The mechanism of receptor-mediated endocytosis.
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Biomedical subjects
Publications and source records attributed to G Warren.
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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.
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.
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.
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.
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.
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.
The development of neighbourhood forums in North Staffordshire is the result of a simple belief that, to deliver cost-effective services of quality, care needs to be provided through a partnership with those who live and work in that neighbourhood. Glenn Warren outlines the role and function of forums.
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.
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Facial palsy is cosmetically unacceptable, whether affecting eyes or mouth. It endangers the vision. Both upper and lower facial paralysis can be surgically dealt with, using the temporal muscle and fascia. This produces satisfactory results in a relatively short period of time.
The consensus sequence of three bacterial ice nucleation proteins was determined by extrapolation from the nucleotide (nt) sequences of three ice nucleation-encoding genes, iceE (presented here), inaW and inaZ. The three proteins possess considerable similarity, so that a preferred amino acid is shown in most positions of the consensus. The corresponding genes show considerable divergence in the third nt positions of synonymous codons, suggesting that the proteins' conserved features have been maintained by selection. Therefore, the consensus sequence is likely to represent the components of primary structure most important to the ice nucleation function.
Certain bacteria possess proteins that enable them to nucleate crystallization in supercooled water. These ice-nucleation proteins are thought to produce templates for the assembly of very small seed crystals of ice. The proteins from different species have related, internally repetitive primary structures, which may be directly responsible for aligning the water molecules of the seed crystal.
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