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1.
Artigo em Inglês | MEDLINE | ID: mdl-27692294

RESUMO

Good cell culture practice and characterization of the cell lines used are of critical importance in in vitro genotoxicity testing. The objective of this initiative was to make continuously available stocks of the characterized isolates of the most frequently used mammalian cell lines in genotoxicity testing anywhere in the world ('IVGT' cell lines). This project was organized under the auspices of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing. First, cell isolates were identified that are as close as possible to the isolate described in the initial publications reporting their use in genotoxicity testing. The depositors of these cell lines managed their characterization and their expansion for preparing continuously available stocks of these cells that are stored at the European Collection of Cell Cultures (ECACC, UK) and the Japanese Collection of Research Bioresources (JCRB, Japan). This publication describes how the four 'IVGT' cell lines, i.e. L5178Y TK+/- 3.7.2C, TK6, CHO-WBL and CHL/IU, were prepared for deposit at the ECACC and JCRB cell banks. Recommendations for handling these cell lines and monitoring their characteristics are also described. The growth characteristics of these cell lines (growth rates and cell cycles), their identity (karyotypes and genetic status) and ranges of background frequencies of select endpoints are also reported to help in the routine practice of genotoxicity testing using these cell lines.


Assuntos
Técnicas de Cultura de Células/normas , Dano ao DNA/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfoma/tratamento farmacológico , Testes de Mutagenicidade/normas , Mutagênicos/toxicidade , Padrões de Referência , Animais , Células CHO , Células Cultivadas , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Cariotipagem Espectral , Proteína Supressora de Tumor p53/metabolismo
2.
J Mol Biol ; 267(5): 1247-57, 1997 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-9150409

RESUMO

Specific molecular interactions involved in catalysis by antibody 6D9 were investigated by site-directed mutagenesis. The catalytic antibody 6D9, which was generated against a transition state analog (III), hydrolyzes a non-bioactive chloramphenicol monoester derivative (I) to produce chloramphenicol (II). Construction of a three-dimensional molecular model of 6D9 and sequence comparison within a panel of related antibodies suggested candidates for catalytic residues, His (L27d), Tyr (L32), Tyr (H58) and Arg (H100b); these were targeted for the site-directed mutagenesis study. The Y-H58-F and R-H100b-A mutants possessed catalytic activities comparable to that of the wild-type, and the Y-H58-H and Y-L32-F mutant displayed an approximately fivefold decrease in k(cat)/Km. In the transition state analysis, the plots of logK(TSA) versus log(k(cat)/Km) for the mutants are linear, with a slope of approximately 1.0, indicating that the entire hapten-binding energy in the mutants is also utilized to bind the transition state and to accelerate the catalysis. In addition, a dramatic change in the catalytic activity was observed when the histidine residue (27d) in the CDR1 light chain was replaced with alanine. The H-L27d-A mutant had no detectable catalytic activity. This mutation led to a large, 40-fold reduction in transition state binding, with no change in substrate binding. Coupled with the previous kinetic studies and chemical modifications of the intact 6D9 antibody, this mutagenesis study has demonstrated that His L27d plays an essential role in stabilization of the transition state, the mechanism of catalysis by the 6D9 antibody.


Assuntos
Anticorpos Catalíticos/metabolismo , Sítios de Ligação de Anticorpos , Histidina/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Sequência de Aminoácidos , Anticorpos Catalíticos/genética , Sítios de Ligação de Anticorpos/genética , Cloranfenicol/biossíntese , Análise Mutacional de DNA , Ésteres/metabolismo , Histidina/genética , Hidrólise , Fragmentos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pró-Fármacos/metabolismo
3.
FEBS Lett ; 311(3): 226-30, 1992 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-1397319

RESUMO

Processing of human lysozyme with artificially designed signal sequences was examined in an in vitro translation-translocation system and compared with their secretory capabilities in yeast. It has been shown that the conformation of the C-terminal region of the signal sequence and the length of the hydrophobic segment are important factors for efficient cleavage of the signal sequence.


Assuntos
Muramidase/genética , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/química , Sinais Direcionadores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Sistema Livre de Células , Humanos , Cinética , Dados de Sequência Molecular , Muramidase/biossíntese , Biossíntese de Proteínas , Conformação Proteica , Coelhos , Proteínas Recombinantes/biossíntese , Reticulócitos/metabolismo , Transcrição Gênica , Tripsina
4.
Neurochem Int ; 31(5): 715-22, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9364457

RESUMO

We investigated kainate-induced excitotoxicity in embryonic rat hippocampal cells cultured in a chemically defined medium. Treatment with kainate for 24 h resulted in neuronal death, as assessed by the release of lactate dehydrogenase into the culture media. This neurotoxic effect was kainate dose- and culture age-dependent. EC50 of kainate was 127 +/- 11 microM. 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (f)quinoxaline (NBQX) completely blocked the toxicity, while MK801, an N-methyl-D-aspartate (NMDA) receptor antagonist, also blocked it but not completely. Furthermore, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) attenuated the kainate injury, while the selective and noncompetitive AMPA-preferring receptor antagonist 1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2,3-benzo-diazepine (GYKI 52466) blocked it completely. Concanavalin A (ConA), which potentiates the response to kainate at kainate-preferring receptors, had little effect on kainate toxicity. Further, AMPA alone induced little toxicity, but produced remarkable toxicity when cyclothazide was used to block the desensitization of AMPA-preferring receptors. These results indicate that kainate excitotoxicity in hippocampal cultures is mediated by AMPA- but not kainate-preferring receptors, and that it involves NMDA-receptor-mediated toxicity. The non-desensitizing response at AMPA-preferring receptors may play an important role in kainate-induced excitotoxicity.


Assuntos
Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Ácido Caínico/farmacologia , Neurotoxinas/farmacologia , Receptores de AMPA/efeitos dos fármacos , Receptores de Ácido Caínico/efeitos dos fármacos , Animais , Células Cultivadas , Meios de Cultivo Condicionados , Embrião de Mamíferos , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Ácido Caínico/metabolismo , N-Metilaspartato/farmacologia , Neurotoxinas/metabolismo , Ratos , Ratos Wistar , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
5.
Eur J Pharmacol ; 309(3): 299-306, 1996 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-8874154

RESUMO

The inhibitory potencies of 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K), 2-3,dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) and 1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5H-2,3-benzodiazep ine (GYKI 52466) at excitatory amino acid receptors were examined in rat cortical mRNA-injected Xenopus oocytes using a two-electrode voltage clamp. Schild analysis of YM90K and NBQX inhibition of kainate currents yielded pA2 values of 6.83 +/- 0.01 and 7.24 +/- 0.01, respectively. GYKI 52466 reduced the maximum kainate response and increased the kainate EC50 in a dose-dependent manner, suggesting that the antagonism of AMPA receptors by GYKI 52466 is mixed competitive and non-competitive for kainate. Schild analysis of YM90K and NBQX inhibition of kainate currents in the presence of 30 microM cyclothiazide yielded pA2 values of 6.62 +/- 0.03 (slope: 1.02 +/- 0.01) and 7.10 +/- 0.02 (slope: 1.00 +/- 0.02), respectively, consistent with competitive antagonism. Cyclothiazide potentiated the AMPA response as well as the kainate response and increased the apparent Hill coefficients in a concentration-dependent manner. The potency of YM90K to inhibit AMPA-induced current could be reduced by increasing the concentration of cyclothiazide. We showed that YM90K is a potent and competitive antagonist for AMPA receptors and the apparent affinity of competitive antagonists was reduced by cyclothiazide. Cyclothiazide can affect the interaction between receptors and both agonists and antagonists, suggesting that it might allosterically alter the affinity of agonists and competitive antagonists for their binding site on the AMPA receptor complex.


Assuntos
Anticonvulsivantes/farmacologia , Córtex Cerebral/efeitos dos fármacos , Quinoxalinas/farmacologia , Receptores de AMPA/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Ácido Caínico/farmacologia , Masculino , Oócitos , Ratos , Ratos Wistar , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
6.
Mutat Res ; 441(2): 205-13, 1999 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-10333534

RESUMO

Some 16 nitroquinolines (NQs) and their fluorinated derivatives were tested for mutagenicity in Salmonella typhimurium TA100 without S9 mix to investigate the effect of fluorine-substitution on the mutagenicity. These NQs consist of 5-NQs, 5-nitroquinoline N-oxides (5-NQOs), N-methyl-5-nitroquinolinium methanesulfonates (N-Me-5-NQs) and 8-NQs, including three ortho-F-NQs, one meta-F-NQ, four para-F-NQs and four 3-F-NQs. For this purpose, eight F-NQs were newly synthesized. The data indicated that the ratio of the mutagenic activities (revertants/plate/nmol) of fluorinated NQs to those of the corresponding parent non-fluorinated compounds ranged from 0.6- to 119-fold. The fluorine atom located para to the nitro group markedly enhanced the mutagenicity (24-fold and more), while three ortho-fluorinated derivatives showed no significant increase in mutagenicity (enhancement ratio were 0.6, 0.8 and 1.7). With respect to 8-NQs, its meta-fluorinated derivative also had an enhanced mutagenicity over the parent compound (53-fold). In addition, although N-Me-5-NQ was less mutagenic than 5-NQ and 5-NQO, the mutagenicity of N-Me-5-NQ was most significantly enhanced by fluorine-substitution. These results suggest that introduction of a fluorine atom to the molecule in question may be a useful tool to modify their mutagenic potency and to better understand the mechanism of mutation.


Assuntos
Compostos de Flúor/toxicidade , Nitroquinolinas/toxicidade , Cromatografia em Gel , Compostos de Flúor/síntese química , Espectroscopia de Ressonância Magnética , Testes de Mutagenicidade , Nitroquinolinas/síntese química , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Relação Estrutura-Atividade
7.
Mutat Res ; 491(1-2): 211-20, 2001 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-11287313

RESUMO

The o-aminoazotoluene (AAT) has been evaluated as a possible human carcinogen by the International Agency for Research on Cancer. In rodents, it is carcinogenic mainly in the liver, and also in lung following long term administration. We previously examined in lambda/lacZ transgenic mice for the induction of lacZ mutations in liver, lung, urinary bladder, colon, kidney, bone marrow, and testis. AAT induced gene mutations strongly in the liver and colon. In the present report, we reveal the molecular nature of mutations induced by AAT in the lambda cII gene (the cII gene, a phenotypically selectable marker in the lambda transgene, has 294bp, which makes it easier to sequence than the original target, the 3kb lacZ gene). The cII mutant frequency in liver and colon was five and nine times higher, respectively, in AAT-treated mice than in control mice. Sequence analysis revealed that AAT induced G:C to T:A transversions, whereas spontaneous mutations consisted primarily of G:C to A:T transitions at CpG sites.


Assuntos
Óperon Lac , Mutagênicos/toxicidade , Mutação , Fatores de Transcrição/genética , o-Aminoazotolueno/toxicidade , Animais , Sequência de Bases , Primers do DNA , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Virais
8.
Mutat Res ; 456(1-2): 73-81, 2000 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-11087898

RESUMO

Quinoline is carcinogenic to the liver in rodents, but it is not clear whether it acts by a genotoxic mechanism. We previously demonstrated that quinoline does induce gene mutation in the liver of lambda/lacZ transgenic mice. In the present report, we reveal the molecular nature of the mutations induced by quinoline in the lambda cII gene, which is also a phenotypically selectable marker in the lambda transgene. (The cII gene has 294bp, which enables much easier sequence analysis than the original lacZ gene (3kb)). The liver cII mutant frequency was nine times higher in quinoline-treated mice than in control mice. Sequence analysis revealed that quinoline induced primarily G:C to C:G transversions (25 of 34). Thus, we have confirmed that quinoline is genotoxic in its target organ, and the G:C to C:G transversion is the molecular signature of quinoline-induced mutations.


Assuntos
Carcinógenos/toxicidade , Fígado/efeitos dos fármacos , Mutação , Quinolinas/toxicidade , Fatores de Transcrição/genética , Animais , Bacteriófago lambda/genética , Sequência de Bases , Primers do DNA/genética , DNA Recombinante/genética , Óperon Lac , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Troca de Cromátide Irmã/efeitos dos fármacos , Troca de Cromátide Irmã/genética , Proteínas Virais
9.
J Pharm Pharmacol ; 50(7): 795-801, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9720630

RESUMO

The in-vitro pharmacological properties of (2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl)-acetic acid monohydrate, YM872, a novel and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor antagonist were investigated. YM872 is highly water soluble (83 mg mL(-1) in Britton-Robinson buffer) compared with 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). YM872 potently inhibits [3H]AMPA binding with a Ki (apparent equilibrium dissociation constant) value of 0.096 +/- 0.0024 microM. However, YM872 had very low affinity for other ionotropic glutamate receptors, as measured by competition with [3H]kainate (high-affinity kainate binding site, concentration resulting in half the maximum inhibition (IC50) = 4.6 +/- 0.14 microM), [3H]glutamate (N-methyl-D-aspartate (NMDA) receptor glutamate binding site, IC50 > 100 microM) and [3H]glycine (NMDA receptor glycine-binding site, IC50 > 100 microM). YM872 competitively antagonized kainate-induced currents in Xenopus laevis oocytes which express rat AMPA receptors, with a pA2 value of 6.97 +/- 0.01. In rat hippocampal primary cultures, YM872 blocked a 20-microM AMPA-induced increase of intracellular Ca2+ concentration with an IC50 value of 0.82 +/- 0.031 microM, and blocked 300-microM kainate-induced neurotoxicity with an IC50 value of 1.02 microM. These results show that YM872 is a potent and highly water-soluble AMPA antagonist with great potential for treatment of neurodegenerative disorders such as stroke.


Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/efeitos dos fármacos , Imidazóis/farmacologia , Fármacos Neuroprotetores/farmacologia , Quinoxalinas/farmacologia , Receptores de AMPA/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , 6-Ciano-7-nitroquinoxalina-2,3-diona/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Ligação Competitiva , Soluções Tampão , Células Cultivadas , Antagonistas de Aminoácidos Excitatórios/metabolismo , Hipocampo/metabolismo , Ácido Caínico , Masculino , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Quinoxalinas/metabolismo , Ratos , Ratos Wistar , Solubilidade , Xenopus laevis
11.
Biosci Biotechnol Biochem ; 58(4): 779-81, 1994 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7764869

RESUMO

We examined the secretion of human growth hormone in yeast cells with the artificial signal sequence L8LP, which is functional for human lysozyme secretion. The precursor was cleaved efficiently and the mature protein was secreted into the periplasmic space, but the protein aggregated. These results suggest that L8LP is also functional for human growth hormone. pI precipitation might be responsible for the aggregation.


Assuntos
Hormônio do Crescimento/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/genética , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Hormônio do Crescimento/genética , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Mutagênese , Plasmídeos , Conformação Proteica , Precursores de Proteínas/metabolismo , Sinais Direcionadores de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética
12.
J Biol Chem ; 266(30): 20363-8, 1991 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-1939091

RESUMO

Signal sequences play a central role in the initial membrane translocation of secretory proteins. Their functions depend on factors such as hydrophobicity and conformation of the signal sequences themselves. However, some characteristics of mature proteins, especially those of the N-terminal region, might also affect the function of the signal sequences. To examine this possibility, several mutants of human lysozyme modified in the N-terminal region of the mature protein were constructed, and their secretion in yeast as well as in vitro translocation into canine pancreatic microsomes were analyzed using an idealized signal sequence L8 (MR(L)8PLAALG). Our results show the following. (1) Change in the charge at the N-terminal residue of the mature protein does not affect secretion drastically. (2) Substitution of a proline residue at the N terminus prevents cleavage of the signal sequence, although translocation itself is not impaired. (3) Excessive positive charges in the N-terminal region delay translocation of the precursor protein across the membrane. (4) Polar and negatively charged residues introduced into the N-terminal region affect the secretion of the mature protein by preventing its correct folding.


Assuntos
Microssomos/metabolismo , Muramidase/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Membrana Celular/metabolismo , Cães , Glicosilação , Humanos , Dados de Sequência Molecular , Muramidase/metabolismo , Mutação , Biossíntese de Proteínas , RNA Fúngico/genética , RNA Mensageiro/genética , Transcrição Gênica , Translocação Genética
13.
Biol Pharm Bull ; 20(8): 838-42, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9300127

RESUMO

A cytotoxic factor (CF) appeared in murine serum after the intravenous injection of the dehydrogenation polymers (DHPs) of p-coumaric acid (DHP-pCA), caffeic acid (DHP-CA), and ferulic acid (DHP-FA), which are categorized as a class of synthetic lignins. The highest CF activity was observed 15 min after the i.v. injection of DHP-pCA. CF is likely to be cytocidal through an apoptotic mechanism accompanied by nucleosome-sized DNA fragmentation. CF is extractable with aqueous ethanol and highly stable against heat, proteases, and acid/alkali treatments. The ethanol extract showed cytotoxicity toward various cultured cell lines and also ascites carcinoma cells in vivo. The parent molecules DHPs did not show any appreciable cytotoxicity. After the induction of CF activity, the activity quickly diminished and completely disappeared from the blood stream within an hour or so. The cytotoxicity was observed only when the target cells were exposed to CF for longer than 10 h.


Assuntos
Derivados de Benzeno/toxicidade , Citotoxinas/química , Lignina/toxicidade , Polímeros/toxicidade , Animais , Derivados de Benzeno/química , Linhagem Celular , Fragmentação do DNA , Feminino , Hidrogenação , Injeções Intravenosas , Leucemia L1210 , Lignina/química , Camundongos , Camundongos Endogâmicos ICR , Testes de Mutagenicidade , Extratos Vegetais/química , Polímeros/química
14.
Biol Pharm Bull ; 21(10): 1098-101, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9821818

RESUMO

A cytotoxic factor (CF) toward cultured murine leukemia L1210 cells was induced in mouse serum by intravenous injection of a dehydrogenation polymer of p-coumaric acid (DHP-pCA). When the serum from the treated mice was diluted with ethanol, CF was preserved in its supernatant (EtOH-sup). An EtOH-sup prepared from untreated control mice also showed cytotoxicity, although at much higher concentrations. The CF activity of EtOH-sups from both treated and untreated mice was completely eliminated by acid treatment at pH 2 at 90 degrees C for 30 min but kept intact by alkali treatment. In addition, the CF activity of both EtOH-sups was not affected by digestion with chymotrypsin. CF was recovered in a neutral MeOH-eluate from a DEAE-cellulofine column but not in HCI-MeOH eluate, in which lignified materials including DHP-pCA should have been recovered. These findings strongly suggest that CF is not a metabolite of DHP-pCA but an endogenous component of the normal serum which is augmented by DHP-pCA administration.


Assuntos
Ácidos Cumáricos/farmacologia , Citotoxinas/biossíntese , Animais , Cromatografia , Citotoxinas/sangue , Estabilidade de Medicamentos , Etanol/química , Concentração de Íons de Hidrogênio , Leucemia L1210/tratamento farmacológico , Camundongos
15.
Agric Biol Chem ; 54(1): 131-7, 1990 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1368515

RESUMO

Silkworm antitrypsin (sw-AT) isolated from larval hemolymph was limitedly digested by Achromobacter lysylendopeptidase, alpha-chymotrypsin, subtilisin BPN', subtilisin Carlsberg, papain, or Pseudomonas elastase. Each proteinase could cleave specific site(s) around the reactive site identified for the reaction of sw-AT and bovine trypsin. Among these proteinases, only subtilisin BPN' was inhibited by sw-AT, although weakly. By the cleavable amino acid sequence in sw-AT, it was suggested that whether or not these proteinases were inhibited by sw-AT did not solely depend on their substrate specificities. The susceptibility to the attack of proteinase should indicate that this region is exposed on the molecular surface. The amino acid sequence in the COOH-terminal region slightly away from the reactive site in sw-AT had homology with that in the corresponding region of the serine proteinase inhibitor (serpin) group.


Assuntos
Peptídeo Hidrolases/metabolismo , alfa 1-Antitripsina/metabolismo , Sequência de Aminoácidos , Animais , Bombyx , Eletroforese em Gel de Poliacrilamida , Hemolinfa , Larva , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Inibidores de Proteases , Homologia de Sequência do Ácido Nucleico
16.
Agric Biol Chem ; 54(1): 139-45, 1990 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1368516

RESUMO

Silkworm antitrypsin (sw-AT), which was thought to belong to serpin family, changed its behavior against denaturation after chymotryptic cleavage of a single peptide bond (Tyr-Val) two amino acids away from the reactive site for trypsin (Lys-Val). This chymotrypsin-modified sw-AT became resistant to denaturation by heat, sodium dodecyl sulfate, or guanidine hydrochloride, and this characteristic was evident in its circular dichroism spectrum. The modified sw-AT was also indigestible by S. aureus V8 protease. These facts should indicate a structural change from a stressed, unstable state to a stable one accompanying the cleavage of the single peptide bond in sw-AT. The stabilizing factor was in part attributed to the interaction of a COOH-terminal fragment (5 kDa) and an NH2-terminal one (36 kDa) in modified sw-AT.


Assuntos
Quimotripsina/metabolismo , Desnaturação Proteica , Serina Endopeptidases/metabolismo , alfa 1-Antitripsina/metabolismo , Animais , Bombyx , Dicroísmo Circular , Guanidina , Guanidinas/farmacologia , Hemolinfa , Cinética , Larva , Conformação Proteica
17.
J Biol Chem ; 266(32): 21709-17, 1991 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-1939195

RESUMO

In the adult dog liver cytosol we identified four glutathione S-transferase (GST) subunits, Yd1 (Mr 26,000), Yd2 (Mr 27,000), Yd3 (Mr 28,000), and Ydf (Mr 27,400), and purified GST forms comprising Yd1, Yd2, and Yd3, to apparent homogeneity. Unlike rat transferases the enzyme activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was not retained on the affinity column. Thus the DCNB-active enzyme, GST YdfYdf, from the flow-through fraction of the affinity column was also purified to homogeneity by gel filtration, DE52 chromatography, chromatofocusing, and hydroxylapatite column chromatography. Immunoblot analysis of dog GSTs revealed that the subunits Yd1, Yd2, and Yd3 belong to the pi, alpha, and mu class, respectively. On the contrary, Ydf had no reactivity with antibodies raised against any of the three classes of GST. Each subunit, Yd1, Yd2, Yd3, and Ydf, was distinguishable by its own retention time on reverse-phase high performance liquid chromatography. N-terminal amino acid sequences of the dog GSTS Yd1Yd1 and Yd3Yd3 revealed a high degree of homology to the pi and mu class transferases from rat, human, and mouse, respectively, while the N terminus of Yd2Yd2 is blocked. N-terminal amino acid sequences of GST YdfYdf showed no homology to any of the three classes of GST. The most significant property noted of GST YdfYdf is the high specific activity toward DCNB, exceeding by 1 order of magnitude the corresponding values for the known mu class GSTs. The present results strongly suggest that dog GST YdfYdf is a unique enzyme distinct from the hitherto characterized GST isozymes.


Assuntos
Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Fígado/enzimologia , Nitrobenzenos/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Citosol/enzimologia , Cães , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/genética , Glutationa Transferase/isolamento & purificação , Humanos , Imunodifusão , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Cinética , Substâncias Macromoleculares , Masculino , Dados de Sequência Molecular , Peso Molecular , Homologia de Sequência do Ácido Nucleico , Especificidade por Substrato
18.
J Biol Chem ; 264(20): 11546-9, 1989 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-2545673

RESUMO

To facilitate the preparation of ribonuclease H from Escherichia coli in an amount sufficient for crystallographic studies, we have constructed an overproduction system for the enzyme. The structural gene for the enzyme was subcloned from pSK750 (Kanaya, S., and Crouch, R. J. (1983) J. Biol. Chem. 258, 1276-1281) to make a plasmid vector pPL801, in which the gene was under the control of bacteriophage lambda PL promoter. Thermal induction of the gene accumulated the enzyme in E. coli N4830-1 to approximately 8% of the total cytosolic protein. The level of production of the enzyme in N4830-1 harboring pPL801 was 14 mg/liter culture, which was 3000 times as high as that in the host cell. The enzyme was purified with a yield of more than 80% and crystallized by utilizing the property that the solubility of the enzyme decreased at pH values close to its isoelectric point (pI = 9). Crystals were grown by successive seeding (hanging drop method) for x-ray crystallographic analysis. The crystals belong to space group P212121 with unit cell dimensions of alpha = 44.1 A, b = 87.0 A, c = 35.5 A and contain one molecule in an asymmetric unit. They diffracted x-rays beyond 2.5 A resolution.


Assuntos
Endorribonucleases/biossíntese , Escherichia coli/enzimologia , Bacteriófago lambda/genética , Cristalografia , Endorribonucleases/genética , Escherichia coli/genética , Hibridização de Ácido Nucleico , Plasmídeos , Regiões Promotoras Genéticas , Ribonuclease H
19.
Plant Cell ; 9(8): 1265-77, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9286105

RESUMO

Previous studies have shown that recessive mutations at the Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEAFY COTYLEDON1 (LEC1) loci lead to various abnormalities during mid-embryogenesis and late embryogenesis. In this study, we investigated whether these loci act in independent regulatory pathways or interact in controlling certain facets of seed development. Several developmental responses were quantified in abi3, fus3, and lec1 single mutants as well as in double mutants combining either the weak abi3-1 or the severe abi3-4 mutations with either fus3 or lec1 mutations. Our data indicate that ABI3 interacts genetically with both FUS3 and LEC1 in controlling each of the elementary processes analyzed, namely, accumulation of chlorophyll and anthocyanins, sensitivity to abscisic acid, and expression of individual members of the 12S storage protein gene family. In addition, both FUS3 and LEC1 regulate positively the abundance of the ABI3 protein in the seed. These results suggest that in contrast to previous models, the ABI3, FUS3, and LEC1 genes act synergistically to control multiple elementary processes during seed development.


Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Genes de Plantas , Ácido Abscísico/farmacologia , Alérgenos , Antocianinas/metabolismo , Antígenos de Plantas , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Clorofila/metabolismo , Expressão Gênica , Modelos Biológicos , Família Multigênica , Mutagênese , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas de Armazenamento de Sementes , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Fatores de Transcrição
20.
Nature ; 383(6595): 89-92, 1996 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-8779726

RESUMO

Stimulation of two metabotropic glutamate-receptor subtypes, mGluR1 and mGluR5, triggers the release of Ca2+ from intracellular stores through the inositol-(1,4,5) trisphosphate (InsP3) pathway. Here we report that glutamate induces single-peaked intracellular Ca2+ mobilization in mGluR1alpha-transfected cells but elicits Ca2+ oscillations in mGluR5a-transfected cells. The response patterns of the intracellular Ca2+ increase depend upon the identity of a single amino acid, aspartate (at position 854) or threonine (at position 840), located within the G-protein-interacting domains of mGluR1alpha and mGluR5a, respectively. Pharmacological and peptide mapping analyses indicated that phosphorylation of the threonine residue at position 840 of mGluR5a by protein kinase C (PKC) is responsible for the generation of Ca2+ oscillations in mGluR5a-expressing cells. To our knowledge this is the first evidence that PKC phosphorylation of G-protein-coupled receptors is important in producing oscillations in intracellular Ca2+ signalling.


Assuntos
Cálcio/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Ácido Aspártico/metabolismo , Linhagem Celular , Proteínas de Ligação ao GTP/metabolismo , Ácido Glutâmico/metabolismo , Camundongos , Dados de Sequência Molecular , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Receptores de AMPA/metabolismo , Receptores de Glutamato Metabotrópico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Treonina/metabolismo , Transfecção
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