gram-positive bacteria and archaebacteria. A detailed phylogenetic analysis based on Hsp70 sequences was also performed. In neighbor-joining and parsimony trees based on Hsp70 sequences, both mycoplasma species branched with the low-G + C-content gram-positive group of bacteria (e.g., Lactobacillus and Erysipelothrix species) in 87% and 96% of the bootstrap replicates, respectively, indicating their close evolutionary relationship to this group. The phylogenetic trees based on Hsp70 sequences show a polyphyletic branching of archaebacteria with the gram-postive species, which is statistically strongly favored.

Amino Acid Sequence↗
PubMed1997

Sequencing of heat shock protein 70 (DnaK) homologs from Deinococcus proteolyticus and Thermomicrobium roseum and their integration in a protein-based phylogeny of prokaryotes.

The 70-kDa heat shock protein (hsp70) sequences define one of the most conserved proteins known to date. The hsp70 genes from Deinococcus proteolyticus and Thermomicrobium roseum, which were chosen as representatives of two of the most deeply branching divisions in the 16S rRNA trees, were cloned and sequenced. hsp70 from both these species as well as Thermus aquaticus contained a large insert in the N-terminal quadrant, which has been observed before as a unique characteristic of gram-negative eubacteria and eukaryotes and is not found in any gram-positive bacteria or archaebacteria. Phylogenetic analysis of hsp70 sequences shows that all of the gram-negative eubacterial species examined to date (which includes members from the genera Deinococcus and Thermus, green nonsulfur bacteria, cyanobacteria, chlamydiae, spirochetes, and alpha-, beta-, and gamma-subdivisions of proteobacteria) form a monophyletic group (excluding eukaryotic homologs which are derived from this group via endosybitic means) strongly supported by the bootstrap scores. A closer affinity of the Deinococcus and Thermus species to the cyanobacteria than to the other available gram-negative sequences is also observed in the present work. In the hsp7O trees, D. proteolyticus and T. aquaticus were found to be the most deeply branching species within the gram-negative eubacteria. The hsp70 homologs from gram-positive bacteria branched separately from gram-negative bacteria and exhibited a closer relationship to and shared sequence signatures with the archaebacteria. A polyphyletic branching of archaebacteria within gram-positive bacteria is strongly favored by different phylogenetic methods. These observations differ from the rRNA-based phylogenies where both gram-negative and gram-positive species are indicated to be polyphyletic. While it remains unclear whether parts of the genome may have variant evolutionary histories, these results call into question the general validity of the currently favored three-domain dogma.

Amino Acid Sequence↗
PubMed1997

Epidemiology of endemic viral hepatitis in an urban area of India: a retrospective community study in Alwar.

In a community study during a reference period of 1 year, 192 cases of jaundice were detected in an urban population of 69,440 in Alwar, Rajasthan. Detected by paramedics and confirmed by physicians, these cases gave an annual incidence of 2.76 (95% CI: 2.37-3.15) per 1000 population. At least one of these patients died, giving a case fatality ratio of 0.6%. The jaundice cases occurred in all areas investigated, and affected all socioeconomic strata. About 94% of the affected families had only single cases. Although cases occurred throughout the year, more than 59% occurred during June-September, which are the summer and monsoon months. The incidence was highest (5.23 per 1000) among under-5-year-olds and declined progressively and significantly thereafter. Males had a higher incidence than females at all ages; the differences were not significant. Blood samples from 56 cases who had jaundice in the last 3 months of the reference period were tested for markers of viral hepatitis. Of these, 18 (32.1%), 1 (1.8%), 0, 2 (3.6%), and 4 (7.1%) were found to have hepatitis A, B, C, D and E, respectively. The etiology of the remaining 31 cases (55%) could not be established; previously, they would have been included in the NANB (non-A, non-B) category, inflating its proportion. Hepatitis A (HA) was the predominant type; being comparatively mild, it is perhaps underrepresented in hospital-based data. Many HA cases were in adults, which may be the beginning of an age shift of HA to the right owing to improvements in living standards of the study population. Five cases were carriers of hepatitis B virus (HBV), indicating the importance of HBV infection in India as well. Finally, the study found the annual incidence of laboratory-supported cases of viral hepatitis to be 1.24 (95% CI: 0.98-1.5) per 1000 population, which suggests that it is a major public health problem in India.

Adolescent↗
PubMed1996

Cloning and characterization of cDNA for adenosine kinase from mammalian (Chinese hamster, mouse, human and rat) species. High frequency mutants of Chinese hamster ovary cells involve structural alterations in the gene.

The enzyme adenosine kinase constitutes the major purine nucleoside phosphorylating activity in mammalian cells. In view of its central role in adenosine metabolism, which is an important physiological regulator, an understanding of the primary structure of adenosine kinase is of much interest. Using microsequence information from peptides derived from purified Syrian hamster liver enzyme, we have succeeded in isolating full length cDNA clones encoding adenosine kinase from Chinese hamster ovary cells and mouse 3T3 cells. The open reading frames in these clones consist of 334 and 335 amino acids and encode proteins of molecular masses 37364 Da and 37489 Da, respectively. In addition, the coding and upstream sequences for adenosine kinase from human (HeLa cells) and rat liver have also been cloned and sequenced. Transfection of an adenosine-kinase-deficient mutant (selected for resistance to the adenosine analog toyocamycin) of Chinese hamster ovary cells with a plasmid containing the cloned adenosine kinase cDNA, leads to regaining of adenosine kinase activity in the transformed cell. The adenosine kinase transformants also simultaneously lost their toyocamycin resistance and became similarly sensitive to the analog as the parental wild-type Chinese hamster ovary cells. The cloned adenosine kinase cDNA was also used to examine structural changes in mutants affected in adenosine kinase. In Chinese hamster ovary cells, one type of mutant that lacks adenosine kinase activity and displays high degree of resistance to various adenosine analogs, is obtained at an unusually high spontaneous frequency (10(-4)-10(-3)). Results of Southern and northern-blot analysis provide evidence that this group of mutants involves gross structural alterations affecting the adenosine kinase gene. Such structural alterations are not observed in another type of mutant which exhibits increased resistance only to C-adenosine analogs. Sequence similarity searches indicate that several of the bacterial and yeast sugar kinases (ribokinase, fructokinase and inosine-guanosine kinase) exhibit limited but significant similarity to the mammalian adenosine kinase. The sequence similarity data support the possibility that adenosine kinase shares a common evolutionary ancestor with these protein sequences.

Adenosine Kinase↗
PubMed1996

Immunoelectron microscopic localization of the 60-kDa heat shock chaperonin protein (Hsp60) in mammalian cells.

The subcellular distribution of the 60-kDa heat shock protein (Hsp60) was examined in a variety of mammalian cells and tissues, including Chinese hamster ovary cells, human fibroblasts, B-SC-1 kidney cells, Daudi Burkitt's lymphoma cells, and rat liver, by immunogold electron microscopy employing six different monoclonal and polyclonal antibodies that are specific for Hsp60. In cryosections or LR Gold sections of different cultured cells, intense labeling of mitochondria was obtained, typically 200-500 gold particles per mitochondrion and accounting for 80-85% of the total gold particles. In addition, however, in all cell types and using all of the antibodies, about 15-20% of the labeling due to Hsp60 was seen at discrete extramitochondrial sites. Such sites included those in close proximity to mitochondrial outer membranes, foci on endoplasmic reticulum, on the cell surface, and in unidentified vesicles. In cryosections of rat liver, specific labeling due to Hsp60 antibodies was also observed within peroxisomes. Labeling of all cellular components by these antibodies could be prevented by preadsorption with purified recombinant mitochondrial Hsp60 indicating that the labeling is specific for Hsp60. Biotin labeling of cell surface proteins results in biotinylation of Hsp60 as analyzed by immunoprecipitation and Western blots, providing further evidence for Hsp60 presence on the plasma membrane. Immunoprecipitation experiments with Hsp60 antibodies show that under normal conditions no detectable precursor Hsp60 protein is present in cells. However, in cells treated with the potassium ionophore nonactin, which blocks mitochondrial import, only the precursor form of Hsp60 accumulates, providing evidence that at least partial mitochondrial import of Hsp60 is necessary for its maturation. These results also provide evidence that no other 60-kDa protein other than mitochondrial Hsp60 is recognized by the antibodies used for electron microscopy. These findings raise interesting questions concerning the possible role of Hsp60 at extramitochondrial sites.

Animals↗
PubMed1996

Regression of arteriosclerotic lesions induced by immunization with heat shock protein 65-containing material in normocholesterolemic, but not hypercholesterolemic, rabbits.

Previous studies in our laboratory have shown that arteriosclerotic changes can be induced in normocholesterolemic rabbits by immunization with mycobacterial heat shock protein 65 (hsp 65). To investigate the possible regression of such vascular lesions, 63 male New Zealand White rabbits were treated either by triple immunization with fortified Freund's complete adjuvant containing 5 mg/ml Mycobacterium tuberculosis, a hsp 65-rich material, by administration of a 0.2% cholesterol-rich diet only or by a combination of both immunization and cholesterol-rich diet. Sixteen weeks after the first immunization, half of the animals of each group were sacrificed, and as expected arteriosclerotic lesions in the intima of the aortic arch were found in 8 of 10 immunized animals. The remaining animals were sacrificed 16 weeks thereafter, having been maintained on a normal, non-cholesterol-enriched diet from week 16 to 32. Only 3 of 10 rabbits immunized showed moderate lesions in their aortae 32 weeks after the first immunization. On the other hand, atherosclerotic lesions induced by cholesterol-rich diet, or by immunization plus cholesterol-rich diet, showed no significant regression between 16 and 32 weeks. In conclusion, the early inflammatory stages of arteriosclerotic lesions induced by immunization with hsp 65 can regress in the absence of additional risk factors for atherosclerosis, such as a cholesterol rich diet.

Animals↗
PubMed1996

Adenosine-AMP exchange activity is an integral part of the mammalian adenosine kinase.

Purified adenosine kinase (AK) from Syrian hamster and bovine liver was examined for the presence of adenosine (Ad)-AMP exchange activity. The enzyme from both sources, in addition to catalyzing the conventional ATP-dependent phosphorylation of adenosine, supported an Ad-AMP exchange reaction that required ADP. Under optimal conditions both these reactions were found to occur at comparable rates. Several observations strongly indicate that the Ad-AMP exchange activity is an integral part of AK and it is likely associated with its catalytic mechanism. These observations include: (i) Both AK and Ad-AMP exchange activities show a nearly complete dependence upon the presence of pentavalent ions such as phosphate, arsenate or vanadate for catalysis; (ii) Both activities show similar heat-lability and inhibition by 5-iodotubercidin (5-ITu); (iii) In a Chinese hamster cell mutant resistant to adenosine analogs that lacked AK activity, the Ad-AMP activity was also found to be absent. The presence of a phosphoryl-enzyme intermediate, or any exchange between free 32Pi and any of the reactants, however, was not detected under the reaction conditions. Some implications of these observations regarding the catalytic mechanism of AK are discussed.

Adenosine↗
PubMed1996

Autoantibodies to human heat shock protein (hsp)60 may be induced by Escherichia coli groEL.

The 65-kD hsp from Mycobacterium tuberculosis has been reported to induce an autopathogenic subset of T cells in at least two animal models of autoimmune disease. Reports of increased expression of human hsp60 in the inflamed synovial tissue of rheumatoid arthritis (RA) patients, increased proliferation of RA synovial fluid T cells to mycobacterial hsp65, and increased levels of anti-mycobacterial hsp65 antibody in synovial fluid, have suggested that the highly homologous human (hu) hsp60 may be recognized as an autoantigen iin RA patients. In the present study, we have examined by ELISA the serum IgG antibody levels to mycobacterial hsp65 and hu hsp60, as well as to the Escherichia coli hsp60, groEL, in patients with RA, systemic lupus erythematosus (SLE), Reiter's syndrome, active tuberculosis, and normal controls. In all these groups, the levels of anti-groEL and anti-hu hsp60 were significantly higher than the anti-mycobacterial hsp65. Anti-hu hsp60 was positively correlated with anti-groEL, but not with anti-mycobacterial hsp65. Anti-hu hsp60 was competitively inhibited by either soluble groEL or hu hsp60, but little or none by mycobacterial hsp65. Reiter's sera were found to have somewhat higher levels of anti-groEL and anti-hu hsp60 than did normal controls. We conclude that IgG anti-hu hsp60 autoantibodies arise primarily as a consequence of the humoral immune response to E. coli groEL through the recognition of cross-reactive epitopes.

Antibodies, Bacterial↗
PubMed1996

Identification of endoplasmic reticulum in the primitive eukaryote Giardia lamblia using cryoelectron microscopy and antibody to Bip.

Giardia lamblia trophozoites contain a complex endomembrane system as demonstrated by fluorescence and cryoelectron microscopy. The endomembrane system was weakly detected in live cells using the fluorescent membrane dye 3,3'-dihexyloxacarbocyanine iodide. The definitive identification of endoplasmic reticulum required the development of a molecular label. We expressed Giardial Bip in Escherichia coli and raised a polyclonal antibody to the purified protein. In western blots, the antibody was specific for Giardial Bip and did not react with human, monkey and rodent homologs. By immunofluorescence microscopy in methanol fixed cells the antibody visualized tubular structures and other subcellular components that required characterization by electron microscopy. Using cryotechniques we directly demonstrate the presence of a complex endomembrane system at the ultrastructural level. In conjunction with Bip immunogold labeling of cryosections we identify: (1) endoplasmic reticulum cisternae and tubules; (2) stacked perinuclear membranes; and (3) Bip presence in the nuclear envelope. Both the endoplasmic reticulum and nuclear envelope were found either with or without a cleft region suggesting each may contain common specialized sub-regions. In stacked perinuclear membranes, which may represent either multilamellar endoplasmic reticulum or a Golgi apparatus, Bip labeling was restricted to peripheral layers, also suggesting specialized sub-regions. Labeled endomembrane systems could be observed associated with microtubule structures, including axonemes and the adhesive disk. The presence of an extensive endomembrane system in Giardia lamblia, which represents one of the earliest diverging eukaryotic species, supports the view that both the nucleus and endomembrane system co-evolved in a common ancestor of eukaryotic cells.

Animals↗
PubMed1996

Prokaryotic homolog of tubulin? Consideration of FtsZ and glyceraldehyde 3-phosphate dehydrogenase as probable candidates.

The bacterial FtsZ protein has recently been suggested as a probable prokaryotic homolog of the tubulin family of proteins (Cell 80, 1995, 367-370). We have compared the sequence similarity of tubulins to FtsZ and another protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Both these proteins exhibited similar levels of sequence identity to the tubulins, which in a few cases was indicated to be significant. We report that incubation of the GAPDH in microtubule assembly buffer causes its polymerization into filamentous structures. In eukaryotic cells, GAPDH is known to be associated with cytoskeletal structures and it binds specifically to both tubulins and colchicine. The latter is a distinctive characteristic of the tubulin family of proteins. These observations indicate that similar to the FtsZ proteins, GAPDH also exhibits a number of intriguing similarities to the tubulins. Whether any of these proteins truly represent the prokaryotic homolog of tubulin, however, is unclear at present.

Amino Acid Sequence↗
PubMed1996

The origin of the eukaryotic cell.

Molecular sequence data are beginning to provide important insights into the evolutionary origin of eukaryotic cells. Global phylogenies of numerous protein sequences indicate that the eukaryotic cell nucleus is a chimera, which has received major contributions from both a Gram-negative eubacterium and an archaebacterium. Recent studies also indicate that the formation of the nuclear envelope and the endoplasmic reticulum was accompanied by duplication of genes for the molecular chaperone proteins (e.g. hsp70, hsp90), which facilitate protein transport across membranes. Based on these observations, it is suggested that the ancestral eukaryotic cell arose by a unique endosymbiotic event involving engulfment of an eocyte archaebacterium by a Gram-negative eubacterial host.

Amino Acid Sequence↗
PubMed1996

Pentavalent ions dependency of mammalian adenosine kinase.

The enzyme adenosine kinase (AK) has been purified to homogeneity from Syrian hamster and bovine livers. The purified enzymes from both these sources have a Mr of approximately 38 kDa, as determined by gel-filtration and SDS-polyacrylamide gel electrophoresis. A novel characteristic of AK observed here is that its catalytic activity shows a nearly complete dependence upon the presence of pentavalent ions such as phosphate (P(i)), arsenate or vanadate. Maximal AK activity was observed in the presence of either 2-3 mM P(i), or 5-10 mM arsenate, or 10-20 mM vanadate. A low basal level of AK activity (1-5% of maximal) observed in the absence of these ions is attributed to P(i) contamination in the adenine nucleotides preparations. The presence of P(i) had no effect on the K m for ATP (0.4 mM), but it markedly increased the affinity of the enzyme for adenosine. The K(m) of AK for adenosine in presence of 0, 0.1 mM and 2 mM P(i) was estimated to be 1.4 mu M, 0.77 mu M and 0.095 mu M, respectively. Free P(i) showed no exchange with any of the reactants during the assay conditions, and its presence had no effect on the thermostability of the enzyme. These observations suggest that the pentavalent ions such as phosphate may be playing an important role in the enzyme's catalytic mechanism by facilitating either binding of adenosine to the enzyme or in the formation of an enzyme-ATP-adenosine complex.

Adenosine Kinase↗
PubMed1995

Purification of recombinant human Hsp60: use of a GroEL-free preparation to assess autoimmunity in rheumatoid arthritis.

A 65 kDa mycobacterial heat shock protein has been implicated in the development or perpetuation of the inflammatory diseases rheumatoid arthritis (RA) and insulin dependent diabetes mellitus (IDDM). An homology of the mycobacterial hsp65 with human hsp60 (HuHsp60) has been thought to constitute a cross reactive autoimmunizing pathogenetic potential. Study of this cross reactivity with recombinant reagents has been complicated by the fact that recombinant HuHsp60 might be contaminated by the E. coli homologue of HuHsp60, groEL. GroEL and HuHsp60 are very similar in isoelectric point and molecular weight and therefore difficult to separate by classical physicochemical means. Therefore, the HuHsp60 gene was subcloned into the vector, pRSET-B, which resulted in recombinant HuHsp60 protein fused to a 4.5 kDa peptide containing a polyhistidine hexamer. Metal ion affinity for the polyhistidine allowed the rapid and efficient chromatographic separation of the HuHsp60 from groEI. Rabbit antisera were developed to linear peptide epitopes unique to either HuHsp60 or groEL and utilized to discriminate between these proteins during their separation. With the newly prepared HuHsp60 we show that the amount of anti-HuHsp60 autoantibody in both RA and normal sera was too great to be accounted for by cross reacting anti-MbHsp65.

Amino Acid Sequence↗
PubMed1995

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

A biochemical investigation was carried out on the relative presence of some enzymes of the Krebs cycle and of the associated energy metabolism in various fractions (namely, cyst wall, cyst fluid and zoites) of sarcocysts of Sarcocystis fusiformis from the oesophageal muscles of naturally infected Indian water buffalo (Bubalus bubalis). Except for malate dehydrogenase, the activities of aconitase, isocitrate dehydrogenase, succinate dehydrogenase and fumarase were beyond detectable limits, pointing to a non-functional Krebs cycle in the cysts of this parasite. The activities of adenosine triphosphatase and cytochromes were lowest in cyst fluid and were maximally depicted by cyst wall and zoites.

Aconitate Hydratase↗