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Biomedical subjects

G Warren

Publications and source records attributed to G Warren.

At least 19 recordsLinked to original sources
PubMed1992

Sphingolipid transport in mitotic HeLa cells.

Mitotic and interphase HeLa cells were labeled with [3H]serine. Ceramide and its derivatives, lactosylceramide and sphingomyelin, were biosynthetically labeled under both conditions. Only in the absence of nocodazole, as the cells entered telophase, was an additional glycosphingolipid synthesized which was identified as GA2 (GalNAc(beta 1,4)Gal(beta 1,4)Glc(beta 1,1)Cer). Ceramide, the basic sphingolipid precursor, is synthesized in the endoplasmic reticulum, whereas its immediate derivatives are synthesized in early Golgi compartments. Transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi is inhibited in mitotic cells while ceramide acquires early Golgi modifications under the same conditions, suggesting that ceramide can be delivered to the Golgi by a different route. Since GA2 is synthesized in late Golgi, its absence in mitotic cells strongly argues for an in vivo inhibition of intra-Golgi transport, an observation with important implications for the mechanism of Golgi division.

Biological Transport↗
PubMed1992

Mitosis and inhibition of intracellular transport stimulate palmitoylation of a 62-kD protein.

Recent studies suggest that a cycle of acylation/deacylation is involved in the vesicular transport of proteins between intracellular compartments at both the budding and the fusion stage (Glick, B. S., and J. E. Rothman. 1987. Nature (Lond.). 326:309-312). Since a number of cellular processes requiring vesicular transport are inhibited during mitosis, we examined the fatty acylation of proteins in interphase and mitotic cells. We have identified a major palmitoylated protein with an apparent molecular weight of 62,000 (p62), whose level of acylation increases 5-10-fold during mitosis. Acylation was reversible and p62 was no longer palmitoylated in cells that have exited mitosis and entered G1. p62 is tightly bound to the cytoplasmic side of membranes, since it was sensitive to digestion with proteases in the absence of detergent and was not removed by treatment with 1 M KCl. p62 is removed from membranes by nonionic detergents or concentrations of urea greater than 4 M. The localization of p62 by subcellular fractionation is consistent with it being in the cis-Golgi or the cis-Golgi network. A palmitoylated protein of the same molecular weight was also observed in interphase cells treated with inhibitors of intracellular transport, such as brefeldin A, monensin, carbonylcyanide m-chlorophenylhydrazone, or aluminum fluoride. The protein palmitoylated in the presence of brefeldin A was shown to be the same as that palmitoylated during mitosis using partial proteolysis. Digestion with two enzymes, alkaline protease and endoprotease lys-C, generated the same 3H-palmitate-labeled peptide fragments from p62 from mitotic or brefeldin A-treated cells. We suggest that the acylation and deacylation of p62 may be important in vesicular transport and that this process may be regulated during mitosis.

Acylation↗
PubMed1992

Cell-free fusion of endocytic vesicles is regulated by phosphorylation.

Okadaic acid and microcystin-LR, both potent inhibitors of protein phosphatases (PP), blocked vesicle fusion in a cell-free system. The effect of okadaic acid was reversed by the purified catalytic subunit of PP2A, but not PP1. Inhibition was gradual, required Mg-ATP, and was reduced by protein kinase inhibitors, indicating that it was mediated via protein phosphorylation. A candidate protein kinase would be cdc2 kinase, which normally is active in mitotic extracts and has been shown to inhibit endocytic vesicle fusion (Tuomikoski, T., M.-A. Felix, M. Dorée, and J. Gruenberg. 1989. Nature (Lond.). 342:942-945). However, it would appear that cdc2 kinase is not responsible for inhibition by okadaic acid. When compared to cytosol prepared from mitotic cells, okadaic acid did not increase cdc2 kinase activity sufficiently to account for the inhibition. In addition, inhibition was maintained when cdc2 protein was depleted from cytosol.

CDC2 Protein Kinase↗
PubMed1991

The membrane spanning domain of beta-1,4-galactosyltransferase specifies trans Golgi localization.

Chimeric cDNAs were constructed so as to generate hybrid proteins in which different parts of the N-terminal domain of the human invariant chain were replaced by equivalent sequences from the trans Golgi resident enzyme, beta-1,4-galactosyltransferase. The cytoplasmic and membrane spanning domains of galactosyltransferase were found to be sufficient to retain all of the hybrid invariant chain in trans Golgi cisternae as judged by indirect immunofluorescence, treatment with brefeldin A and immuno-electron microscopy. As few as ten amino acids corresponding to the lumenal half of the membrane spanning domain of the Golgi enzyme sufficed to localize most of the hybrid invariant chain to the trans cisternae. A cytoplasmic domain was necessary for complete retention as assessed by flow cytofluorometry but could be provided either by galactosyltransferase or by invariant chain. This suggests that the cytoplasmic domain plays a role accessory to the membrane spanning domain, the latter mediating compartmental specificity.

Amino Acid Sequence↗
PubMed1991

Isolation of functional, coated, endocytic vesicles.

Brief internalization of [125I]transferrin was used to label coated endocytic vesicles, which were then purified using a combination of 2H2O and 2H2O/Ficoll density gradients. Purification was monitored using an assay measuring fusion of endocytic organelles, so as to isolate functional vesicles. Isolated vesicles had all the properties of clathrin-coated vesicles, being enriched for the major components of clathrin coats and uncoated by either 1 M Tris-HCl or an uncoating ATPase. Nearly half of the labeled vesicles were able to participate in subsequent fusion events, as measured by the cell-free assay. Fusion was specific, requiring energy and cytosol, and being sensitive to N-ethyl maleimide.

Cell Fractionation↗
PubMed1991

Mitotic cytosol inhibits invagination of coated pits in broken mitotic cells.

Receptor-mediated endocytosis is inhibited during mitosis in mammalian cells and earlier work on A431 cells suggested that one of the sites inhibited was the invagination of coated pits (Pypaert, M., J. M. Lucocq, and G. Warren. 1987. Eur. J. Cell Biol. 45: 23-29). To explore this inhibition further, we have reproduced it in broken HeLa cells. Mitotic or interphase cells were broken by freeze-thawing in liquid nitrogen and warmed in the presence of mitotic or interphase cytosol. Using a morphological assay, we found invagination to be inhibited only when mitotic cells were incubated in mitotic cytosol. This inhibition was reversed by diluting the cytosol during the incubation. Reversal was sensitive to okadaic acid, a potent phosphatase inhibitor, showing that phosphorylation was involved in the inhibition of invagination. This was confirmed using purified cdc2 kinase which alone could partially substitute for mitotic cytosol.

CDC2 Protein Kinase↗
PubMed1991

Mirizzi's syndrome: a contraindication to coelioscopic cholecystectomy.

An impacted gallstone in the cystic duct with subsequent inflammation and edema resulting in extrinsic compression of the common bile or common hepatic duct with obstructive jaundice is known as Mirizzi's syndrome. An uncommon complication of cholelithiasis, Mirizzi's syndrome should be included in the differential diagnosis of any patient who has extrahepatic biliary obstruction. We present a case of a patient who underwent open rather than coelioscopic cholecystectomy based upon the preoperative diagnosis of Mirizzi's syndrome. A multidisciplinary approach to such patients facilitates the decision between open and endoscopic cholecystectomy. Mirizzi's syndrome may represent a contraindication to endoscopic cholecystectomy.

Adult↗
PubMed1991

Molecular aspects of microbial ice nucleation.

Certain organisms nucleate the crystallization of ice. This requires a small volume of water to be induced, probably by lattice-matching with a solid template, to form an 'ice embryo'--a region sharing at least some of the characteristics of macroscopic ice. It is of particular interest to understand the structure and function of biological structures capable of lattice-matching (or otherwise inducing a quasi-crystalline state). Some strains of the Gram-negative eubacterial genera Erwinia, Pseudomonas, and Xanthomonas, and the mycobionts of certain lichens, display ice-nucleating activity. In bacteria, the activity is conferred by a protein that contains three nested periodicities of repetition, which probably reflects a hierarchy of three motifs of structural repetition. Thus the tertiary structure of the ice-nucleation protein is likely to be regular, consistent with the expectation of its forming a template for lattice-matching. Even within a clonal culture, the nucleating sites formed by bacteria and lichens vary considerably in the threshold temperatures at which they display activity; this indicates wide variations in either the size of the template, or its structural regularity, or both. However, ice-nucleating sites of lichen and bacterial origin are clearly differentiated by their sensitivities to experimental treatments.

Amino Acid Sequence↗
PubMed1991

Okadaic acid induces Golgi apparatus fragmentation and arrest of intracellular transport.

The specific phosphatase inhibitor okadaic acid (OA) induced fragmentation of the Golgi apparatus in interphase HeLa cells. Immunoelectron microscopy for galactosyltransferase identified a major Golgi fragment composed of a cluster of vesicles and tubules that was morphologically indistinguishable from the 'Golgi cluster' previously described in mitotic cells. The presence of homogeneous immunofluorescence staining for galactosyltransferase in OA-treated cells also suggested that isolated Golgi vesicles, previously found in mitotic cells, existed along with the clusters. After removal of OA, both clusters and vesicles appeared to participate in a reassembly pathway that strongly resembled that occurring during telophase. OA also induced inhibition of intracellular transport, another feature of mitotic cells. OA treatment prevented newly synthesised G protein of vesicular stomatitis virus (VSV) from acquiring resistance to endoglycosidase H and from arriving at the cell surface. In addition, fluid phase endocytosis of horseradish peroxidase (HRP) was reduced to less than 10% of control values. All these effects were dose-dependent and reversible. OA should be a useful tool to study the Golgi division and membrane traffic.

Biological Transport↗
PubMed1991

Neighbourhood health forums: local democracy at work.

People in the North Staffordshire district in the United Kingdom have planned and implemented a community service project designed to increase local participation in health matters. Neighbourhood Forums meet to examine local needs and make decisions involving available resources and services. Self-observation by the Forums has revealed a potential for initiating positive change. Such interventions are applicable in other communities worldwide.

Community Participation↗