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T Evans

Publications and source records attributed to T Evans.

At least 145 records · Page 8Linked to original sources
PubMed1992

Visceral leishmaniasis: a model for infection-induced cachexia.

Parasitic infections and malnutrition coexist in many tropical and subtropical areas. Studies of Leishmania donovani and of experimentally infected Syrian hamsters have provided important insights into the complex interrelationships between malnutrition and this parasitic disease. Malnutrition, which adversely affects cell-mediated immunity, is associated with the development of visceral leishmaniasis (kala-azar) in children living in endemic areas. In turn, L. donovani can cause wasting as well as hepatosplenomegaly, fever, and anemia. Syrian hamsters infected with L. donovani develop a disease that is comparable to that of humans with kala-azar. Weight loss in infected hamsters is associated with splenic macrophage secretion of potentially catabolic cytokines as measured by the D10.G4.1 assay for interleukin-1 and the L929 cytotoxicity assay for tumor necrosis factor/cachectin. Although decreased food intake contributes to wasting in infected hamsters, studies of skeletal muscle function indicate that it is not the sole factor. Leishmania donovani-infected hamsters have also been used to study drugs with the potential to prevent or reverse cachexia.

Adipose Tissue↗
PubMed1991

Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2.

A major action of the microbicidal system of human neutrophils is the formation of superoxide anion (O2-) by a multicomponent oxidase that transfers electrons from the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to molecular oxygen. The mechanism of assembly and activation of the oxidase from its cytosolic and membrane-bound components is unknown, but may require the activity of a guanosine 5'-triphosphate (GTP)-binding component. A cytosolic GTP-binding protein (Gox) that regulates the NADPH oxidase of neutrophils was identified. Gox was purified and shown to augment the rate of O2- production in a cell-free oxidase activation system. Sequence analysis of peptide fragments from Gox identified it as Rac 2, a member of the Ras superfamily of GTP-binding proteins. Antibody to a peptide derived from the COOH-terminus of Rac 2 inhibited O2- generation in a concentration-dependent manner. These results suggest that Rac 2 is a regulatory component of the human neutrophil NADPH oxidase, and provide new insights into the mechanism by which this oxygen radical-generating system is regulated.

Amino Acid Sequence↗
PubMed1991

Catalysis of guanine nucleotide exchange on the CDC42Hs protein by the dbl oncogene product.

THE superfamily of low molecular mass GTP-binding proteins, for which the ras proteins are prototypes, has been implicated in the regulation of diverse biological activities including protein trafficking, secretion, and cell growth and differentiation. One member of this family, CDC42Hs (originally referred to as Gp or G25K), seems to be the human homologue of the Saccharomyces cerevisiae cell-division-cycle protein, CDC42Sc. A second S. cerevisiae protein, CDC24, which is known from complementation studies to act with CDC42Sc to regulate the development of normal cell shape and the selection of nonrandom budding sites in yeast, contains a region with sequence similarity to the dbl oncogene product. Here we show that dbl specifically catalyses the dissociation of GDP from CDC42Hs and thereby qualifies as a highly selective guanine nucleotide exchange factor for the GTP-binding protein. Although guanine nucleotide exchange activities have been previously described for other members of the Ras-related GTP-binding protein family, this is the first demonstration, to our knowledge, of the involvement of a human oncogenic protein in catalysing exchange activity.

Enzyme Activation↗
PubMed1991

Identification of the human platelet GTPase activating protein for the CDC42Hs protein.

The CDC42Hs protein appears to be an isoform of the ras-related GTP-binding protein G25K and is an apparent human homolog of the Saccharomyces cerevisiae cell-division-cycle protein, CDC42Sc. In this study, we report the identification of a GTPase-activating protein (GAP) for CDC42Hs from human platelets (designated from here on as CDC42Hs-GAP). The CDC42Hs-GAP activity was solubilized from platelet membranes, recovered through successive chromatography steps (the final step being Mono-Q chromatography), and purified approximately 3500-fold. The CDC42Hs-GAP activity appeared to correspond to a polypeptide with an apparent Mr of approximately 25,000. The GTPase activities of the purified human platelet CDC42Hs, the Escherichia coli-recombinant CDC42Hs, and the Spodoptera frugiperda-recombinant GTP-binding proteins are all stimulated by the CDC42Hs-GAP to identical extents, which indicates that the recombinant CDC42Hs proteins are as effective as the native human platelet protein in coupling to the GAP. However, a mutant form of the E. coli-recombinant CDC42Hs which contains a valine residue at position 12 (CDC42HsVal-12) has a significantly reduced intrinsic GTPase activity (relative to the wild type CDC42HsGly-12) which is not stimulated by the CDC42Hs-GAP. The CDC42Hs-GAP also does not stimulate the GTPase activities of the ras or rap GTP-binding proteins; however, it is capable of a weak stimulation of the GTPase activity of mammalian rho. Based on the apparent similarities in the molecular size of the CDC42Hs- and rho-GAPs (i.e. 25-30 kDa), and the cross-reactivity of rho with the CDC42Hs-GAP, it seems likely that the CDC42Hs- and rho-GAPs will constitute a specific subclass of the ras-related GAP superfamily.

Blood Platelets↗
PubMed1991

Mutant alpha subunits of Gi2 inhibit cyclic AMP accumulation.

One or more of three Gi proteins, Gi1-3, mediates hormonal inhibition of adenylyl cyclase. Whether this inhibition is mediated by the alpha or by the beta gamma subunits of Gi proteins is unclear. Mutations inhibiting the intrinsic GTPase activity of another G protein, the stimulatory regulator of adenylyl cyclase (Gs), constitutively activate it by replacing either of two conserved amino acids in its alpha subunit (alpha s). These mutations create the gsp oncogene which is found in human pituitary and thyroid tumours. In a second group of human endocrine tumours, somatic mutations in the alpha subunit of Gi2 replace a residue cognate to one of those affected by gsp mutations. This implies that the mutations convert the alpha i2 gene into a dominantly acting oncogene, called gip2, and that the mutant alpha i2 subunits are constitutively active. We have therefore assessed cyclic AMP accumulation in cultured cells which stably or transiently express exogenous wild-type alpha i2 complementary DNA or either of two mutant alpha i2 cDNAs. The results show that putatively oncogenic mutations in alpha i2 constitutively activate the protein's ability to inhibit cAMP accumulation.

Adenylate Cyclase Toxin↗
PubMed1991

G-protein beta gamma forms: identity of beta and diversity of gamma subunits.

Signal-transducing G-proteins are heterotrimers composed of GTP-binding alpha subunits in association with a tightly bound complex of beta and gamma subunits. While the alpha subunits are recognized as a family of diverse structures, beta and gamma subunits have also been found as heterogeneous isoforms. To investigate the diversity and tissue specificity of the beta gamma complexes, we have examined homogeneous oligomeric G-proteins from a variety of sources. The beta and gamma subunits isolated from the major-abundance G-proteins from bovine brain, bovine retina, rabbit liver, human placenta, and human platelets were purified and subjected to biochemical and immunological analysis. Protease mapping and immune recognition revealed an identical profile for each of the two distinctly migrating beta isoforms (beta 36 and beta 35) regardless of tissue or G-protein origin. Digestion with V8 protease revealed four distinct, clearly resolved terminal fragments for beta 36 and two for beta 35. Trypsin and chymotrypsin digestion yielded numerous bands, but again each form had a unique profile with no tissue specificity. Tryptic digestion was found to be conformationally specific with the most resistant structure being the native beta gamma complex. With increasing trypsin, the complex was digested but in a pattern distinct from that for denatured beta. In contrast to the two highly homologous beta structures, examination of this set of proteins revealed at least six distinct gamma peptides. Two unique gamma peptides were found in bovine retinal Gt and three gamma peptides in samples of bovine brain derived Go/Gi. Human placental and platelet Gi samples each contained a unique gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Structure and promoter activity of the gene for the erythroid transcription factor GATA-1.

We have cloned the gene for the chicken erythroid transcription factor GATA-1 (formerly Eryf1, NF-E1, or GF-1). The gene is composed of six exons, two of which encode the two finger domains of the protein. Transcription of GATA-1 in chicken embryonic erythroid cells initiates from multiple sites clustered approximately 200 base pairs upstream from the start of protein-coding sequence. A number of sequence motifs for known DNA-binding proteins are found to be protected in DNase I-footprinting experiments by either erythroid or brain nuclear extracts or by both. Notably, a cluster of three GATA-1 sites is protected by the erythroid extract, as well as by purified GATA-1. We find that the upstream region of the gene functions as a powerful promoter when transfected into embryonic erythroid cells. In primary chicken embryo fibroblasts the promoter exhibits lower activity, which is increased when the cells are cotransfected with a second plasmid expressing the GATA-1 cDNA. The results suggest that GATA-1 protein plays an autoregulatory role in its own expression.

Animals↗
PubMed1991

Purification of a plasma membrane-associated GTPase-activating protein specific for rap1/Krev-1 from HL60 cells.

rap1/Krev-1 is a p21ras-related GTP-binding protein that has been implicated in the reversion of the ras-transformed cell phenotype. We have identified a GTPase-activating protein (GAP) specific for rap in plasma membranes isolated from differentiated HL60 cells. The rap GAP activity remained quantitatively associated with the membrane following washes with buffered 1 M LiCl containing 20 mM EDTA but was solubilized with the detergents Nonidet P-40 and deoxycholate. On the basis of size-exclusion chromatography, the membrane-associated rap GAP (rap GAPm) appeared distinct from the rap GAP detected in the cytosolic fraction from HL60 cells. The molecular sizes of the membrane and cytosolic forms were estimated to be 36 and 54 A, respectively. rap GAPm was solubilized and purified to near homogeneity by successive column chromatographies in the presence of detergent. The rap GAPm activity corresponded to a single polypeptide that migrated with a molecular mass of approximately 88 kDa on SDS/polyacrylamide gels. The purified rap GAPm was inactive toward the GTP-bound forms of p21ras, rho, G25K, and rac-1 and did not stimulate dissociation of guanine nucleotide from rap.

Amino Acid Sequence↗
PubMed1991

Membrane-binding domain of the small G protein G25K contains an S-(all-trans-geranylgeranyl)cysteine methyl ester at its carboxyl terminus.

We showed previously that a 23-kDa guanine nucleotide-binding protein (G protein) purified from bovine brain membranes is carboxyl methylated and that this modification occurs at or near the membrane-binding domain. In the present study, we identified this small G protein as G25K (formerly termed Gp). We demonstrated that proteolytic digests of 3H-methylated G25K contained radiolabeled material that coeluted with synthetic S-(geranylgeranyl)cysteine methyl ester on reversed-phase HPLC. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the isoprenoid moiety and carboxyl methyl ester are localized on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G25K by Raney nickel treatment positively identified the covalently bound lipid as an all-trans-geranylgeranyl (C20) isoprenoid moiety. These results suggest that geranylgeranyl modification and perhaps methyl esterification function in the membrane localization of this small G protein.

Amino Acid Sequence↗
PubMed1991

The Ras superfamilies: regulatory proteins and post-translational modifications.

The Ras-like GTP-binding proteins comprise a large superfamily of proteins that play key roles in a wide variety of cellular activities, including cell growth, differentiation, secretion, and protein trafficking. During the past few years, it has become clear that these GTP-binding proteins are regulated by a variety of manners, including interactions with specific types of regulatory proteins and post-translational modification events.

Amino Acid Sequence↗
PubMed1991

trans-Activation of a globin promoter in nonerythroid cells.

We show that expression in fibroblasts of a single cDNA, encoding the erythroid DNA-binding protein Eryf1 (GF-1, NF-E1), very efficiently activates transcription of a chicken alpha-globin promoter, trans-Activation in these cells occurred when Eryf1 bound to a single site within a minimal globin promoter. In contrast, efficient activation in erythroid cells required multiple Eryf1 binding sites. Our results indicate that mechanisms exist that are capable of modulating the trans-acting capabilities of Eryf1 in a cell-specific manner, without affecting DNA binding. The response of the minimal globin promoter to Eryf1 in fibroblasts was at least as great as for optimal constructions in erythroid cells. Therefore, the assay provides a very simple and sensitive system with which to study gene activation by a tissue-specific factor.

Animals↗
PubMed1990

Beta gamma dimers of G proteins inhibit atrial muscarinic K+ channels.

It has been proposed that beta gamma dimers of signal-transducing G proteins mediate muscarinic activation of atrial K+ channels. We examined this hypothesis by testing the effects of beta gamma dimers from four sources (human erythrocytes, human placenta, bovine brain, and bovine retina) on single channel muscarinic K+ (K+[acetylcholine (ACh)]) currents in inside-out membrane patches of adult guinea pig atria. None of the four beta gamma dimer preparations stimulated K+[ACh] currents; on the contrary, each inhibited the currents whether the currents were activated with GTP alone (agonist-independent activity) or with GTP plus a muscarinic agonist (agonist-dependent activity). Detergents at concentrations used to suspend erythrocyte, brain, and placental beta gamma dimers had no effect by themselves, and detergents were not used with the retinal beta gamma dimers. We conclude that beta gamma dimers do not mediate stimulatory effects of the endogenous G protein that regulates the K+ channels. In fact beta gamma dimers appear to inhibit activation by the endogenous G alpha subunits. Further insight into the role of beta gamma dimers came from the observation that agonist-independent GTP-activated K+[ACh] currents were inhibited by beta gamma dimers at about one-tenth the concentration required to inhibit agonist-dependent activation. One possibility is that dimeric beta gamma may have a higher affinity for free alpha subunits than for alpha subunits associated with agonist-occupied receptors. Thus, in addition to the known requirement of beta gamma dimers for the interaction of alpha subunits with receptors, beta gamma dimers may also improve the signal-to-noise ratio for agonists by reducing agonist-independent background activities.

Acetylcholine↗
PubMed1990

The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system.

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.

Animals↗