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W S Trimble

Publications and source records attributed to W S Trimble.

At least 55 records · Page 3Linked to original sources
PubMed1995

Identification of a second homolog of N-ethylmaleimide-sensitive fusion protein that is expressed in the nervous system and secretory tissues of Drosophila.

N-Ethylmaleimide-sensitive fusion protein (NSF) is an ATPase known to have an essential role in intracellular membrane transport events. Recently, cDNA clones encoding a Drosophila melanogaster homolog of this protein, named dNSF, were characterized and found to be expressed in the nervous system. We now report the identification of a second homolog of NSF, called dNSF-2 within this species and report evidence that this ubiquitous and widely utilized fusion protein belongs to a multigene family. The predicted amino acid sequence of dNSF-2 is 84.5% identical to dNSF (hereafter named dNSF-1), 59% identical to NSF from Chinese hamster, and 38.5% identical to the yeast homolog SEC18. The highest similarity was found in a region of dNSF-2 containing one of two ATP-binding sites; this region is most similar to members of a superfamily of ATPases. dNSF-2 is localized to a region between bands 87F12 and 88A3 on chromosome 3, and in situ hybridization techniques revealed expression in the nervous system during embryogenesis and in several imaginal discs and secretory structures in the larvae. Developmental modulation of dNSF-2 expression suggests that quantitative changes in the secretory apparatus are important in histogenesis.

Amino Acid Sequence↗
PubMed1994

Botulinum G neurotoxin cleaves VAMP/synaptobrevin at a single Ala-Ala peptide bond.

Similarly to other serotypes, botulinum neurotoxin serotype G (BoNT/G) contains the zinc binding motif of zinc endopeptidases. Highly purified preparations of BoNT/G show a zinc-dependent protease activity specific for VAMP/synaptobrevin, a membrane protein of synaptic vesicles. The two neuronal VAMP isoforms are cleaved with similar rates at one Ala-Ala peptide bond present in the same region, out of the several such peptide bonds present in their sequences. This site of cleavage is unique among the eight clostridial neurotoxins. VAMP proteolysis is displayed only after reduction of the single interchain disulfide bond present in the toxin, and it is inhibited by EDTA, o-phenanthroline and captopril.

Alanine↗
PubMed1994

Tetanus toxin light chain cleaves a vesicle-associated membrane protein (VAMP) isoform 2 in rat pancreatic zymogen granules and inhibits enzyme secretion.

Pancreatic acinar cells, a model cell type for the study of exocytosis in non-excitable cells, were used here to test the hypothesis that molecular mechanisms regulating exocytosis from neuronal and neuroendocrine cells may also be utilized in exocrine cells. Using specific antisera raised against vesicle-associated membrane protein (VAMP) isoforms 1 and 2, we have identified VAMP-2, but not VAMP-1, immunoreactive proteins on rat pancreatic acinar cell secretory (zymogen) granules. This 18-kDa protein co-migrated with rat brain synaptic vesicle VAMP-2. Tetanus toxin (TeTx) light chain caused complete cleavage of this protein, which was prevented by the addition of EDTA. This toxin also inhibited in a dose-dependent manner Ca(2+)-stimulated enzyme secretion by up to approximately 30% in streptolysin O-permeabilized acini. This effect of TeTx on secretion was prevented by the zinc endopeptidase inhibitor captopril or by boiling the toxin. Incomplete inhibition of secretion by TeTx may suggest that TeTx-insensitive or VAMP-2-independent mechanisms also regulate exocytosis. In support, TeTx light chain was without effect on Rab3AL (effector domain peptide of Rab3)-mediated potentiation of Ca(2+)-stimulated secretion. These results indicate that a TeTx-sensitive VAMP-2-like protein on zymogen granules participates in Ca(2+)-regulated pancreatic enzyme secretion and that undefined Rab3AL-activated mechanisms may act independently to regulate exocytosis.

Amino Acid Sequence↗
PubMed1994

Synaptic regulation of glial protein expression in vivo.

We investigated signaling between individual nerve terminals and perisynaptic Schwann cells, the teloglial cells that cover neuromuscular junctions. When deprived of neuronal activity in vivo, either by motor nerve transection or tetrodotoxin injection, perisynaptic Schwann cells rapidly up-regulated glial fibrillary acidic protein. Addition of transcription or translation inhibitors to excised muscles prevented this increase. Stimulation of cut nerves prevented glial fibrillary acidic protein increases even when postsynaptic nicotinic receptors were blocked, but not when neurotransmitter release was blocked with omega-conotoxin GVIA. We conclude that there is a nerve terminal to glial signal, requiring presynaptic neurotransmitter release, which regulates perisynaptic Schwann cell genes. This may be a general principle since many types of glial are sensitive to transmitters applied in vitro or released in situ.

Action Potentials↗
PubMed1993

Analysis of the structure and expression of the VAMP family of synaptic vesicle proteins.

VAMP/synaptobrevin proteins were first discovered as small integral membrane proteins in synaptic vesicles of vertebrates and invertebrates. At least two isoforms are expressed in the central nervous system of mammals in non-overlapping patterns. Biochemical studies have revealed that the VAMP synaptic vesicle proteins are the specific target in the presynaptic nerve terminal of botulinum B neurotoxin and tetanus toxin metalloendoprotease activities. The fact that these toxins rapidly and completely abrogate neurotransmission suggests that VAMP proteins play an essential role in this process. More recently, immunologically related proteins have been identified in non-neuronal cells such as adipocytes. In addition, molecular genetic studies of yeast secretion have identified VAMP-related proteins as playing important roles in vesicular transport between the endoreticulum and Golgi. Taken together, these results suggest that the VAMP proteins found on synaptic vesicles might represent specialized forms of proteins which participate in general aspects of cell membrane trafficking.

Amino Acid Sequence↗
PubMed1992

Members of the VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes.

Existing data support the hypothesis that insulin triggers the exocytosis of small vesicles containing the GluT4 isoform of the glucose transporter. The data also suggest that these vesicles reform through endocytosis of GluT4. These processes resemble those described for synaptic vesicles after depolarization of nerve cells. To determine whether GluT4 vesicles are related to synaptic vesicles, rat adipocyte low density microsomes (LDM), which are rich in GluT4 vesicles, were screened for the synaptic vesicle proteins synaptotagmin, synaptophysin, SV2, p29, rab3, and VAMP (synaptobrevin) by immunoblotting. Two polypeptides that reacted with antibodies against the VAMPs were identified, one with the same apparent size as the two isoforms of VAMP in the brain (18 kDa) and one that was slightly smaller (17 kDa). These members of the VAMP family were highly enriched in GluT4 vesicles isolated by immunoadsorption and translocated from the LDM to the plasma membrane in response to insulin. With the exception of rab3, which was observed in the LDM but was not localized in the GluT4 vesicles, the other synaptic vesicle proteins were not detected. The presence of the VAMPs in both GluT4 and synaptic vesicles suggests that the genesis and/or exocytosis of these two types of vesicles involve shared processes.

Adipose Tissue↗
PubMed1990

Distinct patterns of expression of two VAMP genes within the rat brain.

VAMPs are synaptic vesicle-specific proteins composed of a carboxy-terminal hydrophobic membrane anchor and an approximately 100 amino acid domain oriented towards the cytoplasm. In rat, two 77% homologous VAMP genes are expressed in the CNS. To precisely localize the neurons expressing these 2 forms of VAMP, we have used RNA blotting and in situ hybridization histochemistry with RNA probes specific for the 3' untranslated regions of the transcripts. These experiments revealed that the 2 genes are expressed in distict, but slightly overlapping, patterns in the rat brain. VAMP-1 expression is localized to a limited number of nuclei, particularly those involved in modulating somatomotor functions, while VAMP-2 expression is more ubiquitous, being found in nuclei associated with autonomic, sensory, and integrative roles. These data suggest that the specific structural features of individual VAMPs may play an important role in synaptic vesicle metabolism.

Animals↗
PubMed1989

Two vesicle-associated membrane protein genes are differentially expressed in the rat central nervous system.

Vesicle-associated membrane protein (VAMP) 1 is a 120-amino acid protein which co-purifies with cholinergic synaptic vesicles from the marine ray Torpedo californica. We used the Torpedo gene to isolate two independent classes of VAMP cDNA clones from rat brain. Nucleotide sequence analysis of the cDNAs predicts proteins which are 84 and 75% homologous to Torpedo VAMP-1. The amino-terminal 24-28 amino acid residues which comprise the proline-rich head are only about 50% homologous between the different VAMPs, yet the proline-rich character is maintained. The 69 amino acids which comprise the hydrophilic core are highly homologous to Torpedo VAMP-1, with only 2 amino acid substitutions in rat VAMP-1 and 6 in rat VAMP-2. The carboxyl-terminal 23 amino acids of all of the VAMP proteins maintain the hydrophobic character necessary to serve as a membrane anchor. Both VAMP transcripts are expressed differentially in the rat central nervous system. Whereas VAMP-2 is more highly expressed in the whole brain, VAMP-1 is expressed at a higher level in the spinal cord.

Amino Acid Sequence↗
PubMed1988

VAMP-1: a synaptic vesicle-associated integral membrane protein.

Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters.

Amino Acid Sequence↗
PubMed1987

Deletion analysis of the c-Ha-ras oncogene promoter.

Using transient assays of chloramphenicol acetyltransferase expression in CV-1 cells, we have localized the human c-Ha-ras oncogene promoter to a 550 bp fragment located about 1 kb upstream from the ras coding sequence. Deletion analysis has revealed that a 100 nucleotide region 200 base pairs upstream of the putative initiation sites for transcription is essential for high levels of expression. Within this sequence lie two Spl binding sites and a consensus CCAAT box.

Acetyltransferases↗
PubMed1987

Enhanced lytic susceptibility of Ha-ras transformants after oncogene induction is specific to activated NK cells.

C3H 10T1/2 mouse fibroblasts were transfected with a plasmid vector composed of EJ, the mutated c-Ha-ras, and a metallothionein promotor that induced amplified ras expression when activated by culture in the presence of zinc. Experiments were conducted to compare the effect of induction on killing by activated natural killer (NK) cells, cytotoxic T lymphocytes, activated macrophages, and antibody plus complement. The only effector that recognized increased ras expression and exhibited high-inducible cytolysis was an activated NK cell. The effectors from spleen were poly I.C. boostable, Lyt-1.1 negative, NK 1.2 positive, and asialo GM1 positive. Spleen cells from T cell-deficient nude mice, but not NK-deficient beige mice, exhibited high levels of killing activity, and experiments with NK cell clones demonstrated that these lines were also highly cytolytic and killed Ha-ras transfectants in parallel to YAC. Transfection of the same fibroblast line with c-myc did not alter the level of activated NK sensitivity. Cold target competition experiments revealed that Ha-ras-transfected and non-transfected 10T1/2 fibroblasts competed equally for lysis of either YAC or Ha-ras transfectants. Rat-1 fibroblasts did not compete, but gained this capacity when transformed with the v-Ki-ras oncogene but not v-fps. These data suggest that Ha-ras acts in target cells at a post-binding step, whereas Ki-ras may affect expression of target-effector binding structures. The findings that activated NK cell lysis may be specifically influenced by ras expression support a role for NK cells in host surveillance against early neoplastic changes.

Animals↗
PubMed1986

Analysis of hybridoma mutants defective in synthesis of immunoglobulin M.

Hybridoma mutants defective in the expression of IgM have been analyzed by molecular and somatic cell hybridization techniques. The frequency of kappa light-chain mutants in the hybridoma PC7 was much higher than for other cell lines. In contrast to the mutations which we observed previously, the kappa mutants examined here resulted from complete or partial deletion of the kappa gene. Mutants defective in mu chain synthesis were of more diverse types including deletions, gross rearrangements, and more subtle changes. One mutant containing a cis-acting mutation resulting in reduced expression of the mu chain had an associated partial duplication of the mu gene, while others making low or undetectable levels of mu had no gross alterations in genetic structure. The usefulness of this approach to the study of gene structure and expression is discussed.

Animals↗
PubMed1984

Transposition of intracisternal A-particle genes in mouse hybridomas.

A nonfunctional immunoglobulin kappa-chain gene previously shown to contain intracisternal A-particle (IAP) sequences within one of its introns was further characterized by DNA and RNA blot analysis. The results of these experiments indicate that the joining of the IAP sequences to the kappa-chain gene was a consequence of an insertion event which presumably involved a complete IAP gene. It is postulated that the insertion of the IAP gene has altered the secondary or tertiary structure of the kappa-chain pre-mRNA, resulting in the utilization of a "cryptic" 5' splice site. The detection of numerous IAPs in isogenous wild-type hybridoma cells by electron microscopy suggests that transposition of IAP genes might proceed via the reverse transcription of IAP-associated RNA. These findings raise the possibility that transposition of IAP genes might also occur in other IAP-positive mouse cells. In particular, insertional mutagenesis by IAP genes in IAP-producing tumor cells could play a role in tumor cell heterogeneity or tumor progression. We have also investigated the possibility that IAP-related sequences might be present in the human genome. Using nonstringent hybridization conditions, multiple discrete restriction fragments could be detected in human DNA with several mouse IAP-specific probes.

Animals↗
PubMedDate not supplied

Phosphatidylinositol polyphosphate binding to the mammalian septin H5 is modulated by GTP.

BACKGROUND: Septins are members of a conserved family of GTPases found in organisms as diverse as budding yeast and mammals. In budding yeast, septins form hetero-oligomeric filaments that lie adjacent to the membrane at the mother-bud neck, whereas in mammals, they concentrate at the cleavage furrow of mitotic cells; in both cases, septins provide a required function for cytokinesis. What directs the location and determines the stability of septin filaments, however, remains unknown. RESULTS: Here we show that the mammalian septin H5 is associated with the plasma membrane and specifically binds the phospholipids phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P(2)) and phosphatidylinositol 3,4, 5-trisphosphate (PtdIns(3,4,5)P(3)). Deletion analysis revealed that this binding occurs at a site rich in basic residues that is conserved in most septins and is located adjacent to the GTP-binding motif. Phosphoinositide binding was inhibited by mutations within this motif and was also blocked by agents known to associate with PtdInsP(2) or by a peptide corresponding to the predicted PtdInsP(2)-binding sequence of H5. GTP binding and hydrolysis by H5 significantly reduced its PtdInsP(2)-binding capability. Treatment of cells with agents that occluded, dephosphorylated or degraded PtdInsP(2) altered the appearance and localization of H5. CONCLUSIONS: These results indicate that the interaction of septins with PtdInsP(2) might be an important cellular mechanism for the spatial and temporal control of septin accumulation.

3T3 Cells↗