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Biomedical subjects

R S Gupta

Publications and source records attributed to R S Gupta.

At least 235 records · Page 13Linked to original sources
PubMed1979

Antagonism by some beta-adrenoceptor-blocking agents to cholinergic stimulation of skeletal muscle in vitro.

Antiacetylcholine activity some beta-adrenoceptor-blocking drugs was investigated using isolated guinea pig cremaster muscle and frog fectus abdominis muscle. On the cremaster muscle, the antagonism to acetylcholine was non competitive in K0 1313, (+/-)-INPEA and (--)-INPEA, competitive in (+)-INPEA and functional in practolol; All three INPEA isomers, practolol and propranolol behaved as noncompetitive antagonists of acetylcholine on frog rectus muscle. Caffeine-induced contractions of this muscle were partially inhibited by propranolol but not by the other drugs. It is suggested that the beta-adrenoceptor-blocking drugs produce their antiacetylcholine action by interaction with sites on the muscle which are different from the cholinceptor, and which vary between compounds and species.

Acetylcholine↗
PubMed1978

An in vitro analysis of the dominance of emetine sensitivity in Chinese hamster ovary cell hybrids.

The behavior of ribosomes derived from EmtR X EmtS hybrid cells in in vitro protein synthesis is similar to that observed with a 1:1 mixture of ribosomes from EmtR and EmtS cells. When mRNA (BM virus RNA) is present in limiting amounts (RNA/ribosome molar ratio = 0.1), protein synthesis in either mixture is sensitive to emetine. In contrast, when mRNA is present in excess (RNA/ribosome molar ratio = 2), the emetine resistant as well as the sensitive components are both expressed in the mixtures. These results strongly indicate that emetine resistant and sensitive ribosomes are present in the hybrid cells in about equal amounts and that the dominance of emetine sensitivity is best explained by assuming that emetine acts by blocking ribosome movement along mRNA by inhibiting the translocation step. The observed time lag in the expression of EmtRI and EmtRII mutations following mutagenesis is consistent with the above hypothesis for the mechanism of action of emetine.

Cell Line↗
PubMed1978

Evidence for variation in the number of functional gene copies at the AmaR locus in Chinese hamster cell lines.

The hypothesis of functional hemizygosity has been examined for the alpha-amanitin resistant (AmaR, a codominant marker) locus in a series of Chinese hamster cell lines. AmaR mutants were obtained from different cell lines, e.g., CHO, DHW, M3- 1 and CHO-Kl, at similar frequencies. After fractionation of different RNA polymerase activities in the extracts by chromatographic procedures, the sensitivity of the mutant RNA polymerase II towards alpha-amanitin was determined. While all of the RNA polymerase II activity in mutant CHO and CHO-Kl lines became resistant to alpha-amanitin inhibition, only about 50% of the activity is highly resistant in AmaR mutants of CHW and M3- 1 cell lines. The remaining activity in the latter cell lines shows alpha-amanitin sensitivity similar to that seen with the wild-type enzyme. This behaviour is similar to that observed with a 1:1 mixture of resistant and sensitive enzymes from CHO cells. These results, therefore, strongly indicate that while only one functional copy of the gene affected by alpha-amanitin is present in CHO and CHO-Kl cells, two copies of this gene are functional in the CHW and M3-1 cell lines.

Amanitins↗
PubMed1978

Genetic and biochemical characterization of mutants of CHO cells resistant to the protein synthesis inhibitor trichodermin.

Mutants resistant to the protein synthesis inhibitor trichodermin have been selected in Chinese hamster ovary (CHO) cells. The mutants vary in their stability from those which rapidly lose their resistance to others which are relatively stable after prolonged growth in nonselective medium. Protein synthesis in extracts from the latter class of mutants (Trir) is resistant to the inhibitory action of trichodermin as compared to similar extracts from wild-type cells. After dissociation into subunits, the ability of the 60S ribosomal subunits from Trir cells to function in a protein-synthesizing system is greatly diminished. This subunit also shows reduced binding of [acetyl-14C]TRICHODERMIN. The lesion in Trir mutants therefore seems to have affected this ribosomal subunit. Trir X Tris hybrids are sensitive to trichodermin indicating that the Trir mutation behaves recessively to Tris in hybrids. The Emtr and Trir markers segregate independently from hybrid cells showing that the Trir mutation is probably not linked to the Emtr locus, which as we have shown earlier affects the 40S ribosomal subunit.

Cell Line↗
PubMed1978

Diphtheria-toxin-resistant mutants of CHO cells affected in protein synthesis: a novel phenotype.

Stable mutants highly resistant to the protein-synthesis-inhibitor diphtheria toxin have been selected in Chinese hamster ovary (CHO) cells. Protein synthesis in extracts of mutant cells is resistant to the inhibitory action of diphtheria toxin, indicating that the lesion has affected the protein-synthesis machinery. However, about 50% of the elongation factor-2 (EF-2) activity in the mutant cells can still be ADP-ribosylated by diphtheria toxin, and this remaining EF-2 activity is similar to that present in the wild-type cells. We suggest that this result is best explained by assuming that our CHO cells contain two functional copies of the EF-2 gene, and that only one of the copies is altered in the mutants. According to this view, the mutated allele produces EF-2 resistant to ADP-ribosylation which is capable of supporting cell growth in the presence of diphtheria toxin. Although the Dipr marker seems to act dominantly in the parental CHO cells, its behavior in Dipr X Dips hybrids (CHO X CHO) is recessive as measured by cell survival in presence of the toxin. This paradoxical behavior may be due to a gene dosage effect. Segregation studies from hybrids show that the Dipr marker segregates independently of the Emtr and Thgr markers indicating that the Dipr locus is not linked to either the Emtr locus or to the X chromosome.

Adenosine Diphosphate Sugars↗
PubMed1978

Genetic and biochemical studies with the adenosine analogs toyocamycin and tubercidin: mutation at the adenosine kinase locus in Chinese hamster cells.

The pyrrolopyrimidine nucleosides toyocamycin and tubercidin show several unique features of growth inhibition in Chinese hamster ovary (CHO) cells. Stable mutants which are more than 600-fold resistant to these drugs are obtained in CHO cells at a strikingly high frequency of approximately 10(-3), in the absence of mutagenesis. The mutants resistant to toyocamycin (Toyr) and tubercidin (Tubr) exhibit similar cross-resistance patterns to the two selective drugs as well as to adenosine and 6-methyl mercaptopurine riboside, indicating that the same lesion is probably involved in all cases. The mutants examined were found to be deficient in the enzyme adenosine kinase (AK), indicating that the phosphorylation of these analogs is an essential first step in their toxic action. The above mutants (AK-) behaved recessively in cell hybrids, and segregation studies indicate that the AK locus is not linked to the X chromosome. The frequencies of similar Toyr mutants in other Chinese hamster lines, e.g., V79, CHW, M3-1, GM7, and CHO-K1, varied from similar to more than three logs less than that observed for CHO cells, indicating that various cell lines probably differ in the number of functional gene copies for this locus.

Adenosine Kinase↗
PubMed1978

Mutants of CHO cells resistant to the protein synthesis inhibitor emetine: genetic and biochemical characterization of second-step mutants.

Second-step mutants highly resistant to the protein synthesis inhibitor emetine (Emt(RII) have been selected from emetine resistant (Emt(RI)) Chinese hamster ovary cells described earlier. The frequency of the Emt(RII) mutants was increased 50- to 75-fold after mutagenesis, and none of these highly resistant mutants could be selected in one step using wild-type cells. Like the Emt(RI) mutants, the increased resistance of Emt(RII) mutants results from another lesion in the polyribosomal fraction, as measured by the effects of emetine in fractionated extracts. As with the first-step mutants, the Emt(RII) isolates behave recessively in somatic cell hybrids. Segregation studies have shown that the Emt(R) lesions are not on the X chromosome, and in at least one isolate there is evidence that the Emt(RI) and Emt(RII) mutations may occur at different sites.

Animals↗
PubMed1978

Evidence for functional hemizygosity at the Emtr locus in CHO cells through segregation analysis.

The hypothesis of functional hemizygostiy at the emetine-resistant (Emtr, a non-X-linked recessive marker) locus in Chinese hamster ovary (CHO) cells has been examined by segregation analysis. The frequencies and the rates of segregation of the Emtr and Thgr (thioguanine-resistant, an X-linked recessive mutation) markers were determined from hybrids constructed between an Emtr-Thgr CHO cell line and various other Chinese hamster lines (V79, M3-1, CHO, GM7S, CHW and CHL). Thgr segregants were obtained at similar frequencies (10(-2)-10(-3)) from all the hybrids. The frequency of segregation of the Emtr marker, however, was similar to that of Thgr only in the CHO x CHO hybrids and was much lower (10(-4)-10(-6)) than the CHO x other Chinese hamster hybrids. Similar results were obtained when the segregation rates for the two markers from various hybrids were determined. These results are consistent with the hypothesis that in CHO cells, the gene responsible for Emtr is present in a single (functional) copy, whereas two copies of this gene are present in other Chinese hamster lines examined.

Cell Line↗
PubMed1978

Isolation and characterization of mutants of human diploid fibroblasts resistant to diphtheria toxin.

Stable mutants highly resistant to the protein synthesis inhibitor diphtheria toxin (dipr) have been selected in human diploid fibroblast cells at a frequency of 1-8 X 10(-6). Treatment of cells with mutagens, (e.g., ethylmethanesulfonate, nitrosoguanidine, and ICR-170), increased the frequencies of dipr mutants by 50- to 500-fold in different experiments, and the optimal expression time for dipr mutation was about 5 days. All mutants examined thus far have bred true, and no effects of cell density or cross feeding have been observed on the selection. Fluctuation analysis showed that the dipr mutation occurs in these fibroblasts at the rate of 5-6 X 10(-7) mutations per cell per generation. Protein synthesis in mutant extracts was resistant to diphtheria toxin, indicating that the dipr lesion in such mutants lies in the protein synthesis machinery. The characteristics of the dipr marker should make this system particularly useful for studies of quantitative mutagenesis in human diploid cells.

Cell Line↗
PubMed1978

Characterization of the adrenoceptor in the isolated cremaster muscle of the guinea-pig.

1 Contractions of the isolated cremaster muscle of the guinea-pig obtained in response to direct electrical stimulation were augmented by salbutamol, isoprenaline, adrenaline, noradrenaline and orciprenaline (in that order of potency), and by potassium chloride. Phenylephrine and xylometazoline had no effect. 2 The augmenting effect of isoprenaline on the twitch response was not altered by yohimbine or tolazoline, but was totally prevented by butoxamine, (--)-1-(4-nitrophenyl)-2-isopropylamino-ethanol (INPEA), (+/-)-INPEA, sotalol, metalol and practolol; (+)-INPEA was much less effective. The augmenting effect of potassium was not altered by beta-adrenoceptor blockade. 3 The results are consistent with the view that the augmenting effect of the sympathomimetic agents is mediated through beta2-type of adrenoceptor in the skeletal muscle fibres.

Animals↗
PubMed1977

Purification and some novel properties of Escherichia coli RNase II.

RNase II of Escherichia coli (EC 3.1.4.23) has been purified to apparent homogeneity. The K+-activated diesterase activity against poly(U), which defines RNase II, cochromatographs with activity against T4 mRNA or pulse-labeled E. coli RNA successively on DEAE-cellulose, hydroxyapatite or phosphocellulose, and Sephadex G-150 columns. Activities with both substrates are selectively reduced to less than 2% of the wild type level in a newly isolated mutant strain, S296, or after thermal inactivation in a mutant strain with temperature-sensitive RNase II. RNase II releases 5'-XMP without a lag as its only detectable alcohol-soluble produce from all substrates and has an apparent molecular weight of 80,000 to 90,000 in both nondissociating and sodium dodecyl sulfate-polyacrylamide gels. The pure enzyme shows the standard K+ activation against poly(A), poly(U), or poly(C), but only a slight preference for K+ over Na+ ions with T4 mRNA or pulse labeled E. coli RNA as substrate. Uniformly labeled E. coli rRNA or tRNA is degraded little if at all.

Enzyme Activation↗
PubMed1977

Mutants of CHO cells resistant to the protein synthesis inhibitors, cryptopleurine and tylocrebrine: genetic and biochemical evidence for common site of action of emetine, cryptopleurine, tylocrebine, and tubulosine.

Stable mutants resistant to the protein synthesis inhibitors cryptopleurine and tylocrebine can be isolated in Chinese hamster ovary (CHO) cells, in a single step. The frequency of occurrence of cryptopleurine (CryR) and tylocrebrine (TylR) resistant mutants in normal and mutagenized cell populations is similar to that observed for emetine resistant (EmtR) mutants. The CryR, TylR, and EmtR mutants exhibit strikingly similar cross-resistance to the three drugs used for selection, to tubulosine and also to two emetine derivatives cephaeline and dehydroemetine, based on assays of in vivo cytotoxicity and on assays of protein synthesis in cell-free extracts. The identity of cross-resistance patterns of the CryR, TylR, and EmtR mutants indicates that the resistance to all these compounds results from the same primary lesion, which in the case of EmtR cells has been shown to affect the 40S ribosomal subunit. This conclusion is strongly supported by the failure of EmtR, TylR, and CryR mutants to complement each other in somatic cell hybrids. Based on these results it is suggested that the above group of compounds possesses common structural determinants which are responsible for their activity. The above mutants, however, do not show any cross-resistance to other inhibitors of protein synthesis such as cycloheximide, trichodermin, anisomycin, pactamycin, and sparsomycin, either in vivo or in vitro, indicating that the site of action of these inhibitors is different from that of the emetine-like compounds.

Alkaloids↗