Phosphorylation of the glucose transporter in vitro and in vivo by protein kinase C

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) is present in many mammalian tissues, and its important physiological protein substrates are only now beginning to be identified. A useful advance in identifying these intracellular substrates has been the recognition that the kinase is the receptor for phorbol esters, which stimulate phosphotransferase activity. Phorbol ester-induced changes in protein phosphorylation in intact cells may thus be taken, in part, as a probable indication of protein kinase C activation. The many cellular effects of phorbol esters include the stimulation of glucose uptake, although the response of glucose uptake to phorbol esters appears to be complex, apparently varying in response time and requirement for protein synthesis. Such observations prompted us to explore one possible explanation for the alteration of glucose uptake, namely, phosphorylation of the glucose transporter by protein kinase C. We report here that incubation of purified human erythrocyte glucose transporter with rat brain protein kinase C results in the phosphorylation of a protein of relative molecular mass (Mr) 50,000-60,000 which has subsequently been identified as the glucose transporter by specific immunoprecipitation with a monoclonal antibody. Immunoprecipitation of membrane proteins from 32P-labelled human erythrocytes revealed a phorbol ester-stimulated phosphorylation of the transporter. This covalent modification of the glucose transporter may thus, in part, underlie the ability of phorbol esters and certain hormones to stimulate glucose uptake.     

Demonstration of retinoic acid isomers in human urine under physiological conditions

Untransformed retinoic acid has never been demonstrated in human excreta under normal physiological conditions. We have developed a two-step liquid chromatographic system for the demonstration of subnanogram amounts of this compound in human urine without administration of any precursor.     

Uninterrupted protein synthesis is essential for survival in the early stages of carbontetrachloride-induced hepatocellular necrosis in the mouse

Summary The fatal syndrome produced by cycloheximide given 6 h after a hepatonecrogenic dose of CCl₄ is due neither to direct toxic synergism between CCl₄ and cycloheximide nor to transient sinusoidal thrombosis. It is suggested that survival in the presence of unknown factors released from dying liver cells requires uninterrupted protein synthesis. The life-saving effect of sterilization of the intestine by antibiotics indicates that the gut flora or its products play a vital role in pathogenesis.Acknowledgments. I wish to thank Mr C. R. West for carrying out the statistical analysis, Mrs Brenda Brooks for histological processing, and Berk Pharmaceuticals Ltd for information on Ancrod defibrination in mice.     

Escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae

The migration of genetic information from ancestral prokaryotic endosymbionts into eukaryotic nuclei is thought to have had an important role in the evolution of mitochondria and chloroplasts. Here we describe an assay for the detection of movement of DNA between mitochondria and the nucleus in yeast. Because recombinant plasmid DNA replicates after transformation into mitochondria of yeast strains lacking endogenous mitochondrial DNA we were able to propagate the nuclear genetic marker URA3 in mitochondria. As expected, the wild-type URA3 gene in mitochondria failed to complement the uracil auxotrophy (Ura-) caused by a nuclear ura3 mutation. But selection of Ura+ prototrophs from a Ura- strain carrying URA3 on a plasmid in its mitochondria enabled us to detect plasmid movement to the nucleus. Conversely, as the plasmid used also contained the mitochondrial gene COX2 required for respiratory growth, we were able to set up corresponding selections to detect migration of DNA from the nucleus to the mitochondria. Our results show that, in yeast, DNA escapes from mitochondria and appears in the nucleus at a surprisingly high frequency (approximately 2 x 10(-5) per cell per generation). But the rate at which DNA makes the journey in the opposite direction--nucleus to mitochondria--is apparently at least 100,000 times less.     

No evidence for expression of the insulin-regulatable glucose transporter in endothelial cells

A major effect of insulin is to increase glucose transport in muscle and fat. A family of genes encoding distinct mammalian glucose transporters has recently been elucidated. One of these, the insulin-regulatable glucose transporter (IRGT), is primarily expressed in muscle and fat, tissues that exhibit insulin-dependent glucose transport. Insulin promotes glucose transport in these tissues by stimulating movement of the glucose transporter from an intracellular location to the plasma membrane. Recent studies, however, suggest that an additional effect of insulin in these tissues may be the facilitation of glucose transport, presumably across capillary endothelium. This hypothesis is based on the localization of the IRGT in endothelial cells specific to muscle and adipose tissue. We report here, however, on morphological and biochemical studies using several different IRGT-specific antibodies in which we could not reproduce these results.     

The effect of guanethidine treatment of testicular blood flow and testosterone production in rats

Testicular blood flow was measured by means of Xenon-133 clearance in control rats and rats treated with guanethidine for 3 weeks. Plasma and testis testosterone concentrations were also measured, and the effect of hCG-treatment was examined. No difference in testicular blood flow between the control group and the guanethidine-treated group was found. However, in guanethidine-treated rats, plasma and testis testosterone concentrations after hCG-treatment were significantly decreased. The results may indicate that adrenergic nerves are involved in the regulation of Leydig cell function.     

Urushiol components as mediators in DNA strand scission

Summary Poison oak urushiol, a mixture of 3-alk(en)ylcatechol derivatives was found to mediate DNA strand scission in the presence of oxygen and with copper(II) chloride as a catalyst. The reactionis believed to occur via activated reduced oxygen produced during oxidation of the catechol into itso-quinone derivative.