Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0038362 (stomatitis)
8,852 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a highly efficient in vitro-transport assay that couples translocation across the ER membrane and transport to the Golgi complex using the secreted pheromone alpha-factor as a marker protein. Radiolabeled prepro-alpha-factor of high specific radioactivity is obtained by in vitro-translating this protein in a yeast lysate. Prepro-alpha-factor synthesized in vitro is then translocated directly into microsomes or the ER of permeabilized yeast cells. Conversion of the 26-kDa ER form of pro-alpha-factor to the high molecular weight Golgi form is dependent on the presence of ATP and soluble and membrane-bound factors. Differential centrifugation and fractionation on a sucrose gradient have shown that the ER and Golgi forms of alpha-factor are enriched in separate compartments after the transport reaction. These and other findings (see Ruohola et al., 1988, for a more complete discussion) indicate that conversion to the high molecular weight form of alpha-factor is the result of authentic intercompartmental transport. Permeabilized mammalian cells have been used to reconstitute transport from the ER to the Golgi complex. In these systems (Becker et al., 1987; Simons and Virta, 1987), a viral membrane glycoprotein protein (vesicular stomatitis virus G protein) is used as the marker protein. This protein is radiolabeled with [35S]methionine during virus infection, either before or after the cells are permeabilized. Radiolabeled G protein, residing in the ER, is then transported to the Golgi complex in the presence of an ATP-regenerating system. In the mammalian system the donor and acceptor compartments are retained within the permeabilized cells (Simons and Virta, 1987); however, on occasion the addition of an exogenous acceptor compartment is required (Beckers et al., 1987). The assay we developed (Ruohola et al., 1988) differs from the mammalian assay (Beckers et al., 1987) in that we introduce radiolabeled marker protein into the ER in vitro during translocation rather than during virus infection. In addition, in our assay the acceptor Golgi compartment is always provided exogenously to the permeabilized cells. Therefore, if acceptor membranes are present in the PYC, they are not utilized. Because the permeabilized cells and the S3 fraction are prepared differently, the conditions used to prepare the cells may lead to inactivation or loss of the acceptor compartment. The in vitro assay will enable us to purify components involved in transporting proteins from the lumen of the ER to the Golgi complex. Antibody prepared to purified components can be used to clone the genes that code for these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Reconstitution of transport from the ER to the Golgi complex in yeast using microsomes and permeabilized yeast cells. 267 24

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.
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PMID:Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins. 282 Oct 12

The interaction of the mouse hepatitis virus (MHV) nucleocapsid protein (N) and viral RNA was examined. Monoclonal antibody specific for N protein coimmunoprecipitated MHV genomic RNA as well as all six MHV subgenomic mRNAs found in MHV-infected cells. In contrast, monoclonal antibodies to the MHV E2 or E1 envelope glycoproteins, an anti-I-A monoclonal antibody, and serum samples from lupus patients did not immunoprecipitate the MHV mRNAs. Moreover, the anti-N monoclonal antibody did not coimmunoprecipitate vesicular stomatitis virus RNA or host cell RNA under conditions which immunoprecipitated all MHV RNAs. These data suggest a specific interaction between the N protein and the virus-specific mRNAs. Both the membrane-bound and cytosolic small MHV leader-specific RNAs of greater than 65 nucleotides long were immunoprecipitated only by anti-N monoclonal antibody. These data suggest that an N binding site is present within the leader RNA sequences at a site at least 65 nucleotides from the 5' end of genomic RNA and all six subgenomic mRNAs. The larger leader-containing RNAs originating from mRNA 1 and mRNA 6, as well as the MHV negative-stranded RNA, were also immunoprecipitated by the anti-N monoclonal antibody. These data indicate that the MHV N protein is associated with MHV-specific RNAs and RNA intermediates and may play an important functional role during MHV transcription and replication.
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PMID:Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription. 284 40

Transport of the vesicular stomatitis virus-encoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (man GlcNAc2) ER form of G protein to the man5GlcNAc5 form by the cis Golgi enzyme alpha-mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the man5GlcNAc2 form without the appearance of the intermediate man GlcNAc2 oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi alpha-mannosidase I inhibitor 1-deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supernatant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro.
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PMID:Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system. 302 44

Membrane-bound polysomes from vesicular stomatitis virus (VSV)-infected HeLa cells synthesize predominantly three proteins in an in vitro protein synthesizing system. These three proteins have different molecular weights than the viral structural proteins, i.e., 115,000, 88,000, and 72,000. Addition of preincubated L or HeLa cell S10 or HeLa cell crude initiation factors stimulates amino acid incorporation and, furthermore, alters the pattern of proteins synthesized. Stimulated membrane-bound polysomes synthesize predominantly viral protein G and lesser amounts of N, NS, and M. In vitro synthesized proteins G and N are very similar to virion proteins G and N based on analysis of tryptic methionine-labeled peptides. Most methionine-labeled tryptic peptides of virion G protein contain no carbohydrate moieties, since about 90% of sugar-labeled peptides co-chromatograph with only about 10% of methionine-labeled peptides. Sucrose gradient analysis of the labeled RNA present in VSV-infected membrane-bound polysomes reveals a relative enrichment in a class of viral RNA sedimenting slightly faster than the total population of the 13 to 15S mRNA, as compared to a VSV-infected crude cytoplasmic extract. A number of proteins, other than the viral structural proteins, are synthesized in the cytoplasm of five lines of VSV-infected cells. One of these proteins has the same molecular weight as the major in vitro synthesized protein, P(88). In vitro synthesized protein P(88) does not appear to be a precursor of viral structural proteins G, N, or M based on pulse-chase experiments and tryptic peptide mapping. Nonstimulated membrane-bound polysomes from uninfected HeLa cells synthesize the same size distribution of proteins as nonstimulated VSV-infected membrane-bound polysomes.
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PMID:In vitro synthesis of proteins by membrane-bound polyribosomes from vesicular stomatitis virus-infected HeLa cells. 436 99

The entry of many animal viruses into their host cells often proceeds via a specialized internalization pathway involving clathrin-coated regions of the plasma membrane. We have examined the effect of dansylcadaverine and amantadine on the entry of vesicular stomatitis virus (VSV) into mouse cells. Both compounds inhibit VSV entry as determined by fluorescence and electron microscopy, 3H-labeled VSV uptake, and VSV-dependent RNA synthesis assays. They also inhibit the uptake of alpha 2-macroglobulin, a protein that binds to specific membrane receptors and follows the same route of internalization. Dansylcadaverine is 20-fold more potent than amantadine in blocking virus and alpha 2-macroglobulin uptake. One cellular target for both of these amine-containing compounds appears to be the clustering of membrane-bound ligands or particles in clathrin-coated pits.
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PMID:Amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of alpha 2-macroglobulin. 617 94

Simple, sensitive, and reproducible assay systems for measurement of the biological activity of interferon are described. The methods used are based on the quantification of cell membrane-bound viral and cellular antigens in interferon-treated cells by enzyme immunoassays. To measure the antiviral activity, samples of human interferon are titrated in microplates with human or bovine cells. After incubation with challenge virus (vesicular stomatitis or herpes simplex virus) the cells are fixed with glutaraldehyde and assayed for viral antigens by enzyme-labeled antibodies. This assay permits the detection of less than 0.1 unit of interferon per milliliter, after optimization of several factors, such as type of cell, multiplicity of infection, temperature, and period of incubation. The effect of interferon on cellular antigens is measured in a similar way, by using peroxidase-labeled antibodies directed against beta 2-microglobulin. The two types of assays described appear suitable for kinetic experiments and for detection of interferons of different specificities in body fluids.
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PMID:Bioimmunoassays (BIAs) of human interferon. 619 51

Human hepatoma cells, infected by vesicular stomatitis virus, offer a good system to study simultaneously the intracellular localization of a well defined transmembrane glycoprotein (VSV-G), a secretory glycoprotein (transferrin), and a nonglycosylated secretory protein (albumin). We used monospecific antibodies in combination with 5- and 8-nm colloidal gold particles complexed with protein A to immunolabel these proteins simultaneously in thin frozen sections of hepatoma cells. VSV-G, transferrin, and albumin are present in the same rough endoplasmic reticulum cisternae, the same Golgi compartments, and the same secretory vesicles. In the presence of the ionophore monensin intracellular transport is blocked at the trans cisternae of the Golgi complex, and VSV-G, transferrin, and albumin accumulate in dilated cisternae, which are apparently derived from the trans-Golgi elements. Glycoproteins, synthesized and secreted in the presence of monensin, are less acidic than those in control cultures. This is probably caused by a less efficient contact between the soluble secretory proteins and the membrane-bound glycosyltransferases that are present in the most monensin-affected (trans) Golgi cisternae.
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PMID:Vesicular stomatitis virus glycoprotein, albumin, and transferrin are transported to the cell surface via the same Golgi vesicles. 631 44

In eukaryotic cells, secretory proteins and glycoproteins migrate from the rough endoplasmic reticulum, their site of synthesis, through Golgi vesicles before being released from the cell. Cellular and viral integral plasma membrane glycoproteins are co-translationally inserted into the rough endoplasmic reticulum membrane and follow a similar pathway to the cell surface. Previous studies using endoglycosidase H (Endo H) suggested that in rat hepatoma cells the vesicular stomatitis virus (VSV) G protein, albumin and transferrin migrate from the rough endoplasmic reticulum to the Golgi apparatus at different rates. Here we show directly that in human hepatoma HepG2 cells, five secreted proteins mature from the rough endoplasmic reticulum to Golgi vesicles at characteristic rates which differ at least threefold. The results are incompatible with bulk-phase movement of the luminal contents of the endoplasmic reticulum, and suggest that there is a membrane-bound receptor that selectively mediates the transport of secretory proteins from the rough endoplasmic reticulum to the Golgi.
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PMID:Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates. 686 94

Using a pulse-chase approach combined with immunoprecipitation, we showed that newly synthesized influenza virus hemagglutinin (HA) and vesicular stomatitis virus G protein associate transiently during their folding with calnexin, a membrane-bound endoplasmic reticulum (ER) chaperone. Inhibitors of N-linked glycosylation (tunicamycin) and glucosidases I and II (castanospermine and 1-deoxynojirimycin) prevented the association, whereas inhibitors of ER alpha-mannosidases did not. Our results indicated that binding of these viral glycoproteins to calnexin correlated closely with the composition of their N-linked oligosaccharide side chains. Proteins with monoglucosylated oligosaccharides were the most likely binding species. On the basis of our data and existing information concerning the role of monoglucosylated oligosaccharides on glycoproteins, we propose that the ER contains a unique folding and quality control machinery in which calnexin acts as a chaperone that binds proteins with partially glucose-trimmed carbohydrate side chains. In this model glucosidases I and II serve as signal modifiers and UDP-glucose:glycoprotein glucosyltransferase, as a folding sensor.
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PMID:Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. 830 66


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