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)

Other workers have reported that vesicular stomatitis virus makes aberrantly long polyadenylic acid [poly(A)] tracts in the presence of S-adenosylhomocysteine (S-Ado-Hcy). In the work reported in this paper, the effects of various analogues of S-adenosylmethionine (S-Ado-Met) and ATP on polyadenylation in an in vitro transcription system were examined to determine whether S-Ado-Hcy exerted its effect on polyadenylation due to its relationship to S-Ado-Met or to ATP. It appeared that compounds which affected polyadenylation were those which were closely related to S-Ado-Met and that had the same L-aminoacyl side chain [(COOH)-CH(NH)2-CH2-CH2-]; the nature of the substituent at the -S+(CH3)- position of S-Ado-Met was less important. These analogues appeared to compete with S-Ado-Met for a binding site(s). These data support a model whereby compounds binding at an S-Ado-Met-binding site may have allosteric effects by causing or preventing conformational changes which are involved in polyadenylation reactions, perhaps by affecting the rate of polyadenylation or of termination.
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PMID:Effect of analogues of S-adenosylmethionine on in vitro polyadenylation by vesicular stomatitis virus. 256 41

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 characterized the process by which the vesicular stomatitis virus (VSV) G protein acquires its final oligomeric structure using density-gradient centrifugation in mildly acidic sucrose gradients. The mature wild-type VSV G protein is a noncovalently associated trimer. Trimers are assembled from newly synthesized G monomers with a t1/2 of 6-8 min. To localize the site of trimerization and to correlate trimer formation with steps in transport between the endoplasmic reticulum (ER) and Golgi complex, we examined the kinetics of assembly of the temperature-sensitive mutant VSV strain, ts045. At the nonpermissive temperature (39 degrees C), ts045 G protein is not transported from the ER. The phenotypic defect that inhibited export from the ER at the nonpermissive temperature was found to be the accumulation of ts045 G protein in an aggregate. After being shifted to the permissive temperature (32 degrees C), the ts045 G protein aggregate rapidly dissociated (t1/2 less than 1 min) to monomeric G protein which subsequently trimerized with the same kinetics as the wild-type G protein. Only trimers were transported to the Golgi complex. Kinetic studies, as well as the finding that trimerization occurred under conditions which block ER to Golgi transport (at both 15 and 4 degrees C), showed that trimers were formed in the ER. Depletion of cellular ATP inhibited both the dissociation of the aggregated intermediate of ts045 G protein as well as the formation of stable trimers. The results indicate that oligomerization of G protein occurs in several steps, is sensitive to cellular ATP, and is required for transport from the ER.
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PMID:Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers. 282 24

We have examined the requirement for ribonucleotides and ribonucleotide triphosphate hydrolysis during early events in the membrane integration of two membrane proteins: the G protein of vesicular stomatitis virus and the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus. Both proteins contain a single transmembrane-spanning segment but are integrated in the membrane with opposite orientations. The G protein has an amino-terminal signal sequence and a stop-transfer sequence located near the carboxy terminus. The HN glycoprotein has a single sequence near the amino terminus that functions as both a signal-sequence and a transmembrane-spanning segment. Membrane insertion was explored using a cell-free system directed by transcribed mRNAs encoding amino-terminal segments of the two proteins. Ribosome-bound nascent polypeptides were assembled, ribonucleotides were removed by gel filtration chromatography, and the ribosomes were incubated with microsomal membranes under conditions of defined ribonucleotide content. Nascent chain insertion into the membrane required the presence of both the signal recognition particle and a functional signal recognition particle receptor. In the absence of ribonucleotides, insertion of nascent membrane proteins was not detected. GTP or nonhydrolyzable GTP analogues promoted efficient insertion, while ATP was comparatively ineffective. Surprisingly, the majority of the HN nascent chain remained ribosome associated after puromycin treatment. Ribosome-associated HN nascent chains remained competent for membrane insertion, while free HN chains were not competent. We conclude that a GTP binding protein performs an essential function during ribosome-dependent insertion of membrane proteins into the endoplasmic reticulum that is unrelated to protein synthesis.
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PMID:Integration of membrane proteins into the endoplasmic reticulum requires GTP. 283 21

Phosphorylation of membrane-associated proteins by protein kinases in the membrane fraction from HeLa S3 cells was rapidly increased when the cells were infected with vesicular stomatitis virus (VSV). SDS-PAGE followed by autoradiography revealed polypeptides with molecular sizes of Mr. 53,000, 44,000, 42,000, 35,000, 30,000 and 27,000 in the kinase fraction from uninfected cells to be highly phosphorylated. Virus-coding NS protein (Mr. 40,000) was phosphorylated when the membrane fraction from virus-infected cells was incubated with [gamma-32P]ATP in the presence of histone H1 and Mg2+. Under these conditions, histone H1 functioned as a stimulator for NS protein phosphorylation by the kinases. One (kinase III) of the membrane-associated kinases was partially purified from HeLa S3 cells using FPLC (type Mono Q) after DEAE-cellulose column chromatography. The enzymatic properties of kinase III were similar to those reported for a polypeptide-dependent protein kinase (protein kinase P), because (a) both kinases highly phosphorylated beta-casein, although no phosphorylation was observed with histones; (b) several endogenous substrates from HeLa S3 cell membrane were phosphorylated by the kinases in the presence of basic proteins, such as histones, protamine and poly-Lys; (c) their activity was insensitive to a low concentration (19 micrograms/ml) of heparin, which highly inhibited casein kinase II activity; and (d) the kinases were extractable from the plasma membrane using Triton X-100. In addition, provided evidence suggests that kinase III may play an important role in an early stage of VSV replication through its specific phosphorylation of NS protein and membrane proteins in virus infected cells.
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PMID:Characterization of a polypeptide-dependent membrane protein kinase that specifically phosphorylates NS protein of vesicular stomatitis virus in vitro. 284 89

In a continuation of previous efforts to study the modified ATP requirements for RNA synthesis by poIR mutants of vesicular stomatitis virus (VSV), we have used a novel reconstitution assay to show that it is the template moiety of the mutants, not the polymerase proteins, which governs both the increased utilization of the ATP analog, beta, gamma-imido ATP (AMP-PMP), and the loss of a positive cooperativity-like response to varying ATP concentrations. Assays utilized uv-irradiated virus as a source of polymerase proteins and purified N-RNA as templates. Homologous and heterologous transcriptase reactions were carried out with wild-type (wt) virus and each of the two independently isolated poIR mutants. We show that in the presence of wt N-RNA template, substitution of AMP-PNP for ATP resulted in only approximately 5% of control RNA synthesis regardless of which source of polymerase was used. Furthermore, all reactions containing wt N-RNA template responded to varying ATP concentrations with a concave, upward-shaped Lineweaver-Burke plot generally indicative of positive cooperativity effects. In contrast, all reactions which utilized N-RNA templates from the poIR mutants showed an increased utilization of AMP-PNP (greater than 20%) and a more characteristic Michaelis-Menten response to changing ATP concentrations. These findings strongly support the notion that the template-associated nucleocapsid protein modulates the utilization of an ATP site which is directly or indirectly involved in VSV RNA synthesis.
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PMID:Altered ATP utilization by the poIR mutants of vesicular stomatitis virus maps to the N-RNA template. 284 22

A fibroblast mutant cell line lacking the Na+/H+ antiporter was used to study the influence of low cytoplasmic pH on membrane transport in the endocytic and exocytic pathways. After being loaded with protons, the mutant cells were acidified at pH 6.2 to 6.8 for 20 min while the parent cells regulated their pH within 1 min. Cytoplasmic acidification did not affect the level of intracellular ATP or the number of clathrin-coated pits at the cell surface. However, cytosolic acidification below pH 6.8 blocked the uptake of two fluid phase markers, Lucifer Yellow and horseradish peroxidase, as well as the internalization and the recycling of transferrin. When the cytoplasmic pH was reversed to physiological values, both fluid phase endocytosis and receptor-mediated endocytosis resumed with identical kinetics. Low cytoplasmic pH also inhibited the rate of intracellular transport from the Golgi complex to the plasma membrane. This was shown in cells infected by the temperature-sensitive mutant ts 045 of the vesicular stomatitis virus (VSV) using as a marker of transport the mutated viral membrane glycoprotein (VSV-G protein). The VSV-G protein was accumulated in the trans-Golgi network (TGN) by an incubation at 19.5 degrees C and was transported to the cell surface upon shifting the temperature to 31 degrees C. This transport was arrested in acidified cells maintained at low cytosolic pH and resumed during the recovery phase of the cytosolic pH. Electron microscopy performed on epon and cryo-sections of mutant cells acidified below pH 6.8 showed that the VSV-G protein was present in the TGN. These results indicate that acidification of the cytosol to a pH less than 6.8 inhibits reversibly membrane transport in both endocytic and exocytic pathways. In all likelihood, the clathrin and nonclathrin coated vesicles that are involved in endo- and exocytosis cannot pinch off from the cell surface or from the TGN below this critical value of internal pH.
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PMID:Low cytoplasmic pH inhibits endocytosis and transport from the trans-Golgi network to the cell surface. 291 22

The structural proteins L and NS of vesicular stomatitis virus were obtained from purified viral ribonucleoprotein complex followed by phosphocellulose column chromatography and assayed for protein kinase activity using [gamma-32P]ATP as the phosphate donor. The fractions containing purified L protein phosphorylated NS protein in vitro. 8-Azido-ATP, a photoreactive analogue of ATP, was also used as the phosphate donor for phosphorylation of NS protein by the L protein. In the presence of ultraviolet light, only L protein was specifically cross-linked with 8-azido-[gamma-32P]ATP. In the absence of u.v. light 8-azido ATP did no inhibit RNA transcription in a reconstituted reaction or substitute ATP for RNA synthesis in vitro. The above results, taken together, suggest that 8-azido-ATP was bound to the kinase site and phosphorylation of NS protein was mediated by the L protein. Exogenous phosphate acceptor proteins such as phosvitin and casein were also phosphorylated by the L protein fraction. However, addition of an excess of phosvitin failed to compete with the phosphorylation of NS by L, indicating that the protein kinase activity possessed higher affinity for NS. The phosphorylation of NS was strongly inhibited by photoreaction of L protein with 8-azido-ATP with concomitant inhibition of transcription in vitro. These results suggest that phosphorylation of NS protein by L may have a role in the regulation of the virus genome transcription in vitro.
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PMID:In vitro phosphorylation of NS protein by the L protein of vesicular stomatitis virus. 298 94

Transport of the vesicular stomatitis virus (VSV)-encoded glycoprotein (G protein) between successive compartments of the Golgi in a cell-free system is measured by the coupled incorporation of N-[3H]acetylglucosamine (GlcNAc). This glycosylation occurs when G protein is transported from a "donor" compartment in Golgi membranes that lack GlcNAc transferase I (from VSV-infected CHO clone 15B cells) to the next "acceptor" compartment in a Golgi population from wild-type CHO cells (containing the GlcNAc transferase but not G protein). Here we present a detailed characterization of the conditions required to achieve transport in vitro. We find that donor and acceptor activities differ markedly in certain of their properties. The donor activity is inhibited by N-ethylmaleimide but the acceptor activity is resistant. Donor activity is unstable in the absence of ATP or the cytosol fraction; acceptor activity is much more stable. This asymmetry may reflect the vectorial nature of the underlying biochemistry of protein transport. Both donor and acceptor are trypsin-sensitive, implying a need for cytoplasmically oriented membrane proteins. Transport occurs only in a restricted range of close to physiological conditions. ATP is absolutely required, although as little as 1 microM is sufficient. Transport is inhibited by ATP-gamma-sulfate and vanadate, suggesting that ATP hydrolysis is needed. By contrast, ionophores that dissipate membrane potentials and proton gradients do not inhibit transport. Monensin was also without effect in the cell-free system.
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PMID:Characterization of protein transport between successive compartments of the Golgi apparatus: asymmetric properties of donor and acceptor activities in a cell-free system. 299 Mar 47

Murine LSTRA lymphoma cells contain a very active tyrosine protein kinase of 56 kDa (p56) which is not related to any of the other known tyrosine kinases. In the past the purification and characterization of the p56 have been hampered because of the low amount of this protein in LSTRA membranes. In this study, we have utilized a different approach for purification which consisted of trapping the protein in the membrane of vesicular stomatitis virus. Incubation of the virions with [gamma-32P]ATP resulted in the phosphorylation of p56 on tyrosine residues. Moreover, the phosphopeptide digest profile of vesicular stomatitis virus-p56 was identical to that observed with authentic LSTRA-p56. The p56 from such virions could be resolved from other proteins by two-dimensional gels, and furthermore, such virions have been used to prepare several antisera directed against the p56.
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PMID:Vesicular stomatitis virus produced from infected LSTRA lymphoma cells bear tyrosine protein kinase activity (p56). 299 8


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