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G Thomas

Publications and source records attributed to G Thomas.

At least 19 recordsLinked to original sources
PubMed1992

Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin.

Proteolytic cleavage of the protective antigen (PA) protein of anthrax toxin at residues 164-167 is necessary for toxic activity. Cleavage by a cellular protease at this sequence, Arg-Lys-Lys-Arg, normally follows binding of PA to a cell surface receptor. We attempted to identify this protease by determining its sequence specificity and catalytic properties. Semi-random cassette mutagenesis was used to generate mutants with replacements of residues 164-167 by Arg, Lys, Ser, or Asn. Analysis of 19 mutant proteins suggested that lethal factor-dependent toxicity required the sequence Arg-Xaa-Xaa-Arg. Based on these data, three additional mutants were constructed with the sequences Ala-Lys-Lys-Arg, Arg-Lys-Lys-Ala, and Arg-Ala-Ala-Arg. Of these mutant proteins, Arg-Ala-Ala-Arg was toxic, confirming that the cellular protease can recognize the sequence Arg-Xaa-Xaa-Arg. The mutant containing the sequence Ala-Lys-Lys-Arg was also toxic but required > 13 times more protein to produce equivalent toxicity. This sequence specificity is similar to that of the ubiquitous subtilisin-like protease furin, which is involved in processing of precursors of certain receptors and growth factors. Therefore we tested whether a recombinant soluble furin would cleave PA. This furin derivative efficiently cleaved native PA and the Arg-Ala-Ala-Arg mutant but not the nontoxic PA mutants. In addition, previously identified inhibitors of furin blocked cleavage of receptor-bound PA. These data imply that furin is the cellular protease that activates PA, and that nearly all cell types contain at least a small amount of furin exposed on their cell surface.

Amino Acid Sequence↗
PubMed1992

PCR assay for chromosome 1p deletion in small neuroblastoma samples.

Deletion of the short arm of chromosome 1 is among the most recurrent cytogenetic alterations found in neuroblastoma and has been associated with short survival. However, this prognostic information, which relies on time-consuming techniques, is not yet routinely exploited. In order to set up a reliable and simple routine test to determine 1p deletion in neuroblastoma, we have used the polymerase chain reaction to genotype neuroblastoma DNA at 2 loci containing a variable number of tandem repeats and located on the distal part of the short arm of chromosome 1. Agarose gel electrophoresis and ethidium bromide staining of the amplification products enable a simple determination of constitutional and tumor genotypes at these loci to be made. A total of 37 samples from 29 patients were studied with this technique. Loss of heterozygosity (LOH) could be identified in 8 cases. In each case the results obtained were in agreement with those achieved by the Southern procedure. This technique will be of particular interest in the pretherapeutic analysis of 1p deletions in small tumor samples obtained by fine-needle aspirates of the primary tumor. It should also enable retrospective studies from paraffin-embedded tumor fragments to be made and provide information for the analysis of tumor heterogeneity in neuroblastoma and in other tumors with 1p deletions.

Base Sequence↗
PubMed1992

Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3.

Experiments using recombinant vaccinia viruses expressing rat proinsulin I coinfected into COS-7 cells with recombinant vaccinia virus expressing human furin, human PC2, mouse PC3 (subtilisin-related proprotein convertases 1-3, respectively), or yeast Kex2 indicate that in this system both Kex2 and furin produce mature insulin, whereas PC2 selectively cleaves proinsulin at the C-peptide-A-chain junction. This is a property consistent with its probable identity with the rat insulinoma granule type II proinsulin processing activity as described by Davidson et al. [Davidson, H. W., Rhodes, C. J. & Hutton, J. C. (1988) Nature (London) 333, 93-96]. PC3 generates mature insulin but cleaves preferentially at the proinsulin B-chain-C-peptide junction. This pattern of cleavage by PC3 is similar, but not identical, to that of the highly B-chain-C-peptide junction-selective type I activity as described by Davidson et al., perhaps due to the presence of a P4 arginine residue near the C-peptide-A-chain junction unique to the rat proinsulins. These results along with data presented on the expression of both PC2 and PC3 in islet beta cells strongly support the conclusion that these proteases are involved in the conversion of proinsulin to insulin in vivo.

Amino Acid Sequence↗
PubMed1992

Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen.

Previous work demonstrated that human furin is a predominantly Golgi membrane-localized endoprotease that can efficiently process precursor proteins at paired basic residues (-Lys-Arg- or -Arg-Arg-) in transfected cells. Anion-exchange chromatography of culture supernatant from cells expressing a soluble truncated form of human furin resulted in a greatly enriched preparation of the endoprotease (approximately 70% pure as determined by protein staining). Enzymatic studies show that furin is a calcium-dependent (K0.5 = 200 microM) serine endoprotease which has greater than 50% of maximal activity between pH 6.0 and 8.5. The inhibitor sensitivity of furin suggests that it is similar to, yet distinct from, other calcium-dependent proteases. Evidence that furin may require a P4 Arg in fluorogenic peptide substrates suggested that this enzyme might cleave the protective antigen (PA) component of anthrax toxin at the sequence -Arg-Lys-Lys-Arg-. Indeed, PA was cleaved by purified furin at the proposed consensus site (-Arg-X-Lys/Arg-Arg decreases-) at a rate (8 mumol/min/mg total protein) 400-fold higher than that observed with synthetic peptides. In addition, the processing of mutant PA molecules with altered cleavage sites suggests that furin-catalyzed endoproteolysis minimally requires an -Arg-X-X-Arg- recognition sequence for efficient cleavage. Together, these results support the hypothesis that furin processes protein precursors containing this cleavage site motif in the exocytic pathway and in addition, raises the possibility that the enzyme also cleaves extracellular substrates, including PA.

Amino Acid Sequence↗
PubMed1992

Molecular characterization of a low-molecular-mass matrix metalloproteinase secreted by glomerular mesangial cells as PUMP-1.

A polymerase chain reaction (PCR)-based homology cloning strategy was used to define the spectrum of stromelysin-like matrix metalloproteinases (MMPs) synthesized by cultured glomerular mesangial cells (MC). Using this technique, cDNAs encoding an unusual, truncated member of the MMP family, punctuated (putative) metalloproteinase (PUMP-1), were exclusively isolated. Incubation with the cytokines interleukin 1 and tumour necrosis factor increased the abundance of PUMP-1 mRNA in mesangial cells. The mesangial PUMP-1 mRNA is processed in a tissue-specific manner, yielding a transcript containing repeated 3'-untranslated region ATTTA motifs commonly found in cytokines with limited mRNA stability. Polyclonal antibodies prepared against the C-terminal region of the PUMP-1 protein documented release of this enzyme by cultures of cytokine-stimulated MC and permitted identification of PUMP-1-expressing mesangial cells within clinical biopsy specimens of acute glomerulonephritis. These findings represent new molecular and clinical evidence that non-malignant cells process and secrete this unusual member of the MMP family in a cytokine-mediated, tissue-specific manner. Mesangial synthesis of PUMP-1 may contribute to the progression of injury during glomerular inflammatory states.

Amino Acid Sequence↗
PubMed1992

Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs.

Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70s6k (M(r) 70,000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were associated with p70s6k activation, the kinase was labeled to high specific activity with 32P(i) in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensional tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide of M(r) 2400. Analysis of the p70s6k protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vivo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p70s6k. In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.

3T3 Cells↗
PubMed1992

pHi controls cytoplasmic calcium in rat parotid cells.

The goal of this investigation was to determine if cytoplasmic pH (pHi) modulated the basal level of the concentration of calcium ions in the cytoplasm (Cai) in rat parotid cells. We investigated the effects of various experimental manipulations on both pHi and Cai as measured with BCECF and the calcium photoprotein aequorin, respectively. We found that various experimental manipulations that increased pHi, such as exposure of the cells to NH4Cl, a decrease of the partial pressure of CO2 or an increase in extracellular pH in the presence of nigericin invariably increased Cai. Moreover, experimental manipulations which lowered Cai, such as a reduction of extracellular [NaHCO3] or the removal of loaded NH4 invariably decreased Cai. Thus pHi and Cai are directly related in parotid cells. Since recent studies have shown that Cai directly influences pHi, we suggest that Cai-handling and pHi-handling are tightly linked in parotid cells.

Aequorin↗
PubMed1992

Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage.

Human furin is a calcium-dependent serine endoprotease that can efficiently cleave many precursor proteins on the carboxyl side of the consensus cleavage sequence, -Arg-X-Lys/Arg-Arg-, both in vivo and in vitro. Analysis of furin proteins in extracts of cells infected with a vaccinia recombinant expressing human furin show that the enzyme is present as two prominent forms of 90 and 96 kDa. Because the structurally related bacterial subtilisins require endoproteolytic removal of the NH2-terminal pro-region by an autocatalytic intramolecular cleavage, we speculated that the size heterogeneity in the furin doublet similarly may result from a proteolytic removal of an NH2-terminal pro-region. Here we report identification of the 90-kDa furin NH2 terminus and, based on the reported sequence of the furin cDNA, demonstrate that this furin protein is derived from a larger precursor by an endoproteolytic cleavage on the COOH-terminal side of a consensus furin cleavage site, -Arg-Thr-Lys-Arg107-. Expression of mutant furin molecules containing an altered cleavage site (Arg104----Ala or Arg107----Gly) resulted in the production of only the 96-kDa furin protein. Assays of furin-dependent cleavage of a protein substrate in vitro showed that proteolytic activity was associated with the 90-kDa and not the 96-kDa furin protein, demonstrating that removal of the NH2-terminal pro-region is required for furin activity. Expression of a third furin construct containing a mutation of the active site aspartate (Asp153----Asn) similarly resulted in the expression of only the 96-kDa protein, suggesting that furin activation occurs by an autoproteolytic cleavage. Finally, the production of 90-kDa furin from either site-directed furin mutant could not be potentiated by overexpressing active furin, suggesting that the autoproteolytic activation was an intramolecular event.

Amino Acid Sequence↗
PubMed1992

A single gene encodes two isoforms of the p70 S6 kinase: activation upon mitogenic stimulation.

Previously, two cDNA clones were isolated from a rat liver or hepatoma cDNA library for the mitogenic-activated p70 S6 kinase (p70s6k). Except for a single amino acid change and a 23-amino acid N-terminal extension in the latter clone, the open reading frames of the two clones are identical. A probe common to both clones also revealed four distinct transcripts. Here, by using specific probes, it was possible to show which transcript corresponds to which clone and that both clones are derived from the same gene. Furthermore, analysis of in vitro translation products using specific antibodies demonstrates that both clones encode the p70s6k but that the clone harboring the 23-amino acid extension also encodes an additional isoform of the kinase, referred to as p85s6k. It could be shown by using the specific antibody to the p85s6k that this isoform of the kinase is present in rat liver and is activated after mitogenic stimulation of quiescent Swiss 3T3 cells.

3T3 Cells↗
PubMed1992

Inhibition or down-regulation of protein kinase C attenuates late phase p70s6k activation induced by epidermal growth factor but not by platelet-derived growth factor or insulin.

The late phase of the time-dependent epidermal growth factor (EGF)-induced biphasic activation of the p70s6k is selectively attenuated by the specific PKC inhibitor, CGP 41,251, a staurosporine derivative. At a 40-fold lower concentration than CGP 41,251, staurosporine inhibits both phases of S6 kinase activation to the same extent, whereas the inactive staurosporine derivative CGP 42,700 shows no effect on either phase. Platelet-derived growth factor (PDGF) and insulin also induce biphasic S6 kinase activation, but in neither case is either phase of activation affected by the presence of CGP 41,251. This finding was unexpected in the case of PDGF, which is a potent activator of PKC and whose receptor directly interacts with phospholipase C gamma 1. However, similar results were obtained following down-regulation of PKC by prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Therefore, even though EGF and PDGF induce PKC activation, PDGF, unlike EGF, does not appear to use this signaling pathway for late phase p70s6k activation.

3T3 Cells↗
PubMed1992

Identification of guanidino succinate as a putative endogenous source of the endothelium derived relaxing factor.

Using a specific HPLC analysis for guanidines, we find that rat aorta contains guanidino succinate (GS), guanidino acetate (GA), guanidino propionate (GP), guanidino butyrate (GB), methyl guanidine (MG) and guanidine. The concentration of L-arginine (0.05 nmol/mg tissue) is significantly lower than the other guanidines. GS is found to be the most potent vasodilator-guanidine in the rat aorta preparation and this vasodilation depends predominantly on the presence of the endothelium. This effect of GS is antagonized by NG-monomethyl L-arginine (L-NMMA), NW-nitro L-arginine benzyl ester (L-NABA), hemoglobin and by methylene blue, all of which are known to block or attenuate endothelium dependent relaxation. Further, the relaxation mediated by GS is accompanied by the formation of cGMP in the rat aorta. From these results we suggest that GS may be a major endogenous source of EDRF.

Animals↗
PubMed1992

Allelic loss on chromosome 22 correlates with histopathological predictors of recurrence of meningiomas.

Meningiomas are common tumors of the nervous system. Although usually benign, they may exhibit variable degrees of aggressiveness. Their probability of recurrence after subtotal resection has been correlated with several histological parameters. Independently, a loss of chromosome 22, as evidenced either by cytogenetics or by somatic loss of alleles, has been observed in about half of the cases studied. In 34 meningiomas we have examined the relationship between loss of chromosome 22 alleles and 6 histological predictors of recurrence. Significant correlations were found for 3 of these, i.e. prominent nucleoli (p less than 0.002), microscope count of mitoses (p less than 0.05) and nuclear pleomorphism (p less than 0.02). Correlation with the other 3, i.e. sheeting of cells, vascularity and micronecrosis, did not reach significance. Total tumor score, defined by the sum of the individual scores for these 6 parameters, was strongly correlated to allelic loss (p less than 0.0001). Thus, the loss of chromosome 22 alleles, which possibly contribute to the inactivation of tumor-suppressor gene(s), might be a potent genetic marker of the aggressiveness of meningiomas.

Aneuploidy↗
PubMed1992

Stomal seeding by percutaneous endoscopic gastrostomy in patients with head and neck cancer.

Presented is the first known case of squamous cell carcinoma that developed at the site of a "pull" type percutaneous endoscopic gastrostomy stoma. The patient had advanced squamous cell carcinoma of the pharynx when the percutaneous endoscopic gastrostomy tube was placed. This article should serve as a warning of this potential complication. Alternate techniques and other precautionary methods are described.

Carcinoma, Squamous Cell↗
PubMed1992

Unicolor and bicolor in situ hybridization in the diagnosis of peripheral neuroepithelioma and related tumors.

The chromosome 22 breakpoint of the t(11;22) translocation of peripheral neuroepithelioma has been located, by fluorescence in situ hybridization, in the proximity of the interface between 22q12.1 and 22q12.2. Use of single cosmids or pools of cosmids of the flanking regions enables the monitoring of the translocation in interphase and in metaphase chromosomes. This method can now be routinely applied for the diagnosis of this translocation in mixed round cell tumors.

Bone Neoplasms↗
PubMed1992

Identification and early activation of a Xenopus laevis p70s6k following progesterone-induced meiotic maturation.

Employing oligonucleotide primers derived from the DNA sequence of rat p70s6k a homologous sequence was shown by polymerase chain reaction (PCR) to be present as a maternal transcript in stage IV-VI Xenopus laevis oocytes. The sequence covered 665 bp of p70s6k and was 97% identical at the amino acid level. When used to probe a Northern blot of the poly(A)+ mRNA from stage VI oocytes, this sequence recognized four transcripts of 1.9, 2.5, 3.2 and 5.2 kb. Specific rat p70s6k antibodies immunoprecipitated active S6 kinase from stage VI oocytes but not unfertilized eggs. The basal level of activity was 3- to 5-fold higher in primed versus non-primed oocytes indicating that p70s6k activation was an early event in maturation. Consistent with this observation, progesterone induced a 10-fold activation of the kinase in non-primed oocytes within 1 h post-induction at a time critical for early activation of protein synthesis. A much smaller, variable peak of activation was observed at 85% germinal vesicle breakdown (GVBD), which was dependent on the rate of maturation. Two members of the pp90rsk family, thought to be the sole S6 kinases present in X.laevis oocytes, exhibited distinct kinetics of activation. Finally, the S6 kinase activity present 1 h post-progesterone stimulation was purified and shown to have a Mr of 70K.

3T3 Cells↗
PubMed1992

Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease.

Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.

Affinity Labels↗