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C Ramachandran

Publications and source records attributed to C Ramachandran.

82 records · Page 5Linked to original sources
PubMed1978

Submitochondrial location of ruthenium red-sensitive calcium-ion transport and evidence for its enrichment in a specific population of rat liver mitochondria.

1. Seven fractions sedimenting at between 3000 and 120000g-min were prepared from a rat liver homogenate by differential centrifugation in buffered iso-osmotic sucrose. The following measurements were carried out on each of these fractions: Ruthenium Red-sensitive Ca(2+) transport in the absence and in the presence of P(i) as well as in the presence of N-ethylmaleimide to prevent P(i) cycling, succinate-supported respiration in the absence and in the presence of ADP, the DeltaE and -59 DeltapH components of the protonmotive force, cytochrome oxidase, uncoupler-stimulated adenosine triphosphatase, alpha-glycerophosphate dehydrogenase, P(i) content and the effect on the ;resting' rate of respiration of repeated additions of a fixed Ca(2+) concentration. 2. Ca(2+) transport either in the presence or in the absence of added P(i) and in the presence of N-ethylmaleimide exhibits significantly higher rates in the fraction sedimenting at 8000g-min. By contrast, respiration in the presence or in the absence of added ADP and the values for DeltaE and -59 DeltapH were similar in those fractions sedimenting between 4000 and 20000g-min, indicating that the driving force for Ca(2+) transport was similar in each of these fractions. 3. Experiments designed to determine the capacity of the individual fractions for Ca(2+), as measured by the effect of repeated additions of Ca(2+) on the resting rate of respiration, showed that fraction 2, i.e. that sedimenting at 8000g-min, also exhibited the greatest tolerance towards the uncoupling action of the ion. 4. Of the three enzyme activity profiles, only that of alpha-glycerophosphate dehydrogenase was similar to that of Ca(2+) transport. Because previous workers have assigned this enzyme to loci in the inner peripheral membrane [Werner & Neupert (1972) Eur. J. Biochem.25, 379-396], it is concluded that the Ruthenium Red-sensitive Ca(2+)- transport system also is located in this domain of the inner membrane. The relation of these findings to the mechanisms of mitochondrial Ca(2+) transport and the biogenesis of mitochondria is discussed.

Animals↗
PubMed1978

Calcium ion cycling in rat liver mitochondria.

1. Addition of N-ethylmaleimide to rat liver mitochondria respiring with succinate as substrate decreases both the initial rate of Ca(2+) transport and the ability of mitochondria to retain Ca(2+). As a result, Ca(2+) begins to leave the mitochondria soon after it has entered. Half-maximal effects occur at an N-ethylmaleimide concentration of about 100nmol/mg of protein. 2. The efflux of Ca(2+) induced by N-ethylmaleimide is not prevented by Mg(2+) or by Ruthenium Red at concentrations known to prevent Ca(2+) efflux when exogenous phosphate also is present. Swelling of mitochondria does not accompany N-ethylmaleimide-induced Ca(2+) efflux. 3. Addition of Ca(2+) to rat liver mitochondria in the presence of N-ethylmaleimide produces an immediate decrease in DeltaE (membrane potential), which decreases further to only a slight extent over the next 8min. Concomitant with this is an immediate increase and then levelling off of the -59DeltapH (transmembrane pH gradient). 4. Preincubation of rat liver mitochondria with p-chloromercuribenzenesulphonate, which by contrast with N-ethylmaleimide is unable to penetrate the inner mitochondrial membrane, also prevents Ca(2+) retention. The DeltaE and -59DeltapH respond to Ca(2+) addition in a manner similar to that which occurs when N-ethylmaleimide is present. Subsequent addition of mercaptoethanol produces an immediate increase in both DeltaE and -59DeltapH. At the same time Ca(2+) is rapidly accumulated by the organelles. 5. The above data are interpreted as indicating that under the conditions of Ca(2+) efflux seen here, the mitochondria retain their functional integrity. This contrasts with the uncoupling effect of Ca(2+) seen in the presence of P(i), which generally leads to a loss of mitochondrial integrity. We suggest that a unique mechanism of Ca(2+) cycling is able to take place when mitochondria have been treated with N-ethylmaleimide.

4-Chloromercuribenzenesulfonate↗
PubMedDate not supplied

Cytogenetic and molecular characterization of a congenital mesoblastic nephroma.

A newborn baby boy was diagnosed with the mixed form of congenital mesoblastic nephroma (CMN) representing both classic and cellular histology features in the renal tumor. Additionally, the patient had skin and bone lesions consistent with multifocal involvement of a generalized infantile fibromatosis (IFS). Both skin and bone lesions were distinctly different from CMN and did not represent metastasis. The primary tumor cell line (MCH-MN-1), established from the resected right kidney tumor, had a diploid DNA content. Cytogenetic studies revealed deletion on the long arm of chromosome 3 (q21q24) and duplication on the short arm of chromosome 11 (p15). MCH-MN-1 cells expressed ETV6-NTRK3 gene fusion transcripts, characteristic of cellular and mixed forms of CMNs. The cells had high p21 and low Bax mRNA expression in the reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The high level of proliferative marker (Ki67) mRNA expression correlated well with the pluripotent nature of MCH-MN-1 in tissue culture (cell doubling time = 12.4 h). Our results showed that MCH-MN-1 might be a good model cell line for investigations on mesoblastic nephroma.

Chromosome Deletion↗
PubMedDate not supplied

Characterization of ATP-dependent Ca2+ transport in the basolateral membrane vesicles from proximal and distal tubules of the rabbit kidney.

Basolateral membrane vesicles were prepared from purified proximal and distal tubules of the rabbit kidney. The properties of the ATP-dependent Ca2+ transport were investigated. In both membranes, there was a high affinity, ATP-dependent Ca2+ transport system (Km = 0.1 microM). The optimal concentration of Mg2+ was 0.5 mM and the optimal concentration of ATP was 1 mM. The nucleotide specificity and pH dependence of the Ca2+ transport in both membranes were similar. In basolateral membrane vesicles, calmodulin had no effect on Ca2+ transport. However, in basolateral membrane vesicles depleted of calmodulin, exogenous calmodulin increased the Ca2+ transport by increasing maximal velocity. There were no major differences in the properties of the ATP-dependent Ca2+ transport system in these two membranes. These findings are discussed in relation to why parathyroid hormone differentially modulates Ca2+ transport in these two segments of the nephron.

Adenosine Triphosphate↗
PubMedDate not supplied

Comparative chemosensitivity profiles in three human breast cancer cell lines with the ATP-cell viability assay.

In this study the dose-response curves for doxorubicin, pirarubicin, 5-fluorouracil, 4-hydroperoxy-cyclophosphamide and taxol were obtained in three breast cancer cell lines (MCF-7, T47D and BT-20). The ATP cell viability assay was chosen to evaluate the chemosensitivity profiles and was a reproducible, practicable method to assess drug response in breast cancer cell lines. The IC50 values were calculated on the median effect principle and indicated that taxol was the most active drug tested in this study with a mean IC50 value of 0.02 microM. This in vitro effect correlated well with clinical observations in metastatic breast cancer where taxol proved to be a vary active drug. Pirarubicin was the second most active drug tested with an IC50 value 10 times less compared to that of doxorubicin. The results obtained with the ATP cell viability assay are promising, therefore further testing with drug combination chemotherapy and fresh human breast cancer tumor testing are warranted and ongoing.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Expression of apoptosis, cell proliferation, and drug resistance genes in pediatric Wilms' tumors.

The expression of genes associated with apoptosis, cell proliferation and drug resistance in tumor cells was investigated in two pediatric Wilms' tumor patients (MCH-WT-1 and MCH-WT-3) for their association with cell cycle, daunorubicin accumulation and clinical data. DNA content, cell cycle and drug accumulation were analyzed immediately after surgery by flow cytometry and mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Primary cell cultures were established from tumor specimens and tumor cells in both cases showed epithelial morphology. Although cell proliferation markers (Ki67 and PCNA) were expressed in both cases, MCH-WT-3 showed higher levels of mRNA expression, which corresponded, with metastatic behavior of the tumor in the patient. While p53 and p21 mRNAs were expressed at low levels in MCH-WT1, MCH-WT-3 showed high levels of p21 mRNA only. The increased expression of cyclin kinase inhibitor (p21) in MCH-WT-3 compared to MCH-WT-1 correlated with a higher percentage of G0/G1 cell population in the tumor specimen. Despite the expression of multidrug resistance markers (MDR1 and LRP) in MCH-WT-1, flow cytometric analysis showed tumor cell populations with very low and high daunorubicin accumulation and with the absence of any effect for verapamil and dipyridamole on daunorubicin accumulation of tumor cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Bcl-2 and mdr-1 gene expression during doxorubicin-induced apoptosis in murine leukemic P388 and P388/R84 cells.

The induction of apoptosis by doxorubicin (DOX) alone or in the presence of efflux blockers, verapamil (VPL) or trifluoperazine (TFP), and of bcl-2 and mdr-1 gene expression was analyzed in murine leukemic P388 and doxorubicin resistant P388/R84 cells. Incubation with DOX (0.1-1 microM) for 24 hours induced apoptosis in sensitive cells but not in the resistant P388/R84 cells. Flow cytometric analysis of apoptosis by terminal dideoxynucleotidyl (TdT) assay showed that 1 microM DOX induced apoptosis in 70% of P388 cells and in less than 5% of P388/R84 cells. When P388/R84 resistant cells were co-incubated with DOX and efflux blockers (10 microM VPL or 15 microM TFP), enhanced cellular DOX accumulation was accompanied by apoptosis. Quantitative analysis of DNA fragmentation by 14C-thymidine incorporation and gel electrophoresis of fragmented DNA confirmed the results from TdT assay and showed enhancement of DOX-induced DNA fragmentation in the presence of efflux blockers (VPL or TFP). DOX + VPL and DOX + TFP combination treatments induced apoptosis in upto 55% and 83% of P388/R84 cells, respectively. mdr-1 mRNA and P-gp expression were not altered during DOX-induced apoptosis. The results suggest that in murine leukemic cells, down-regulation of bcl-2 mRNA expression occurs during DOX-induced apoptosis and it depends on the cellular drug retention determined by mdr-1/P-gp drug efflux.

Animals↗