Biomedical subjects

R S Gupta

Publications and source records attributed to R S Gupta.

At least 145 records · Page 8Linked to original sources

PubMed1990

Cross-resistance and biochemical characteristics of second-step mutants of HeLa cells resistant to cardiac glycosides.

From ouabain-resistant (OuaR) mutants of HeLa cells which do not show any cross resistance to the digoxin analog SC4453, stable second-step mutants resistant to either SC4453 or those exhibiting increased resistance to digoxin have been isolated. The mutants obtained exhibited highly specific cross resistance towards different cardiac glycosides (CGs) and, based on their cross-resistance patterns, contained more than one type of genetic lesion. Biochemical studies with these mutants showed that cellular uptake of 86Rb was inhibited by specific CGs to which they showed increased resistance. The mutants showed reduced binding of [3H]ouabain and [3H]digoxin in comparison with the parental OuaR cells and about 50-60% of the Na+, K(+)-ATPase activity in the mutant cell extract was highly resistant to inhibition by ouabain and digoxin. In contrast to the above changes, these mutants showed no evidence of amplification, enhanced transcription, or gross alterations in the genes for the alpha or beta subunits of Na+, K(+)-ATPase. These observations indicated that these mutants involved a second-specific alteration in Na+, K(+)-ATPase. In contrast to these mutants, Chinese hamster ovary cells, which naturally exhibit comparable levels of resistance to CGs, showed no significant binding of either [3H]ouabain or [3H]digoxin and all of their Na+, K(+)-ATPase activity was resistant to inhibition by CGs.

Animals↗

PubMed1990

Microtubules, mitochondria, and molecular chaperones: a new hypothesis for in vivo assembly of microtubules.

In Chinese hamster ovary cells, a number of independent mutants selected for resistance to antimitotic drugs have been found to be specifically altered in two major cellular proteins, designated P1 (relative mass (Mr) approximately 60-63 kilodaltons (kDa] and P2 (Mr approximately 69-70 kDa), which appeared microtubule related by a number of genetic and biochemical criteria. Antibodies to P1 have been found to bind specifically to mitochondria that showed specific association with microtubules in interphase cells. Biochemical and cDNA sequence studies on P1 showed that this protein, which is localized in the matrix compartment, is the mammalian homolog of the highly conserved chaperonin family of proteins (other members include the GroEL protein of Escherichia coli, the 60-kDa heat-shock protein of yeast, and the rubisco subunit binding protein of plant chloroplasts). The chaperonin proteins in various systems play a transient but essential molecular chaperone role in the proper folding of polypeptide chains and their assembly into oligomeric protein complexes. Our studies on P2 protein established that it corresponds to the constitutive form of the major 70-kDa heat-shock protein of mammalian cells (i.e., hsc70), which also acts as a molecular chaperone in the intracellular transport of nascent proteins to organelles such as mitochondria and endoplasmic reticulum. To account for the above, as well as a number of other observations (e.g., binding of fluorescent-labeled antimitotic drugs to mitochondria, association of tubulin with mitochondria as well as other membranes, and high affinity binding of antimitotic drugs to free tubulin but not to assembled microtubules), a new model for the in vivo assembly of interphase microtubules is proposed. The model ascribes a central role to the mitochondrially localized chaperonin (i.e., P1) protein in the intracellular formation of tubulin dimers and in their addition to the growth sites in microtubules. The proposed model also explains a number of other observations related to microtubule assembly in the literature.

Amino Acid Sequence↗

PubMed1990

Sequence and structural homology between a mouse T-complex protein TCP-1 and the 'chaperonin' family of bacterial (GroEL, 60-65 kDa heat shock antigen) and eukaryotic proteins.

A mammalian cytoplasmic protein TCP-1, encoded by a gene within the mouse t-complex, has been found to exhibit highly significant (p much less than 0.00001) sequence homology to the 'chaperonin' family of bacterial and eukaryotic proteins (viz. groEL protein of E. coli, rubisco subunit binding protein of plant chloroplasts, yeast hsp58 and mammalian P1 proteins and 60-65 kDa mycobacterial antigen). With the introduction of few gaps, the amino acid sequence of TCP-1 shows between 60-63% similarity (17-20% identical residues and 42-45% conserved substitutions) throughout its length to various chaperonin proteins, indicating a common evolutionary origin. The sequence data also suggest that in contrast to the endosymbiotic origin of mitochondrial and chloroplast chaperonins, the cytoplasmic TCP-1 may have directly descended from the common universal ancestor via eukaryotic lineage. The observed similarity between TCP-1 and the 60-65 kDa bacterial 'common antigen' is also of importance from the viewpoint of immune/autoimmune response.

Amino Acid Sequence↗

PubMed1990

Effects of antimitotic and antimitochondrial agents on the cellular distribution of microtubules and mitochondria.

Using antibodies to a mitochondrial molecular chaperone class of protein, which is specifically altered in mutants resistant to microtubule (MT) inhibitors, the effect of a number of MT and mitochondrial inhibitors on the cellular distribution of mitochondria and various cytoskeletal filaments was examined. Treatment of Chinese hamster ovary (CHO) or chicken embryo fibroblast (CEF) cells with the MT inhibitors podophyllotoxin, colchicine, nocodazole and vinblastine caused depolymerization of cellular MTs, but had no significant effect on the distribution patterns of mitochondria. This is attributed to the association of mitochondria with intermediate filaments (IFs) which are not destroyed under these conditions. In contrast to MT inhibitors, treatment of CEFs with the potassium ionophores nonactin and valinomycin caused aggregation of mitochondria towards the perinuclear region of the cells, without having any apparent effect on cellular MTs. This observation suggests that mitochondrial membrane potential, which is abolished by these drugs, play a role in the cellular distribution of mitochondria. In cells recovering from the effects of MT inhibitors, mitochondria have been found to surround the MT organizing complexes and upon complete recovery a realignment of MTs with mitochondria takes place. These observations suggest that MT growth in cells does not occur in a completely random manner but that mitochondria may play some role in their directional growth.

Animals↗

PubMed1989

A novel ubiquitous protein 'chaperonin' supports the endosymbiotic origin of mitochondrion and plant chloroplast.

The deduced amino acid sequences for a major mitochondrial protein (designated P1, related to the 'chaperonin' family of proteins) from human and Chinese hamster cells show extensive similarity (greater than 60% identity observed over the entire length) with a related protein present in evolutionarily as divergent organisms as Escherichia coli, Coxiella burnetii, Mycobacterium species, cyanobacteria as well as in yeast mitochondria and higher plant chloroplasts. Of the different groups of bacteria for which sequence data is available, maximum similarity of the mammalian/yeast P1 protein is observed with the corresponding protein from purple bacteria (especially C. burnetii) while the protein from plant chloroplasts exhibited highest similarity with the corresponding protein from cyanobacteria. The sequence data for this protein thus support the contention that the endosymbiont that gave rise to mitochondrion was a member of purple bacteria, while plant chloroplast originated from a member of the cyanobacterial lineage.

Amino Acid Sequence↗

PubMed1989

Molecular cloning of a Chinese hamster mitochondrial protein related to the "chaperonin" family of bacterial and plant proteins.

The complete cDNA sequence of a mitochondrial protein from Chinese hamster ovary cells, designated P1, which was originally identified as a microtubule-related protein (Gupta, R.S., Ho, T.K.W., Moffat, M.R.K., and Gupta, R. (1982) J. Biol. Chem. 257, 1071-1078), has been determined. The P1 cDNA encodes a protein of 60,983 Da including a 26-amino acid putative mitochondrial targeting sequence at its N-terminal end. The deduced amino acid sequence of Chinese hamster P1 shows 97% identity to the human P1 protein. Most interestingly, the amino acid sequences of mammalian P1 proteins show extensive sequence homology (42-60% identical residues and an additional 15-25% conservative replacements) to the "chaperonin" family of bacterial, yeast, and plant proteins (viz. groEL protein of Escherichia coli, hsp 60 protein of yeast, and ribulose-1,5-bisphosphate carboxylase subunit binding protein of plant chloroplasts) and to the 60-65-kDa major antigenic protein of mycobacteria and Coxiella burnetii. The homology between mammalian P1 and other proteins begins after the putative mitochondrial presequence and extends up to the C-terminal end. Furthermore, similar to the chaperonin family of proteins, P1 appears to exist in cells as a homooligomeric complex of seven subunits and shows ATPase activity. These observations strongly indicate that P1 protein is a member of the chaperonin family and that it may be involved in a similar function in mammalian cells.

Adenosine Triphosphatases↗

PubMed1989

Development of a specific assay for cardiac glycoside-like compounds based on cross-resistance of human cell mutants.

The cross-resistance patterns of single- and two-step mutants of HeLa cells resistant to SC4453 (a digoxin analog) and digoxin, which involve specific alteration in Na+, K+-ATPase towards numerous other compounds, have been examined. The mutants exhibited increased resistance to all of the steroidal compounds known to elicit a digitalis-like positive inotropic response (viz. various cardiac glycosides and their genins, erythrophleum alkaloid cassaine), but they showed no cross-resistance to any of a large number of other compounds which do not show cardiac glycoside (CG)-like biological activity. Based on the above characteristics of the mutants, a new cross-resistance assay for identifying compounds that show CG-like activity has been developed. In this assay, a sample is considered to possess CG-like activity if, in comparison to the parental HeLa cells, the CGR mutants exhibit increased resistance to it. From the known D10 value (drug concentration which reduces cloning efficiency of cells to 10%) of the drug for HeLa cells and the sample dilution necessary to produce equivalent cytotoxicity, the concentration of CGs in a given sample can be estimated. In blind studies the assay correctly identified all of the samples containing CGs; none of the other samples which lacked such activity tested positive. In the blind studies the assay also provided a good estimate of the concentration of CGs (+/- 50% of the actual concentration) that was not affected by the presence of either serum components or a 20-fold excess of various steroidal compounds known to interfere in other assays. In view of the high specificity of the present assay for CG-like compounds, it should prove very useful in establishing/characterizing the presence of such activity in various biological (namely endogenous digitalis-like substances) and other samples.

Cardiac Glycosides↗

PubMed1989

Further evidence that the narrow 1H magnetic resonance signals from malignant cells do not arise from intracellular lipid droplets.

1H magnetic resonance (MR) spectroscopy of intact viable malignant cells yields high resolution spectra from lipid. In previous studies we have provided evidence that these signals are generated by neutral lipid located in the plasma membrane in unique domains. We show that intracellular lipid droplets do not contribute to the MR signal. Two malignant Chinese hamster ovary cell lines, EOT and its parental line WT were studied. The EOT cells have a more highly resolved lipid spectrum than the WT, a result which correlates with slightly increased levels of triglyceride in highly purified plasma membranes. The intracellular lipid droplets of both lines were quantified using both fluorescence and electron microscopy but no significant differences were observed. Together these results provide evidence that narrow 1H MR signals from malignant cells arise from neutral lipid in the plasma membrane, rather than from intracellular lipid droplets.

Animals↗

PubMed1989

Mechanism of action of antimitotic drugs: a new hypothesis based on the role of cellular calcium.

The antimitotic drugs such as colchicine, podophyllotoxin, etc. are currently believed to exert their cytotoxic and antimitotic effects due to binding of the drug-tubulin complex to the growing ends of microtubules (MTs), leading to an "end-capping or poisoning" effect. However, to account for a number of apparently puzzling observations regarding antimitotic drugs (which cannot be readily explained by the current model) and the mitotic process, a new hypothesis regarding the mechanism of action of antimitotic drugs is proposed. The key observations in this context are as follows: (i) The antimitotic drugs bind specifically to free tubulin. (ii) Cell growth by these drugs is specifically blocked in metaphase, and interphase microtubules do not seem to play any role in the drugs' cytotoxic or antimitotic effects. (iii) Tubulin is specifically associated with a number of membranous organelles (viz. mitochondria, plasma membranes, endoplasmic reticulum) which are responsible for intracellular Ca+2 homeostasis. (iv) Fluorescent derivatives of antimitotic drugs also bind to the above membranous organelles and not to MTs. (v) Ca+2 plays a central role in the control of MT assembly/disassembly in vivo and a Ca+2 pulse is necessary for the metaphase to anaphase transition. (vi) Cellular mutants which exhibit specific resistance to various antimitotic drugs are altered in either tubulin(s) or mitochondrial matrix proteins. To account for these observations, it is suggested that free tubulin present in the above membranous organelles serves as the cellular receptor for these drugs and this binding interferes with the Ca+2 regulatory/signalling mechanism essential for anaphase chromosome movement. The effect of these drugs on interphase MTs appears to be a secondary consequence of this alteration in Ca+2 regulation. The observed changes in mitochondrial matrix proteins in many of the mutants resistant to antimitotic drugs further indicate that mitochondria should play an important role in Ca+2 homeostasis, as it relates to mitosis. The possible mechanisms by which these drugs may interfere with the Ca+2 regulation and some implications of this hypothesis are discussed.

Animals↗

PubMed1989

Testicular cell population dynamics following palmitine hydroxide treatment in male dogs.

Palmitine hydroxide isolated from the roots of Berberis chitria administered orally to dogs 30 mg/kg per day for 60 days brings about a consistent impairment of primary and secondary spermatocytes and elongated spermatids (Stages IV-VIII). The primary and secondary spermatocytes were reduced by 60 and 68%, respectively, and the elongated spermatids were decreased by 58%. The number of spermatogonia and Sertoli cells remained unaltered. The production of immature and mature Leydig cells decreased by 66% and 27%, respectively. Protein, sialic acid and glycogen content and acid phosphatase activity of testes and epididymides were lowered to varying extents. Testicular cholesterol was elevated significantly. Weights of the testes and epididymides were significantly reduced. The antispermatogenic action of palmitine hydroxide may be mediated by disturbances in Leydig cell function.

Animals↗

PubMed1989

Immunological characterization of a human homolog of the 65-kilodalton mycobacterial antigen.

A human mitochondrial protein, designated P1 (63 kilodaltons [kDa], shows extensive sequence homology (47% identical residues and an additional approximately 20% conserved changes) to the 65-kDa mycobacterial antigen. To understand the relationship of these proteins, the cross-reactivity of several monoclonal antibodies directed against the 65-kDa Mycobacterium leprae antigen towards human, Chinese hamster, chicken, and bacterial cells has been examined. A number of antibodies (Y1-2, ML 30-A2, and F47-9-1) were found to cross-react with a 63-kDa antigen in vertebrate cell extracts and stained mitochondria in immunofluorescence studies. Some of these antibodies also reacted with a P1-beta-galactosidase fusion protein in recombinant Escherichia coli cells, expressing part of the human P1 protein. These results provide strong evidence that P1 is the mammalian homolog of the 65-kDa antigen. The human P1 protein also shows significant similarity (P less than 0.001) to a number of other bacterial and viral proteins including the pol polyprotein of human immunodeficiency viruses and the penicillin-binding protein of Neisseria gonorrhoeae. The observed similarity between human P1 protein and the major antigenic proteins of pathogenic organisms (e.g., 60- to 65-kDa mycobacterial antigen) suggests its possible involvement in autoimmune diseases (e.g., rheumatoid arthritis) by antigenic mimicry.

Amino Acid Sequence↗

PubMed1989

Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen.

The complete cDNA for a human mitochondrial protein designated P1, which was previously identified as a microtubule-related protein, has been cloned and sequenced. The deduced amino acid sequence of P1 shows strong homology (40 to 50% identical residues and an additional 20% conservative replacements) to the 65-kilodalton major antigen of mycobacteria, to the GroEL protein of Escherichia coli, and to the ribulose 1,5-bisphosphate carboxylase-oxygenase (rubisco) subunit binding protein of plant chloroplasts. Similar to the case with the latter two proteins, which have been shown to act as chaperonins in the posttranslational assembly of oligomeric protein structures, it is suggested that P1 may play a similar role in mammalian cells. The observed high degree of homology between human P1 and mycobacterial antigen also suggests the possible involvement of this protein in certain autoimmune diseases.

Amino Acid Sequence↗

PubMed1989

Experimental model of staphylococcal osteomyelitis in dogs.

Osteomyelitis was induced in 45 male dogs by inoculating hemolytic strain of Staphylococcus aureus alone into the tibial marrow cavity. Clinical, radiological and bacteriological studies were conducted to evaluate the progress of disease up to 15 weeks. Clinical signs consisted of localized soft tissue swelling, pain, pyrexia and lameness which later developed an open wound with purulent exudation. Predominant radiographic features were extensive periosteal reaction, cortical lysis, new bone formation, frequent development of sequestrum and formation of localized abscess pockets in advanced cases. Staphylococci were recovered from the tibial marrow cavity for as long as 15 weeks after onset of the infection.

Animals↗

PubMed1988

Camptothecin-resistant mutants of Chinese hamster ovary cells containing a resistant form of topoisomerase I.

In Chinese hamster ovary cells, stable mutants that exhibit 250- to 350-fold resistance to camptothecin (CptR mutants) have been isolated from mutagen-treated cultures. The CptR mutants exhibited no cross-resistance towards drugs such as colchicine, vinblastine, taxol, or puromycin but showed slightly (2- to 3-fold) enhanced sensitivity towards various drugs that inhibit DNA topoisomerase II (namely teniposide, etoposide, doxorubicin, mitoxantrone, amsacrine, ellipticine), suggesting that the genetic lesion in these mutants was highly specific. In contrast to the wild-type cells, the CptR line was resistant to camptothecin-induced DNA strand breaks as measured by alkaline elution. Biochemical studies revealed that in CptR mutants the cellular activity as well as protein content of DNA topoisomerase I were reduced to about 40-50% of the level in wild-type cells. Normal levels of activity and content were observed for the related enzyme DNA topoisomerase II. Studies with DNA topoisomerase I purified from the wild-type and the mutant cells showed that the enzyme from the CptR cells was markedly resistant to camptothecin as assayed by the drug's effects either on relaxation of supercoiled DNA or on stabilization of the covalent enzyme-DNA intermediate. The presence of a camptothecin-resistant form of DNA topoisomerase I in the mutant cells provides further evidence that this enzyme is the cellular target of camptothecin. Cell hybridization studies between the CptR and CptS cells showed that the hybrids formed between these two cell lines were sensitive to camptothecin. The recessive behavior of the CptR mutation provides a plausible explanation for the reduced topoisomerase I content (about one-half of wild-type cells) of the mutant cells and also for their enhanced sensitivity towards inhibitors of topoisomerase II.

Animals↗

PubMed1988

Species-specific differences in the toxicity of puromycin towards cultured human and Chinese hamster cells.

The toxicity of the protein synthesis inhibitor puromycin towards a number of human and Chinese hamster cell lines has been examined. In comparison to cells of human origin, Chinese hamster cells exhibited about 25-fold higher resistance towards puromycin. These differences appeared to be species related as all the cell lines from any one species showed similar sensitivity towards puromycin. The incorporation of [3H]leucine in the hamster cell lines was accordingly found to be more resistant to the inhibitory effects of puromycin as compared to human cells. Studies on the cellular uptake of [3H]puromycin showed that in comparison to human cells, the drug uptake/binding in the hamster cell lines was greatly reduced. However, protein synthesis in the extracts of hamster and human cells showed no significant differences in sensitivity towards puromycin. These results show that the observed species related differences in cellular toxicity to puromycin are due to differences in the cellular uptake/binding of the drug.

Animals↗

PubMed1988

Intrinsic multidrug resistance phenotype of Chinese hamster (rodent) cells in comparison to human cells.

In comparison to human cells, cell lines of Chinese hamster and mouse origin exhibit between 10-50-fold resistance to a number of different drugs (viz. actinomycin D, daunomycin, chromomycin A3, colchicine, maytansine, mithramycin, puromycin, rhodamine 123, vinblastine and taxol). Studies with a representative Chinese hamster line (CHO) and a single-step multidrug resistant (MDR) mutant of human (HeLa) cells show that: (i) In comparison to the sensitive human cells, both cell lines show a comparable degree of resistance to the above mentioned drugs; (ii) In the presence of non-toxic dosage of verapamil, the drug-resistance phenotype of both cell lines is completely reversed; (iii) Both these cell lines showed greatly reduced uptake/intracellular levels of 3H-daunomycin, 3H-puromycin and 3H-vinblastine, which was restored to sensitive human cell's level in the presence of non-toxic doses of verapamil. The striking similarity in the behaviour of the naturally resistant Chinese hamster cells and a human MDR cell line with regard to the above characteristics, strongly suggests that the species-related differences in sensitivity to the above drugs result from a similar mechanism as that responsible for the MDR phenotype.

Animals↗

PubMed1988

Effect of dietary cholesterol on spermatogenesis.

Cholesterol fed to male rats and rabbits for 120 days impaired testicular function. The production of secondary spermatocytes in rats and rabbits was reduced by 26.1% and 46.9%, respectively. There was significant reduction in spermatid cell population, seminiferous tubules, and Leydig's cell nuclear dimensions. In rats aortic endothelium showed some alterations while in rabbits full-blown atherosclerotic plaque containing a necrotic core and a proliferative fibrous cap was found. Total cholesterol, triglycerides and phospholipids of the testes were increased whereas, testicular glycogen was significantly reduced. Three- and sevenfold increases were noticed in the serum cholesterol levels of rats and rabbits. The LDL cholesterol values were increased while the HDL cholesterol/Total cholesterol ratio was significantly decreased (P less than 0.001). It is concluded that hypercholesterolemia affects the testicular function and aortic endothelium.

Animals↗

PubMed1988

A review and analysis of the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase assay to determine the mutagenicity of chemical agents. A report of phase III of the U.S. Environmental Protection Agency Gene-Tox Program.

Published literature on the Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay from mid-1979 through June 1986 was reviewed and evaluated. Data from the papers considered acceptable include test results on 121 chemicals belonging to 25 chemical classes. A total of 87 chemicals were evaluated positive, 3 negative, and 31 inconclusive. Mutagenicity data on 49 of the 121 chemicals evaluated could also be compared with in vivo animal carcinogenicity data. 40 of the 43 reported animal carcinogens were considered mutagenic. Caprolactam, the only definitive noncarcinogen in the group of 49, was not mutagenic. The CHO/HGPRT assay was concluded to be an appropriate assay system for use in the screening of chemicals for genotoxicity.

Animals↗