Search PubMed⌕ Search

Biomedical subjects

C Kent

Publications and source records attributed to C Kent.

At least 181 records · Page 10Linked to original sources
PubMed1991

The role of protein kinase C in the stimulation of phosphatidylcholine synthesis by phospholipase C.

The role of protein kinase C in the stimulation of phosphatidylcholine (PC) synthesis by phospholipase C was investigated. Phospholipase C treatment of Chinese hamster ovary cells (CHO) generates diacylglycerol, which is an activator of protein kinase C. The protein kinase C activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulated choline incorporation into two CHO cell lines, a wild-type cell line, WTB, and a mutant cell line, DTG 1-5-4. DTG 1-5-4 is a mutant defective in receptor-mediated endocytosis. A 3-h phospholipase C treatment resulted in the activation and translocation of CTP:phosphocholine cytidylyltransferase in both cell lines. TPA treatment, however, resulted in only a slight (20%) translocation of cytidylyltransferase in WTB; no detectable translocation of cytidylyltransferase was observed in DTG 1-5-4. A decrease in the phosphocholine pools was observed in response to TPA treatment in both cell lines, which indicated that the cytidylyltransferase step was being activated. Phospholipase C stimulated choline incorporation into PC even when protein kinase C had been down-regulated in both cell lines. It was concluded that phospholipase C does not activate PC synthesis by activating protein kinase C.

Animals↗
PubMed1991

The immunolocalisation of the neuroendocrine specific protein PGP9.5 during neurogenesis in the rat.

We have examined the immunolocalisation of the protein gene product (PGP) 9.5 during neurogenesis in the rat embryo. PGP9.5 was first present at 11.5 days gestation (E11.5): all morphologically recognisable nerve cell bodies and fibres were immunoreactive. In routinely processed, wax-embedded tissues, using a standard immunocytochemical technique, PGP9.5 polyclonal antibody specifically demonstrated the developing nervous system and primitive adrenal chromaffin cells.

Adrenal Medulla↗
PubMed1991

Biogenesis of the endoplasmic reticulum in activated B lymphocytes: temporal relationships between the induction of protein N-glycosylation activity and the biosynthesis of membrane protein and phospholipid.

An earlier report from this laboratory documented a substantial increase in the rates of dolichol-linked oligosaccharide intermediate synthesis and protein N-glycosylation in purified murine splenic B lymphocytes (B cells) activated by treatment with bacterial lipopolysaccharide (LPS). In this study the developmental patterns for the induction of lipid-mediated protein N-glycosylation, membrane protein, and phosphatidylcholine (PC) biosynthesis were compared during the proliferative response of B cells to LPS. By electron microscopy it could be seen that a distinct endoplasmic reticulum (ER) network began to develop by 24-48 h after exposure of the purified B cells to LPS. The rate of synthesis of membrane protein increased markedly during the first 10 h after activation, reaching a maximum at 30-40 h. The induction of protein N-glycosylation was delayed slightly relative to membrane protein synthesis, with glycoprotein synthesis increasing sharply approximately 20 h after activation. When phospholipid synthesis was monitored by measuring [CH3-3H]choline incorporation into PC, the rate of labeling increased slowly during the first 35 h, but more substantially between 35 and 90 h. The incorporation of labeled choline into PC was drastically reduced by 5'-deoxy-5'-isobutylthio-3-deazaadenosine, an inhibitor of CDP-choline synthesis, indicating that the incorporation of radiolabeled choline is primarily a measurement of the rate of de novo synthesis of PC. In vitro assays revealed that while choline kinase activity was virtually unchanged, CDP-choline synthetase activity increased gradually throughout the activation period. Diacylglycerol cholinephosphotransferase activity, an ER-associated enzyme, was present at low levels between 0 and 35 h, but increased fivefold between 35 and 90 h. On the basis of the developmental patterns for the rates of protein N-glycosylation, membrane protein insertion, and PC biosynthesis determined by metabolic labeling experiments, we tentatively conclude that all of the ER-associated membrane proteins involved in these biosynthetic processes are not induced concurrently during the activation of B cells by LPS.

Animals↗
PubMed1991

Regulation of eukaryotic phospholipid metabolism.

Phospholipids have diverse and critical roles in cellular metabolism and function. Questions about the mechanisms of regulation of phospholipid synthesis are being investigated with a variety of systems and approaches. For example, the yeast Saccharomyces cerevisiae is an organism in which both biochemical and genetic analyses are used. Biochemical approaches have yielded considerable information on the regulatory properties of enzymes of phospholipid biosynthesis. Studies of the activity of purified phosphatidylserine synthase have suggested how that enzyme is influenced by membrane phospholipids in the cell. The enzyme that regulates mammalian phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase, is also influenced by phospholipids. In addition, the activity of this enzyme often correlates with its translocation to membranes. The location of such enzymes in the cell is of particular interest in light of the possibility that the enzymatic reactions may be efficiently coupled in vivo. Techniques to render cultured cells permeable to phosphorylated molecules indicated that the enzymes of phosphatidylcholine biosynthesis may exist in an organized compartment so that the precursors of phosphatidylcholine are efficiently channeled through the pathway. To ask how phospholipids are transported in the cell, a combined biochemical and genetic approach has been used. These studies have revealed that the phosphatidylinositol/phosphatidylcholine transfer protein, considered to mediate intracellular phospholipid transfer, is a critical component of the secretory pathway for proteins. These results have allowed formulation of a number of new questions on the regulation of phospholipid metabolism and its relationship to general membrane processes.

Animals↗
PubMed1991

Time modulation effect of diethyldithiocarbamate (DDC) on radiosensitization by superoxide dismutase (SOD) inhibition.

Superoxide dismutase (SOD) is known to protect cells from the lethal effects of ionizing radiation by the dismutation of oxygen radicals. Diethyldithiocarbamate (DDC) is known inhibitor of SOD and may therefore be useful as a radiosensitizer. DDC however, is also a thiol radioprotector due to its ability to scavenge radiation induced free radicals. We have shown that DDC, if administered to tumours 1 hour prior to x-irradiation exerts a protective effect, whereas if administered 4 hours prior to irradiation, it radiosensitizes. This time modulation effect is not apparent after neutron irradiation where DDC protects in both situations. We have also examined the effect of DDC on the LD50/30 in mice after total body irradiation.

Animals↗
PubMed1990

Altered phosphatidylcholine metabolism in C3H10T1/2 cells transfected with the Harvey-ras oncogene.

The effect of expression of the Harvey-ras oncogene on phosphatidylcholine metabolism in C3H10T1/2 mouse fibroblast cells was examined. There were multiple changes in the CDP-choline pathway for phosphatidylcholine biosynthesis in the ras-expressing cells. The activity of the first enzyme in the pathway, choline kinase, was stimulated 1.9-fold, while the activity of the second enzyme, CTP:phosphocholine cytidylyltransferase, was decreased by one-half. High levels of intracellular phosphocholine measured in the ras cells were consistent with the altered activities of choline kinase and cytidylyltransferase. The overall rate of phosphatidylcholine synthesis appeared to be increased because the turnover rate of phosphocholine from the intracellular pool was higher in the ras-transfected cells. There also appeared to be an increased rate of phosphatidylcholine degradation in ras-expressing C3H10T1/2 cells. Very high levels of glycerophosphocholine (6-fold increased over control cells) suggested that phospholipase A was activated in these cells. These results indicate that the ras oncogene product directly or indirectly causes an increased turnover of phosphatidylcholine in C3H10T1/2 cells.

Animals↗
PubMed1990

Phosphorylation of CTP:phosphocholine cytidylyltransferase in vivo. Lack of effect of phorbol ester treatment in HeLa cells.

The production and characterization of an antibody to rat liver CTP:phosphocholine cytidylyltransferase is described. This antibody quantitatively precipitated cytidylyltransferase from both rat liver and HeLa cell cytosol. Following affinity purification, the antibody was used to demonstrate, for the first time, the phosphorylation of cytidylyltransferase in vivo. Following the immunoprecipitation of cytidylyltransferase from HeLa cells, acid hydrolysis, and thin layer electrophoresis of the amino acids, only [32P]phosphoserine was detected. The phosphorylation state of cytidylyltransferase in HeLa cells was examined following treatment with phorbol ester for 1 h. In agreement with previous studies, the incorporation of [3H]choline into phosphatidylcholine via the CDP-choline pathway was stimulated 5-fold in cultures of HeLa cells following treatment with phorbol ester for 1 h. However, no appreciable translocation of cytidylyltransferase was detected, despite the utilization of two different methods of cell lysis. Furthermore, the inclusion of phosphatase inhibitors and chelators of divalent cations in the homogenization buffers had no effect on the observed distribution or activity of the enzyme. Immunoprecipitated cytidylyltransferase was phosphorylated to the same extent, and on serine residues only, in both control and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-treated cells. Measurement of the pool sizes of the aqueous intermediates of the CDP-choline pathway, following TPA treatment, revealed a modest decrease in the phosphocholine pool only, consistent with an activation of cytidylyltransferase.

Animals↗
PubMed1990

Purification and characterization of choline/ethanolamine kinase from rat liver.

Choline kinase, the first enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis, was purified 26,000-fold from rat liver to a specific activity of 143,000 nmol.min-1.mg-1 protein. The subunit molecular mass was 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the apparent native molecular mass was 160 kDa by size exclusion chromatography, suggesting a tetrameric structure. Two peaks of choline kinase activity were obtained by chromatofocusing. These isoforms eluted at pH 4.7 (CKI) and 4.5 (CKII). CKII appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Peptide mapping of two isoforms indicated a high degree of similarity, although there were peptides not common to both. Ethanolamine kinase activity copurified with both isoforms. The ratio of choline to ethanolamine kinase activity was 3.7 +/- 0.7 throughout the purification procedure. Choline and ethanolamine were mutually competitive inhibitors. The respective Km values, 0.013 and 1.2 mM, were similar to the Ki values of 0.014 and 2.2 mM. An antibody raised against CKII immunoprecipitated both choline and ethanolamine kinase activities from liver cytosol at the same titer. These data suggest that both activities reside on the same protein and occur at the same active site. Similarly, both activities were immunoprecipitated from brain, lung, and kidney cytosols. Western blot analysis showed both purified liver isoforms, as well as brain, lung and kidney enzymes, to have a molecular mass of 47 kDa.

Acetates↗
PubMed1990

Preclinical studies with the Faure high energy neutron facility: response of pig skin to fractionated doses of fast neutrons (66 MeVp----Be).

The early and late responses of pig skin to fractionated doses of both unfiltered and filtered (i.e. hardened) neutrons using the Faure neutron therapy facility (66 MeVp----Be) were determined and compared with those following fractionated doses with 60Co gamma-rays. Dose-effect curves for the quantal responses of moist desquamation (early epithelial response) and dermal necrosis (late response) were fitted by probit analysis and ED50 values obtained. For a neutron fractionation scheme comprised of 12 fractions in 26 days, and using an unfiltered beam, the ED50 values for moist desquamation and dermal necrosis were 18.67 +/- 2.22 and 22.25 +/- 0.48 Gy, respectively, whereas in the case of the filtered beam, the corresponding ED50 values were 24.78 +/- 1.44 and 23.30 +/- 0.47 Gy. In order to provide a comparison, the values for 24 fractions of 60Co gamma-rays given in 39 days (a clinical protocol used in the Groote Schuur Hospital) were 74.02 +/- 2.92 and 66.72 +/- 1.93 Gy for moist desquamation and dermal necrosis, respectively. For the unfiltered beam, values for the comparative biological effectiveness (CBE) were 3.96 and 3.00 for the early and late skin response, respectively. The corresponding CBE values were for the filtered beam 2.99 and 2.86. These results for the Faure neutron therapy facility can be extrapolated to the human situation with a high degree of confidence, so that the neutron dose which would yield acceptable skin damage in patients may be determined using the data presented here.

Animals↗