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Biomedical subjects

G Shanmugam

Publications and source records attributed to G Shanmugam.

At least 55 records · Page 3Linked to original sources
PubMed1988

Age-dependent changes in the level of a 22 KDa plasma membrane phosphoprotein in developing chick embryo brain.

In vitro phosphorylation of brain proteins of developing chick embryos showed a drastic increase in the extent of phosphorylation of a 22 KDa protein from the fourteenth day reaching a peak at seventeenth day of development; the phosphorylation of the 22 KDa protein declined afterwards. Phosphoaminoacid analysis of the 22 KDa protein indicated serine residues as targets of phosphorylation. Isoelectric focusing followed by second dimensional SDS-PAGE indicated that the 22 KDa protein had a pI value of 4.5. Polymyxin B, an inhibitor of Ca2+ and phospholipid dependent protein kinases inhibited the phosphorylation of the 22 KDa protein.

Aging↗
PubMed1987

S phase mouse embryo fibroblasts secrete varying amounts of a 45,000 dalton protein.

Synchronized cultures of mouse embryo fibroblasts upon release from hydroxyurea (HOU) arrest, secreted several proteins of which a polypeptide of molecular weight 45,000 (45K) was barely visible in the conditioned medium of cells that synthesized DNA at peak levels. The quantity of the 45K protein was higher in the medium of HOU arrested cells and the level got progressively reduced as the cells entered into the DNA synthetic phase. Conditioned media containing the 45K protein inhibit DNA synthesis when added to synchronized cultures. These results suggest that the 45K secreted protein may be involved in the autocrine regulation of turning-off of DNA synthesis at the end of S phase.

Animals↗
PubMed1987

Endonucleolytic cleavage of E. coli and pBR 322 DNAs by a placental DNA-binding protein.

A 34,000 dalton DNA-binding protein (DBP) has been purified from human placenta. The purified protein possesses endonuclease activities capable of cleaving plasmid pBR 322 and chromosomal DNAs from E. coli. Maximum endonuclease activity was observed in the pH range of 6-9 and at 30 degrees C. The nuclease activity of the DBP was completely lost at 50 degrees C. Nitrocellulose filter binding assays indicate preferential binding of the DBP to ss DNA. The protein did not bind to apurinic DNA and UV-irradiated ds DNA. Consistent with the lack of binding of the DBP to apurinic DNA, this substrate was not cleaved by the DBP. However, native and UV-irradiated E. coli DNAs which showed poor binding were also cleaved by the DBP.

Chromosomes, Bacterial↗
PubMed1987

Partial purification of a recombinase activity from human placenta and assay of recombination using rec-blot.

We have designed an assay for the detection of recombination intermediates. The assay can also be used for screening for recombinases. In this assay, the nonradioactive plasmid was immobilized on a nylon membrane and the radioactive plasmid counterpart was allowed to recombine in the presence of a DNA binding protein fraction from human placenta. After incubation for 30 min at 30 degrees C, the membrane was washed extensively and dried. The recombination was visualised by autoradiography and quantitated by counting the corresponding areas of the membrane. Recombination was confirmed by transformation using pRDK plasmids and by gel electrophoresis. These results indicate the presence of recombinase activity in the DNA binding protein fraction of human placenta.

DNA Nucleotidyltransferases↗
PubMed1986

Effect of cycloheximide and growth factors on gene expression in quiescent mouse embryo fibroblasts.

Gene expression in quiescent mouse embryo fibroblasts was studied by labelling the cells with [14C] amino acids and analysing the proteins by electrophoresis in polyacrylamide gradient gels containing sodium dodecyl sulfate. Cycloheximide (CH) pretreatment of the cells was found to induce the synthesis of four proteins of molecular weights 72,000, 68,000, 42,000, and 29,000. These proteins were induced by CH both in serum-arrested and serum-stimulated cells. Addition of platelet-derived growth factor to serum-arrested quiescent cells also induced the synthesis of these proteins. Addition of CH and fetal calf serum (20%) to quiescent cells resulted in a dramatic increase in the synthesis of actin and another protein of molecular weight 29,000. The 29,000-dalton protein was present in higher quantities in the nuclei of induced cells. This protein appeared to be an early protein whose synthesis was transiently induced in quiescent cells within 3 hours of addition of 20% fetal calf serum (FCS). The synthesis of this protein was virtually turned off at 5-6 hours after the addition of serum. However, if CH or a combination of CH and FCS was present, a continuous synthesis of the 29 K protein was observed.

Animals↗
PubMed1986

A serum-induced 29 Kd protein of mouse embryo fibroblasts is tightly bound to the chromatin.

Treatment of quiescent cultures of mouse embryo fibroblasts with 20% fetal calf serum (FCS) or cycloheximide (CH) resulted in the induction of a nuclear protein of molecular weight 29 000 daltons. The 29 Kd protein induced by these two agents was found to be tightly bound to the chromatin since it was not released from the chromatin by a combination of 2.5 M NaCl and 3.2 M urea. The chromatin associated CH-induced 29 Kd protein and the 29 Kd protein obtained from serum-induced cells displayed similar N-chlorosuccinimide cleavage patterns. Pulse-chase experiments indicate a half-life of an hour for the 29 Kd protein. The kinetics of induction of the 29 Kd protein in the early hours of mitogen addition, short half-life, nuclear localisation and strong association with chromatin suggest that this protein may have important roles in cell proliferation, possibly as a mediator of mitogen action.

Amino Acids↗
PubMed1985

Age-dependent changes in the level of a 34 kDa DNA-binding protein in developing chick embryo liver.

The relative amounts of a DNA-binding protein of 34 kDa increased during the early stages of development of chick embryo liver. The content of this protein reached a maximum in 18-19-day-old embryonic livers and decreased afterwards in older embryonic and post-natal chick livers. The 34 kDa polypeptide is the major DNA-binding protein (DBP) of embryonic liver and it preferentially binds to single-stranded DNA. The quantity of the 34 kDa DBP was relatively very low in embryonic muscle, heart and brain.

Age Factors↗
PubMed1985

Intracellular and secreted proteins of density-inhibited, serum-arrested and proliferating mouse embryo cells.

Short-term labelling of secondary cultures of mouse embryo fibroblasts with [14-C] aminoacids enabled the identification and quantitation of proteins specific for quiescent and proliferative stages. Intracellular and secreted proteins of cells maintained under different growth conditions were resolved in high resolution SDS-polyacrylamide gradient gels. Two proteins, identified as fibronectin and procollagens and a 34 000 D polypeptide were found to be secreted by all three types (density-arrested, serum arrested and proliferating) of cells. Both types of arrested cells exclusively secreted a 375 000 D protein while the proliferating cells specifically secreted a 48 000 D polypeptide. During progression of cells from quiescence to proliferation, two intracellular proteins showed major variations. A 205 000 D intracellular protein was found to be synthesized in higher amounts by proliferating cells than by arrested cells. Another protein, identified as actin, showed a marked increase in synthesis following the release of cells from serum arrest. The arrested cells showed reduced levels of actin synthesis and the turning-off process in the synthesis of actin was found to be relatively slow as the cells entered into quiescence.

Actins↗
PubMed1985

Action of Pennisetum typhoides double-stranded ribonuclease on viral ds RNAs.

A ribonuclease that preferentially cleaves natural and synthetic double-stranded (ds) RNAs has been partially purified from Pennisetum typhoides. The enzyme degrades [3H]poly(rA) X poly(rU) to acid-soluble products. The ds RNase does not degrade RNA:DNA hybrid but appears to have about 18% activity against single-stranded (ss) RNAs under the assay conditions used for the cleavage of ds RNAs. The RNase has a molecular weight of 35,000 daltons as determined from gel filtration using Sephadex G-200. The ds RNase shows an absolute requirement for divalent cations Mg++ or Mn++ and monovalent cations K+ or Na+. The specificity of the enzyme towards ds RNA template is supported by the inhibition of cleavage of ds RNAs by ethidium bromide and Penicillium chrysogenum viral ds RNA. The enzyme preparation acts on ds RNAs isolated from Saccharomyces cerevisiae and P. chrysogenum virus. The purified ds RNase also cleaves the in vitro transcriptional products of adenovirus DNA and this activity is inhibited by 5 mM ethidium bromide suggesting that ds regions of adenovirus RNA are involved in the cleavage process.

Adenoviridae↗
PubMed1984

DNA-binding proteins of human placenta: purification and characterization of an endonuclease.

DNA binding proteins present in the cytoplasm and nuclei of term placenta were isolated by DNA-cellulose chromatography and analysed by electrophoresis in high resolution polyacrylamide gradient gels. A denatured DNA specific protein of approximate molecular weight 34 000 daltons was the predominant DNA binding protein of the cytoplasm; this protein consisted of over 65% of the total DNA binding proteins of the 0.15 M NaCl eluate of the cytoplasm. The cytoplasmic extracts contained two additional DNA binding proteins of molecular weight 24 000 and 18 000 daltons and these proteins bound preferentially to ds DNA. All the three DNA binding proteins were also present in the nuclei and electrophoresis of histones in adjacent lanes indicated that they are not histones. The 34 000-dalton DNA binding protein has been purified by ammonium sulphate fractionation followed by phosphocellulose (PC) chromatography. The DBP eluted from the PC column between 0.125-0.15 M potassium phosphate. PC fractions containing electrophoretically pure 34 KD DBP showed an endonuclease activity capable of converting plasmid pBR 322 DNA to the linear form. Maximum endonucleolytic activity was observed in the presence of 3-5 mM Mg2+ and the enzyme activity was completely inhibited by 3 mM ethylenediamine tetraacetate.

Cell Nucleus↗
PubMed1984

Double-stranded RNA specific nuclease from germinating embryos of Pennisetum typhoides.

A double-stranded RNA specific nuclease (ds RNase) has been purified from the pearl millet Pennisetum typhoides. The purification involved S-30 preparation from the germinating embryos, DEAE-cellulose and DNA-cellulose chromatography. The partially pure enzyme preferentially solubilized the synthetic double-stranded polynucleotide [3H]poly(rA) . poly(rU); the degradation of [3H]poly(rC) was fourteen fold lower under the same assay conditions. Furthermore, the ds RNase activity was inhibited to an extent of 58% by ethidium bromide, which is known to intercalate with double-stranded RNAs. Active sulfhydryl groups were found to be necessary for the ds RNase activity since the enzyme action was inhibited by N-ethylmaleimide. Ethidium bromide and N-ethyl-maleimide did not significantly inhibit the ss RNase activity. In contrast, diethyl pyrocarbonate inhibited ss RNase activity completely and ds RNase by 58%. Heating the enzyme for 20 min at 50 degrees C resulted in drastic loss of both enzyme activities. The ds RNase showed maximum activity in the pH range of 6.5 to 7.5. The enzyme acts in vitro on E. coli 30S precursor ribosomal RNA and the cleavage products migrated in the region of mature 23S and 16S rRNAs.

Endoribonucleases↗
PubMed1984

Endonucleolytic cleavage of pBR 322 DNA by a Yoshida ascites cell DNA binding protein.

A 34 000 dalton DNA-binding protein has been purified from Yoshida ascites tumor cells to electrophoretic homogeneity. Nitrocellulose filter binding assays showed preferential binding of this protein to single-stranded DNA. This protein cleaved pBR 322 DNA and the cleavage products showed the presence of a major band corresponding to the circular form of the DNA when analysed in agarose gels under non-denaturing conditions. Under denaturing conditions, two major DNA bands, one corresponding to the single-stranded circular form and the other to linear form were obtained. These results indicate that the 34 000 D protein possesses an endonucleolytic activity which cleaves pBR 322 DNA by the introduction of a single cut in one of the DNA strands.

Animals↗
PubMed1983

Partial purification and characterization of a double-stranded RNA-specific nuclease from human placenta.

A double-stranded RNA-specific nuclease (ds RNase) has been isolated and partially purified from human placenta by DEAE-cellulose and DNA-cellulose column chromatography. Denatured DNA-cellulose retained most of the single-stranded RNA-specific nuclease (ss RNase) activity, whereas the ds RNase came out in the void volume. N-ethylmaleimide at a concentration of 5 mM, selectively inhibited ds RNase activity by 60% under the conditions in which the ss RNase activity was inhibited to an extent of 7%. The ds RNase was specifically inhibited by Penicillium chrysogenum viral ds RNA and by ethidium bromide. The partially purified ds RNase showed requirements for Mg+ whereas Mn2+ and NH4+ ions were inhibitory. The DEAE-enzyme cleaved 32P-labelled 45S ribosomal precursor RNAs from Yoshida ascites sarcoma cells into species that had similar electrophoretic mobilities as the mature rRNAs.

Ammonia↗
PubMed1983

Isolation and characterization of DNA-binding proteins of Yoshida ascites tumour cells.

DNA-binding proteins from the cytosol and nuclei of Yoshida ascites sarcoma cells were isolated by affinity chromatography on native and denatured DNA-cellulose columns. The proteins that were retained in the columns were eluted by increasing concentrations of NaCl and analysed by electrophoresis in polyacrylamide gels. Two proteins with approximate molecular weights (MW) of 38 000 (38K) and 34 000 (34K) D showed high affinity for denatured DNA and eluted in large quantities from denatured DNA-cellulose columns. In contrast, only small amounts of 38 000 and 34 000 D proteins were found to be associated with native DNA and the native DNA-associated 38K and 34K proteins showed higher rate of phosphorylation than the corresponding denatured DNA-binding proteins. The 38 000 and 34 000 D DNA-binding proteins were present both in nuclei and cytoplasm. These two proteins together comprised over 70% of the total DNA-binding proteins present in the 0.4 M NaCl eluate of the cytoplasmic extracts. DNA-binding proteins of MW 38 000 and 34 000 D were also present in a methylcholanthrene-induced fibrosarcoma, although in smaller amounts.

Animals↗
PubMed1979

Subcellular distribution of newly synthesized virus-specific polypeptides in Moloney murine leukemia virus infected cells.

Immune precipitation analysis of pulse-labeled proteins present in subcellular fractions of mouse embryo cells infected with Moloney murine leukemia virus showed the presence of anti-gp70 serum-precipitable viral envelope gene products mainly in the microsomal fractions of these cells. In contrast, anti-p30 serum-specific gag (group specific antigen) gene products were found to be distributed in similar amounts in both the microsomal and postmicrosomal supernatant fractions of pulse-labeled cells.

Cell Line↗