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Biomedical subjects

M Shanmugam

Publications and source records attributed to M Shanmugam.

13 recordsLinked to original sources
PubMed2001

A role for protein kinase C delta in the differential sensitivity of MCF-7 and MDA-MB 231 human breast cancer cells to phorbol ester-induced growth arrest and p21(WAFI/CIP1) induction.

The goal of this study was to investigate the differential sensitivity of estrogen receptor (ER) positive MCF-7 and ER negative MDA-MB 231 breast cancer cells to phorbol myristate acetate (PMA)-dependent growth arrest. MCF-7 cells were growth arrested by 80% while MDA-MB 231 cells were arrested by 20% in response to seven days of treatment with 10 nM PMA. Coincident with the increased sensitivity of MCF-7 cells to be growth arrested by the protein kinase C (PKC) activator PMA, PMA induced 9-fold higher levels of the cyclin dependent kinase (Cdk) inhibitor p21(WAF1/GIP1) in MCF-7 compared to MDA-MB 231 cells. A comparison of the PKC isoforms expressed in MCF-7 versus MDA-MB 231 cells showed that only the PMA-sensitive PKC delta and eta isoforms were expressed at markedly (> or =10-fold) elevated levels in MCF7 versus MDA-MB 231 cells. These results suggested that the differential sensitivity to growth arrest and induction of p2l(WAFl/CIPl) could reflect, at least in part, increased expression of PMA-dependent PKC isoforms delta and/or eta. Direct evidence to support this hypothesis was provided by the ability of transient transfections into MCF-7 cells of constitutively active PKC delta but not of PKC's eta or alpha or epsilon to enhance p21(WAFl/CIP1) promoter activity. These results suggest that PKC delta plays a fundamental role in the regulation of growth in estrogen receptor positive breast cancer cells.

Acetophenones↗
PubMed2001

Blood anticoagulant sulphated polysaccharides of the marine green algae Codium dwarkense (Boergs.) and C. tomentosum (Huds.) Stackh.

Cold water extracts of marine green algae Codium dwarkense and C. tomentosum were precipitated with different molar concentrations of KCl and were subjected to anion exchange and gel filtration chromatography. Both the species yielded sulphated arabinan through bioassay-guided purification and both were chemically identified as a polymer of alpha-L-arabinofuranose. Products were assayed for their blood anticoagulant activity using PT, APFT and TT tests and found that they differed in the potency of activity though they are chemically identical. Bioassay-guided purification of cold water extract of C. tomentosum yielded sulphated arabinan and sulphated arabinogalactan.

Anticoagulants↗
PubMed2000

Surgery for stage 5 retinopathy of prematurity: the learning curve and evolving technique.

PURPOSE: To describe our experience with management of eyes with stage 5 retinopathy of prematurity (ROP). METHODS: Closed vitreoretinal surgery was done on 96 eyes of patients with stage 5 ROP. Lens was sacrificed in all but one eye. Surgery involved an attempt to clear all preretinal tissue and open the peripheral trough all round. In most instances bimanual surgery under viscoelastic was performed. RESULTS: At last follow up, anatomical success (defined as attached posterior pole) was achieved in 22.5% cases. Significant postoperative problems included reproliferation and secondary glaucoma. Only two infants obtained mobile vision. CONCLUSION: Late identification of disease, lack of prior treatment such as laser or cryo, and higher incidence of narrow-narrow funnel configuration were responsible for the poor surgical results noted in this series. The poor surgical and functional results reemphasize the need for prompt screening and management of infants at risk.

Clinical Competence↗
PubMed1999

Regulation of protein kinase C delta by estrogen in the MCF-7 human breast cancer cell line.

We have previously shown that estrogen up-regulates expression of protein kinase C (PKC) delta in the rat and rabbit corpus luteum as well as in luteinized rat granulosa primary cell cultures. To determine whether a similar regulation of the PKC delta isoform by estrogen occurred in another estrogen responsive system, we investigated the estrogen receptor positive MCF-7 human breast cancer cells. In a characterization of PKC isoforms in MCF-7 cells we determined that PKC delta was the predominant PKC isoform. However in contrast to the effect of estrogen on PKC delta expression in ovarian cells, estrogen treatment of MCF-7 cells resulted in a significant decrease in PKC delta protein and mRNA expression in a time and dose dependent manner. Treatment of MCF-7 cells with 10(-10)-10(-8) M estrogen for 7 days down-regulated specifically PKC delta mRNA and protein while expression of other PKC isoforms was unchanged. The opposite regulation of PKC delta expression in ovarian and breast cancer cells prompted us to evaluate the type of estrogen receptor present in both cell types. Results showed that luteinized rat granulosa cells expressed predominantly estrogen receptor beta while the MCF-7 cells expressed predominantly estrogen receptor alpha and barely detectable levels of estrogen receptor beta. These results suggest that the differential ability of estrogen to regulate PKC beta expression could potentially be a result of differential signaling through the two estrogen receptor subtypes.

Animals↗
PubMed1998

Heat-shock protein-25/27 phosphorylation by the delta isoform of protein kinase C.

Small heat-shock proteins (sHSPs) are widely expressed 25-28 kDa proteins whose functions are dynamically regulated by phosphorylation. While recent efforts have clearly delineated a stress-responsive p38 mitogen-activated protein-kinase (MAPK)-dependent kinase pathway culminating in activation of the heat-shock (HSP)-kinases, mitogen-activated protein-kinase-activated protein kinase-2 and -3, not all sHSP phosphorylation events can be explained by the p38 MAPK-dependent pathway. The contribution of protein kinase C (PKC) to sHSP phosphorylation was suggested by early studies but later questioned on the basis of the reported poor ability of purified PKC to phosphorylate sHSP in vitro. The current study re-evaluates the role of PKC in sHSP phosphorylation in the light of the isoform complexity of the PKC family. We evaluated the sHSP phosphorylation status in rat corpora lutea obtained from two stages of pregnancy, mid-pregnancy and late-pregnancy, which express different levels of the novel PKC isoform, PKC-delta. Two-dimensional Western blot analysis showed that HSP-27 was more highly phosphorylated in vivo in corpora lutea of late pregnancy, corresponding to the developmental stage in which PKC-delta is abundant and active. Late-pregnant luteal extracts contained a lipid-sensitive HSP-kinase activity which exactly co-purified with PKC-delta using hydroxyapatite and S-Sepharose column chromatography. To determine whether there might be preferential phosphorylation of sHSP by a particular PKC isoform, purified recombinant PKC isoforms corresponding to those PKC isoforms detected in rat corpora lutea were evaluated for HSP-kinase activity in vitro. Recombinant PKC-delta effectively catalysed the phosphorylation of sHSP in vitro, and PKC-alpha was 30-50% as effective as an HSP-kinase; other PKCs tested (beta1, beta2, epsilon and zeta) were poor HSP-kinases. These results show that select PKC family members can function as direct HSP-kinases in vitro. Moreover, the observation of enhanced luteal HSP-27 phosphorylation in vivo, in late pregnancy, when PKC-delta is abundant and active, suggests that select PKC family members contribute to sHSP phosphorylation events in vivo.

Animals↗
PubMed1998

Association of PKC delta and active Src in PMA-treated MCF-7 human breast cancer cells.

Phorbol ester treatment of MCF-7 cells led to the tyrosine phosphorylation and activation of PKC delta. However, through Western blot analysis and in vitro immunecomplex kinase assays, we detected a differential localization of tyrosine-phosphorylated PKC delta and catalytically active PKC delta. Catalytically active PKC delta was concentrated in Triton X-100 solubilized-membrane fractions while tyrosine-phosphorylated PKC delta was localized to the cytosol fraction. Phorbol ester treatment of MCF-7 cells stimulated both the time-dependent in vivo association of Src with PKC delta, evidenced in Src immunoprecipitates by the co-immunoprecipitation of PKC delta, and activation of Src, evidenced in Src immunoprecipitates as an increase in reactivity with a Src antibody (clone 28) reactive only with active Src (dephosphorylated on residue 530) and in Src and PKC delta immunoprecipitates by an increase in Src kinase activity. While our data are consistent with reports in the literature showing the activator/stimulus-dependent tyrosine phosphorylation of PKC delta, our data show that the tyrosine phosphorylation of PKC delta is not essential for kinase activity. These results are the first to demonstrate an in vivo association between PKC delta and active Src in the absence of over-expression of either PKC delta or Src, and support the association of Src and PKC delta towards a physiological function.

Breast Neoplasms↗
PubMed1996

Hormonal regulation of PKC-delta protein and mRNA levels in the rabbit corpus luteum.

We have previously reported that rabbit corpora lutea exhibit a prominent phosphorylated substrate protein at 76 kDa which corresponds to the autophosphorylated form of protein kinase C (PKC) delta and that the expression of PKC-delta protein is increased in rabbit corpora lutea of pseudopregnancy at least 2-fold when serum estrogen levels are raised by the presence of an estrogen implant inserted at the time of human chorionic gonadotropin (hCG)-induced ovulation. The purpose of the experiments described herein was to evaluate further the hormonal regulation of PKC-delta in the rabbit corpus luteum. Results demonstrate that luteal PKC-delta protein and mRNA are concomitantly induced some 5-fold within 48 h in response to an ovulatory surge of hCG; that, as in corpora lutea of pseudopregnancy, luteal PKC-delta expression is relatively constant during the life span of the corpus luteum following a fertile mating; that exogenous estrogen does not modulate the induction of luteal PKC-delta during luteinization but promotes an additional two-fold increase in steady state PKC-delta mRNA (and protein) levels in corpora lutea by day 10 of pseudopregnancy; and that luteal PKC-delta expression can be abruptly and reversibly modulated upon withdrawal and subsequent replacement of an estrogen implant to pseudopregnant rabbits. These results demonstrate that an ovulatory surge of luteinizing hormone induces the expression of PKC-delta mRNA and protein in rabbit corpora lutea, and that once the corpus luteum becomes estrogen responsive, estrogen then regulates expression of PKC-delta mRNA and protein.

Animals↗
PubMed1995

Feasibility of involving literate tribal youths in tuberculosis case-finding in a tribal area in Tamil Nadu.

SETTING: Tribal area in South India with a population of 96,000, where the tuberculosis case-finding activity was very poor. OBJECTIVES: To investigate the feasibility of (1) involving literate (who can read and write) tribal youth volunteers for detecting cases of pulmonary tuberculosis (PT) in their respective hamlets; and (2) antituberculosis drug delivery to sputum-positive patients at their homes by village health nurses (VHNs). DESIGN: One volunteer from each of 61 hamlets was selected and trained in the detection of subjects with chest symptoms, sputum collection and transportation to the Primary Health Centre for smear examination. All smear-positive patients were treated with 2RHZ/6TH and the drugs were supplied by VHNs fortnightly at their homes. RESULTS: During a period of 1 year (1992-93), the total population screened was 9383 persons; of these 5755 were aged 15 years and above. A total of 338 symptomatic subjects were identified; 12 sputum-positive cases were detected and started on treatment. Antituberculosis drugs were supplied by VHNs to patients for the first 9 months of the study and by literate youths for the next 3 months. Spot drug checks revealed that 11 of the 12 patients were regular in drug consumption. CONCLUSION: It is feasible to train literate tribal youth volunteers within a short time to detect subjects with chest symptoms in the community and thereby cases of pulmonary tuberculosis. They can serve as an excellent model for community participation in difficult areas.

Antitubercular Agents↗
PubMed1989

M(III)-facilitated recovery and concentration of enzymes from mesophilic and thermophilic organisms.

Six enzymes isolated from organisms of widely differing thermal growth optima were flocculated from solution at constant pH by addition of Fe(III) solution. In all cases the enzyme concentration was 1 g.l-1 or less. Flocculation profiles were generated for each enzyme over a range of Fe(III) levels. The concentrated enzymes were recovered from the Fe(III)/protein complex by solubilisation with citrate and dithionite followed by precipitation with ammonium sulphate. In all cases approximately 70-80% enzyme recovery was achieved. Enzyme thermal stability did not appear to be important and protein concentration had no effect on the efficiency of enzyme recovery over the range of 0.01-1 g.l-1. Approximately 30 mmol Fe(III)/l of enzyme solution facilitated optimal enzyme recovery for all solutions studied. For protein concentrations up to 1 g.l-1 a 100-fold enzyme concentration factor can be expected.

Aluminum↗