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1.
Mar Pollut Bull ; 73(2): 470-84, 2013 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-23428288

RESUMO

To reduce the negative effect of climate change on Biodiversity, the use of geological CO2 sequestration has been proposed; however leakage from underwater storages may represent a risk to marine life. As extracellular homeostasis is important in determining species' ability to cope with elevated CO2, we investigated the acid-base and ion regulatory responses, as well as the density, of sea urchins living around CO2 vents at Vulcano, Italy. We conducted in situ transplantation and field-based laboratory exposures to different pCO2/pH regimes. Our results confirm that sea urchins have some ability to regulate their extracellular fluid under elevated pCO2. Furthermore, we show that even in closely-related taxa divergent physiological capabilities underlie differences in taxa distribution around the CO2 vent. It is concluded that species distribution under the sort of elevated CO2 conditions occurring with leakages from geological storages and future ocean acidification scenarios, may partly be determined by quite subtle physiological differentiation.


Assuntos
Dióxido de Carbono/análise , Ecossistema , Ouriços-do-Mar/fisiologia , Poluentes Químicos da Água/análise , Adaptação Fisiológica , Animais , Mudança Climática , Fenômenos Geológicos , Concentração de Íons de Hidrogênio , Itália , Água do Mar/química
2.
Z Naturforsch C J Biosci ; 49(7-8): 453-7, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7945671

RESUMO

The interaction of calmodulin with purified guinea pig liver transglutaminase was studied. The nucleotide (ATP and GTP) hydrolysis activity of this tissue transglutaminase was transiently increased and then gradually decreased depending on calmodulin concentration. The peak activation was obtained in the presence of a stoichiometric amount of calmodulin. The effect of calmodulin on the classical transglutaminase activity was minimal. Fluorescence spectroscopy demonstrated that the enzyme produced a significant blue shift in the emission peak of dansylated calmodulin. Interestingly, Ca2+ was not required for the interaction between the two proteins. The results described here give an additional regulatory role to calmodulin.


Assuntos
Trifosfato de Adenosina/metabolismo , Calmodulina/farmacologia , Guanosina Trifosfato/metabolismo , Fígado/enzimologia , Transglutaminases/metabolismo , Animais , Cloreto de Cálcio/farmacologia , Bovinos , Ácido Edético/farmacologia , Ácido Egtázico/farmacologia , Cobaias , Hidrólise , Cinética , Espectrometria de Fluorescência
3.
Biochim Biophys Acta ; 1202(1): 1-6, 1993 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-8104036

RESUMO

Transglutaminases (EC 2.3.2.13) catalyze an acyl-transfer reaction between peptide-bound glutamine residues and primary amines, including the epsilon-amino group of lysine residues in protein. Purified human erythrocyte transglutaminase was found to have another activity, i.e., GTP hydrolysis. Treatment of the enzyme with iodoacetamide, a cysteine-directed reagent, caused a 94% loss of TGase activity within 8 min, but no significant loss of GTPase activity. Cys-277, a known residue which is selectively modified by iodoacetamide, was replaced with Ser by site-directed mutagenesis to assess the role of the Cys-277 in the transglutaminase/GTPase activities. Wild-type cDNA, coding for human endothelial cell transglutaminase, and its C277S-mutated cDNA were cloned into a plasmid vector that contained a promoter from phage T7, and then expressed in Escherichia coli. The wild-type recombinant enzyme was indistinguishable from human erythrocyte transglutaminase in mobility on a SDS-polyacrylamide gel, immunoreactivity and catalytic activities for transglutaminase and GTPase. However, the recombinant enzyme was not blocked at the N-terminal alanine residue, as is the case in the naturally occurring erythrocyte enzyme. The C277S mutant enzyme showed no transglutaminase activity, but had Km and kcat values for GTPase activity that were comparable to those of wild-type recombinant and natural erythrocyte enzymes. These results demonstrate that Cys-277 is essential for transglutaminase activity, but not for GTPase activity, and that N-terminal blocking of tissue-type transglutaminase is not critical for either transglutaminase or GTPase activities.


Assuntos
Cisteína , Eritrócitos/enzimologia , GTP Fosfo-Hidrolases/genética , Transglutaminases/genética , Sítios de Ligação , Ditiotreitol , GTP Fosfo-Hidrolases/isolamento & purificação , GTP Fosfo-Hidrolases/metabolismo , Humanos , Iodoacetamida , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Serina , Transglutaminases/metabolismo
4.
Cancer Res ; 52(17): 4779-86, 1992 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-1511441

RESUMO

A complementary DNA (cDNA) for rat hepatic aryl sulfotransferase IV (AST IV) was isolated, characterized, and used as a hybridization probe to evaluate the molecular basis for the differential expression of AST IV during 2-acetylaminofluorine (2AAF)-induced hepatocarcinogensis. The AST IV cDNA clone was obtained by immunochemical screening of a male Sprague-Dawley rat liver cDNA library. The AST IV cDNA was found to be 1.3 kilobases long and to encode a fusion protein which was reactive with an antibody to AST IV and enzymatically able to generate the sulfuric acid ester of N-hydroxy-2AAF. Sequence analysis of the AST IV cDNA showed it to be 1127 residues in length and to have essentially complete homology with PST-I cDNA, a previously reported (S. Ozawa, et al., Nucleic Acids Res., 18: 4001, 1990), 1028-base cDNA for an uncharacterized rat liver aryl sulfotransferase. Comparison of the PST-I/AST IV cDNA-deduced amino acid sequence with data from a partial (51%) amino acid sequence analysis of purified AST IV showed complete amino acid homology, confirming the identity of the cDNA and establishing that AST IV was an N-blocked, 291-amino acid protein with a molecular mass of 33,909 daltons. The AST IV cDNA sequence differed from the PST-I cDNA in two principal ways: the 5' end lacked 18 coding bases, and the 3' end contained a 190-base extention in the untranslated region, including a consensus sequence for signalling polyadenylation. Studies of AST IV gene transcript levels showed that the livers of rats fed 2AAF for 3 wk (early stage hepatocarcinogenesis) and hyperplastic nodules from the livers of rats fed 2AAF for 19 wk (intermediate stage hepatocarcinogenesis) displayed transcript levels similar to those of livers from normal rats. This contrasted with the 60 to 70% lower than normal capacity of the mRNA fractions to express AST IV observed during in vitro translation. These results indicated that modulation of AST IV expression at early and intermediate stages of hepatocarcinogenesis involved regulatory mechanisms at the translational level. In contrast, mRNA fractions isolated from some 2AAF-induced liver tumors or from known chemical carcinogen-derived rat hepatoma cell lines showed losses of both AST IV transcript level and in vitro translation capacity, suggesting that regulation at the transcriptional level may become important at late stages of 2AAF-induced hepatocarcinogenesis. These results indicated that the molecular mechanisms for the 2AAF-mediated down regulation of AST IV expression during 2AAF-induced hepatocarcinogenesis involved alterations in regulation at both translational and transcriptional levels.


Assuntos
Arilsulfotransferase/genética , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas/genética , 2-Acetilaminofluoreno/toxicidade , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Expressão Gênica , Fígado/enzimologia , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Masculino , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Lesões Pré-Cancerosas/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Ratos , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência
5.
Comput Appl Biosci ; 2(2): 111-4, 1986 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-3450360

RESUMO

A software system utilizing dBASE-II operating on a dual-drive Apple II+ computer is described. Color factors and retention times for 15 amino acids and epsilon-(gamma-glutamyl)lysine dipeptide are calculated following high performance liquid chromatography. The software package produces a listing of acceptable limits for these parameters calculated as plus and minus 2 standard deviations of the mean. The code is distributed in source form.


Assuntos
Sistemas de Gerenciamento de Base de Dados , Dipeptídeos/análise , Fluorescência , Software , Algoritmos , Aminoácidos/análise , Cromatografia Líquida de Alta Pressão , Cor , Microcomputadores
6.
Biochim Biophys Acta ; 763(1): 27-34, 1983 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-6135451

RESUMO

The low level of transglutaminase activity in virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) increased markedly when cells were exposed to sodium butyrate. The effect of sodium butyrate was time- and concentration-dependent and fully reversible. Transformed cells exposed for 5 days to 1 mM sodium butyrate had fewer cells, showed an 8-10 fold higher transglutaminase activity, and stained more abundantly for transglutaminase and pericellular fibronectin than control cells when examined by indirect immunofluorescence. Non-transformed cells (WI-38) showed only a 2-4-fold increase in transglutaminase activity when treated in a similar manner. Studies with metabolic inhibitors revealed the increase in activity was the result of synthesis of new protein. Kinetic studies showed the affinity of putrescine for the enzyme was essentially unchanged but the number of active sites increased 9-fold following exposure to sodium butyrate. Enhanced transglutaminase activity returned to control levels within 7 days after subculture and sodium butyrate removal. These findings suggest that sodium butyrate offers a potential model system to understand better the role of transglutaminase in cells in culture; particularly growth control in transformed cells.


Assuntos
Aciltransferases/genética , Butiratos/farmacologia , Transformação Celular Viral , Aciltransferases/metabolismo , Ácido Butírico , Linhagem Celular , Dactinomicina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Imunofluorescência , Humanos , Cinética , Pulmão/efeitos dos fármacos , Pulmão/embriologia , Puromicina/farmacologia , Vírus 40 dos Símios/genética , Transglutaminases
8.
Proc Natl Acad Sci U S A ; 78(8): 5005-8, 1981 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-6117852

RESUMO

Cystamine inhibited transglutaminase activity (R-glutaminyl-peptide:amine gamma-glutamyltransferase, EC 2.3.2.13) of proliferating WI-38 cells in a dose-dependent manner over the concentration range 0.005-0.25 mM when added to the culture medium. The epsilon-(gamma-glutamyl)lysine content in the cells was decreased and several proliferation markers were enhanced. "Non-mitotic" cells were stimulated by cystamine (about 25% of that observed with 10% fetal bovine serum) to undergo DNA synthesis with subsequent increases in nuclei number. Numerous other disulfides, thiols, and amines were ineffective when added to culture medium. The findings are supportive of the concept that growth control involves a relationship between isopeptide crosslinks and proliferation.


Assuntos
Divisão Celular/efeitos dos fármacos , Cistamina/farmacologia , gama-Glutamiltransferase/antagonistas & inibidores , Células Cultivadas , Replicação do DNA/efeitos dos fármacos , Dissulfetos/farmacologia , Humanos , Compostos de Sulfidrila/farmacologia
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