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Biomedical subjects

C Kent

Publications and source records attributed to C Kent.

At least 217 records · Page 12Linked to original sources
PubMed1984

Phospholipase C from Clostridium perfringens: preparation and characterization of homogeneous enzyme.

A new procedure for the purification of phospholipase C from Clostridium perfringens has been devised that results in essentially pure enzyme. The procedure consists of ammonium sulfate fractionation, ion-exchange chromatography on QAE-Sephadex, and affinity chromatography on phosphatidylcholine linked to Sepharose. The molecular weight of the enzyme, determined by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, and gel filtration, is 43,000; and the isoelectric point is pH 5.4. The enzyme was optimally active with phosphatidylcholine dispersed in sodium deoxycholate, although appreciable activity was observed with either phosphatidylcholine or sphingomyelin dispersed with ethanol. The requirement for metal ions in the assay could be met by a number of different ions. The pure enzyme was found to contain 2 mol zinc per mol enzyme, thus implicating it as a zinc metalloenzyme.

Amino Acids↗
PubMed1984

A functional membrane repair system in Duchenne muscular dystrophy fibroblasts.

Experiments have been performed to determine if fibroblasts from patients with Duchenne muscular dystrophy (DMD) are defective in a process of membrane repair. Normal and DMD fibroblasts were treated with phospholipase C from Clostridium perfringens to degrade plasma membrane phosphatidylcholine, and then phosphatidylcholine synthesis was measured as the incorporation of [3H] choline into lipid. Phosphatidylcholine synthesis was stimulated by phospholipase C treatment to a similar extent in normal and DMD fibroblasts. The activity of CTP: phosphocholine cytidylyltransferase, the enzyme regulating phosphatidylcholine synthesis in phospholipase C-treated mammalian cells, was also stimulated to the same extent in both cell types. The subcellular location of the cytidylyltransferase was changed by phospholipase C treatment from mostly cytosolic to mostly particulate in both normal and DMD fibroblasts. It appears, therefore, that at least one type of membrane repair system functions normally in DMD fibroblasts.

Cell Membrane↗
PubMed1983

Regulation of phosphatidylcholine biosynthesis in mammalian cells. I. Effects of phospholipase C treatment on phosphatidylcholine metabolism in Chinese hamster ovary cells and LM mouse fibroblasts.

Addition of phospholipase C from Clostridium perfringens to cultures of Chinese hamster ovary (CHO) cells resulted in rapid degradation of cellular phosphatidylcholine with concomitant release of phosphocholine. The rate of incorporation of radiolabeled choline into lipids was increased 2-fold in phospholipase C-treated CHO cells as compared to untreated controls. The only enzyme in the pathway of phosphatidylcholine biosynthesis with increased activity in phospholipase C-treated cells was CTP:phosphocholine cytidylyltransferase, indicating that the cytidylyltransferase plays an important role in the stimulation of phosphatidylcholine biosynthesis. The phospholipase treatment was toxic to a CHO mutant cell line with abnormally low cytidylyltransferase activity. Mouse LM fibroblasts were resistant to enzymatic attack by phospholipase C, and cytidylyltransferase activity in LM cells did not change upon phospholipase C treatment.

Animals↗
PubMed1983

Regulation of phosphatidylcholine biosynthesis in mammalian cells. II. Effects of phospholipase C treatment on the activity and subcellular distribution of CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary and LM cell lines.

CTP:phosphocholine cytidylyltransferase was located in both the cytosolic and particulate fractions from Chinese hamster ovary cells. The activity of the cytosolic form of the enzyme was greatly enhanced by incubation with sonicated preparations of several different lipids, although incubations with either phosphatidylcholine or 1,2-sn-diolein did not increase activity. The activation of the cytidylyltransferase in Chinese hamster ovary cells treated with phospholipase C from Clostridium perfringens occurred with a concomitant shift in the subcellular distribution of the enzyme from cytosolic to particulate fractions. This shift was rapid and did not require protein synthesis. Removal of phospholipase C from the cell cultures resulted in a return to basal levels of incorporation of [3H]choline into phosphatidylcholine, a decrease in the activity of cytidylyltransferase, and a loss of the membrane-bound form of the enzyme. Similar experiments with LM cells, which are resistant to exogenous phospholipase C, showed no change in subcellular distribution of cytidylyltransferase, suggesting that the activation of CTP:phosphocholine cytidylyltransferase required a change in membrane phospholipid composition. The results presented are discussed in terms of a mechanism of regulation of phosphatidylcholine production involving monitoring of membrane phospholipid composition.

Animals↗
PubMed1983

Regulation of phosphatidylcholine biosynthesis in mammalian cells. III. Effects of alterations in the phospholipid compositions of Chinese hamster ovary and LM cells on the activity and distribution of CTP:phosphocholine cytidylyltransferase.

The activity and subcellular distribution of CTP:phosphocholine cytidylyltransferase in LM and Chinese hamster ovary cells in which the phospholipid composition had been altered by supplementary feeding with choline analogues were examined. Decreased levels of cellular phosphatidylcholine with corresponding increased levels of either phosphatidylethanolamine, phosphatidylmonomethylethanolamine, or phosphatidyldimethylethanolamine resulted in increased CTP:phosphocholine cytidylyltransferase activity in cell homogenates. In addition, a significantly larger fraction of the total cytidylyltransferase activity was membrane-bound in these cells. The activity of the cytidylyltransferase from cytosolic extracts of both cell types was found to be greatly increased when assayed in the presence of either phosphatidylmonomethyl ethanolamine or phosphatidyldimethylethanolamine. The lysophosphatide forms of these lipids were found to be poor activators of the cytidylyltransferase. These findings suggest that the regulation of phosphatidylcholine biosynthesis is at least partially dependent on information transfer from membranes to CTP: phosphocholine cytidylyltransferase. That is, in the presence of phosphatidylcholine-deficient membranes of cytidylyltransferase becomes activated and associated with the membranes.

Animals↗
PubMed1983

Increased rate of cell-substratum detachment of fibroblasts from patients with Duchenne muscular dystrophy.

When skin fibroblasts from patients with Duchenne muscular dystrophy were treated with trypsin in the presence of divalent cations, they detached more rapidly from the substratum than did fibroblasts from normal individuals of similar age, sex, and passage number. This difference was observed when either the time of incubation or trypsin concentration was varied. The ease of detachment of both normal and dystrophic fibroblasts varied somewhat with culture age and plating density, although detachment was always greater for fibroblasts from dystrophic individuals. If the trypsin treatment was carried out in the absence of divalent cations, both types of fibroblasts detached rapidly from the substratum, suggesting that a divalent-cation dependent cell-substratum adhesion mechanism is altered in Duchenne muscular dystrophy fibroblasts.

Cations, Divalent↗
PubMed1983

Image spread in dry-mounted tritium autoradiographs.

A comparison has been made of autoradiographs prepared by conventional dipping methods and those prepared from the same material using a dry-mounting method developed for the study of receptor labelling. Resolution was found to be inferior, and image spread greater, in the dry-mounted preparations. The extent to which resolution and image spread were affected depended upon the distance between specimen and emulsion.

Animals↗
PubMed1982

Normal function of extra-adrenal chromaffin tissues in the young rabbit and guinea-pig.

An attempt has been made to determine the relative functional activities of adrenal and extra-adrenal chromaffin tissue during the neonatal phase and up to 1 week of age using young rabbits and guinea-pigs whose main extra-adrenal abdominal para-aortic chromaffin bodies are respectively non-innervated and innervated. Amine synthesis and storage were followed by assay and autoradiography after a single intraperitoneal injection of L-[2,5,6-3H]DOPA and the findings correlated with amine content as assessed by high performance liquid chromatography and by volume of tissue. The results indicate that in the guinea-pig, in spite of differences in proportions of adrenaline and noradrenaline in the adrenal gland and para-aortic body (PAB), the loss of labelled catecholamines from the innervated PAB closely follows that from the adrenal medulla, suggesting that both participate in normal sympathoadrenal activity. By comparison, in the rabbit the PAB shows only a minimal decreased labelled amine during the first week of life during which period the amine content and concentration of the PAB doubles: the functional significance of this non-innervated extra-adrenal chromaffin tissue, which persists throughout life, has still to be determined.

Adrenal Cortex↗
PubMed1981

Changes in galactosyltransferase activity in chick pectoral muscle during embryonic development.

The two major vertebrate galactosyltransferases have been investigated in developing chick muscle in ovo and in vitro, and in cultured chick fibroblasts. The two enzymes were UDP-galactose-N-acetylglucosamine galactosyltransferase (galactosyltransferase I) and UDP-galactose-N-acetylgalactosamine galactosyltransferase (galactosyltransferase II). Both activities fell during muscle development in ovo. Galactosyltransferase I activity was constant from day 7 to day 16, after which it declined 5-fold, whereas galactosyltransferase II activity fell markedly from day 9 to 13 and 16 to 20, displaying an overall 8-fold decrease. In primary muscle cultures, galactosyltransferase I activity fell slightly during 7 days in culture, whereas galactosyltransferase II increased 2-fold during the same period. No significant change in activity of either galactosyltransferase was observed during intercellular recognition and fusion. Analysis of muscle cultures treated with cytosine arabinoside and of fibroblast cultures revealed that the majority of galactosyltransferase I activity in primary muscle cultures is associated with fibroblasts, whereas the majority of galactosyltransferase II activity is muscle-associated. The addition of 5-bromodeoxyuridine to primary muscle cultures resulted in a 3-fold rise in activities of both transferases.

Animals↗
PubMed1981

On the uptake of exogenous catecholamines by adrenal chromaffin cells and nerve endings.

Light-microscopic autoradiography has revealed characteristic labelling patterns in adrenal medullary cells following the intravenous administration of different catecholamines. The uptake patterns for [3H] dopa, [3H] dopamine, [3H] noradrenaline and [3H] adrenaline ahve been compared. In all cases A cells were more active than NA cells and cells situated in the zone nearest the cortex demonstrated a markedly higher rate of uptake than central cells. It was concluded that adjacent chromaffin cells with very similar morphology may differ as much as 50 fold in their capacities to incorporate exogenous amines. The adrenergic nature of te innervation of the vessels of the adrenal cortex and capsule in the mouse was confirmed.

Adrenal Medulla↗
PubMed1981

Effect of hydrocortisone, reserpine, propranolol and phentolamine on in vivo uptake of exogenous amines by adrenal chromaffin cells.

An autoradiographic study was performed on the effects of hydrocortisone, reserpine, propranolol and phentolamine on the uptake of tritiated amines by adrenal medullary cells of the mouse. Oral feeding of hydrocortisone ahd no significant effect on the normal uptake pattern of dopamine, noradrenaline or adrenaline by medullary cells of different type (A cells or NA cells) or location (marginal or central), although the overall amounts taken up were markedly reduced. Handling the animals led to similar reductions in the uptake of all three amines and was thus clearly shown to be the important factor in this effect. Reserpine reduced the uptake of [3H] noradrenaline to 25% of the control value although the relative distribution remained unchanged. Propranolol and phentolamine had no observed effect on [3H] noradrenaline uptake. These results are discussed in the light of the previously reported action of ACTH in reversing the effects of hypophysectomy on medullary amine uptake (Hirano and Kobayashi 1978), and it is concluded that ACTH must exert this effect directly on the adrenal medulla rather than through the secretion of adrenal corticosteroids. It is also suggested that reserpine acts, as in neurons, by blocking amine uptake into intracellular granules rather than by blocking uptake into the cell itself.

Adrenal Medulla↗
PubMed1980

Regulation of phosphatidylcholine biosynthesis in cultured chick embryonic muscle treated with phospholipase C.

Cultures of embryonic chick muscle cells grown in medium containing phospholipase C from Clostridium perfringens incorporated [3H]choline into lipid at a rate 3- to 5-fold higher than control cultures. To determine the mechanism by which stimulation of phosphatidylcholine synthesis occurred in phospholipase C-treated cells, activities of enzymes and levels of intermediates in the biosynthetic pathway for phosphatidylcholine were examined. Activities of choline kinase, choline phosphotransferase, glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate acyltransferase, acylglycerol-3-phosphate acyltransferase, and phosphatidic acid phosphatase in phospholipase C-treated cells were the same or only slightly higher than in control cells. CTP:phosphocholine cytidylyltransferase, on the other hand, was 3 times as active in homogenates from phospholipase C-treated cells. Levels of phosphocholine decreased and levels of CDP-choline increased in phospholipase C-treated cells, and a calculation of the disequilibrium ratio indicated that the cytidylyltransferase reaction was not at equilibrium. The cytidylyltransferase was, thus, identified as the regulatory enzyme for choline flux in these cells. The cytidylyltransferase was located in both the cytosolic and particulate fractions from cultured muscle cells and a much larger portion of enzyme activity was associated with the particulate fraction in cells treated with phospholipase C. Sonicated preparations of total chick lipids, phosphatidylethanolamine, and phosphatidylserine greatly stimulated the cytosolic cytidylyltransferase activity but had no effect on the particulate enzyme. Neither stimulation of incorporation of [3H]choline into lipid nor activation of the cytidylyltransferase was dependent on protein synthesis. A model for the mechanism of regulation of phosphatidylcholine synthesis in embryonic chick muscle is presented.

Animals↗
PubMed1979

Stimulation of phospholipid metabolism in embryonic muscle cells treated with phospholipase C.

Phospholipid metabolism is dramatically stimulated in cultured myogenic cells in which cell fusion was inhibited with phospholipase C (phosphatidylcholine choline-phosphohydrolase; EC 3.1.4.3). Phospholipase C was active under the culture conditions as shown by the degradation of exogenous phosphatidylcholine. Rates of incorporation of 32Pi and [methyl-3H]choline into lipids were about 5-fold greater in phospholipase-treated cells than in either untreated fusing cells or untreated cells prevented from fusing by calcium deprivation. The greatest stimulation in the phospholipase C-treated cultures occurred with synthesis of phosphatidylcholine and sphingomyelin; synthesis of phosphatidylinositol and cardiolipin was not stimulated. Degradation of cellular [32P]phosphatidylcholine and appearance in the culture medium of the degradation product [32P]phosphocholine were both increased. Levels of total cellular phospholipids and of individual phospholipid classes were similar in control and phospholipase-treated cells. The results suggest that the membrane phospholipid composition in myogenic cells is controlled by a regulatory mechanism which increases the synthesis of phospholipids that are degraded in the presence of the phospholipase.

Cell Fusion↗
PubMed1978

Observations on the localization of labelled amino acid in mouse adrenal chromaffin cells after the injection of L-[4,5-3H] leucine.

The intracellular localization of L-[4,5-3H] leucine in chromaffin cells has been observed using light and electron microscopic autoradiography and the association of the labelled amino acid with particular cell components confirmed by statistical analysis. By making observations at short intervals after a single intravenous pulse of [3H]leucine it has been possible to follow the movement of the isotope from the endoplasmic reticulum through the Golgi complex to the chromaffin granules. No evidence for movement of the label through the Golgi complex was observed in adjacent cortical cells. The time sequence of transport of the amino acid through the various cell organelles was very similar to that observed by previous workers in protein-secreting exocrine cells.

Adrenal Glands↗