RESUMO
Cerebrovascular disease is a main cause of mortality and one of the big medical problems. After the vascular wall's damage the endothelial cells secrete the von Willebrand factor which then connects with its platelet's receptor GP Ib-V-IX. There are two polymorphisms Thr145Met and T(-5)C of the GP Iba gene associated with arterial thrombosis development. Also the difference in platelets' genes expressions was shown in patients with various clinical course of ischemic heart disease. The aim of this study was to investigate the role of platelet's receptor for von Willebrand factor in platelets' activation in patients with cerebrovascular disease. 123 patients with cerebrovascular disease and 97 healthy donors were included into the study. We analyzed the level of receptor for von Willebrand factor on platelet's membrane by flow cytometry, Thr145Met and T(-5)C GP Iba polymorphiams by PCR-RFLP, the GP Iba gene expression by RT-PCR and ADP-induced platelet aggregation by Born method. We have shown: 1) the 145Met GP Iba allele prevalence in patients with atherotrombotic stroke development due to macroangiopathy; 2) the pre-mRNA transform into the mature mRNA in activated platelets and this process may be stopped by the antiplatelet therapy by acetylsalicylic acid.
Assuntos
Plaquetas/metabolismo , Trombose Coronária/sangue , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Polimorfismo Genético , Acidente Vascular Cerebral/sangue , Difosfato de Adenosina/farmacologia , Alelos , Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Estudos de Casos e Controles , Trombose Coronária/complicações , Trombose Coronária/genética , Trombose Coronária/patologia , Feminino , Expressão Gênica , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Cultura Primária de Células , Precursores de RNA/genética , Precursores de RNA/metabolismo , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
p21/Waf1 protein is one of the main cell cycle arrest regulators and one of the most well-known transcriptional targets of TP53 protein. Here, we demonstrated the activation of expression of the p21/Waf1 gene when the cells were treated to sodium butyrate (NaBu)--one of the natural inhibitors of deacetylase, and investigated whether this phenomenon depends on the presence of functionally active TP53 protein. We compared the effect of the NaBu treatment on the human cell line with different TP53 mutation profile, including: wild-type TP53, single nucleotide substitutions, and the complete absence of TP53 gene. NaBu activated the TP53 protein via hyper acetylation at lysine residue K382, without significant changes in the level of protein expression. Western blotting demonstrated that the addition of NaBu triggers a significant increase in the p21/Waf1 protein level in both the TP53 wild-type cells and in the cells with single nucleotide substitutions in the domain responsible for the binding of TP53 protein to DNA. At the same time, no the p21/Waf1 protein induction was observed in the cells with complete deletion of the TP53 gene. However, NaBu was not able to induce the p2 1/Waf1 production when the expression of TP53 was transiently knocked down by the p53 siRNA. Overall, our results suggest that the NaBu-dependent induction of p21/Waf1 does require the presence of TP53 protein but unexpectedly it can occur regardless of mutational changes in the domain responsible for the TP53 binding to DNA. One of the hypothetical explanations is that NaBu increases the level of TP53 acetylation, and the modified protein is able to establish a new network of protein-protein interactions or trigger some conformational changes affecting the TP53-dependent transcriptional machinery even when its DNA binding ability is impaired.
Assuntos
Ácido Butírico/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Mutação , Proteína Supressora de Tumor p53/genética , Acetilação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/agonistas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inativação Gênica , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
The nucleotide sequence of the ribC gene encoding the synthesis ofbifunctional flavokinase/flavine adenine nucleotide (FAD) synthetase in Bacillus subtilis have been determined in a family of riboflavin-constitutive mutants. Two mutations have been found in the proximal region of the gene, which controls the transferase (FAD synthase) activity. Three point mutations and one double mutation have been found (in addition to the two mutations that were detected earlier) in the distal region of the gene, which controls the flavokinase (flavin mononucleotide (FMN) synthase) activity. On the basis of all data known to date, it has been concluded that the identified mutations affect riboflavin and ATP binding sites. No mutations have been found in the PTAN conserved sequence, which forms the magnesium and ATP common binding site and is identical for organisms of all organizational levels, from bacteria too humans.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Genes Bacterianos/genética , Mutação , Nucleotidiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Análise Mutacional de DNA , Nucleotidiltransferases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Among Bacillus subtilis riboflavin-resistant mutants we identified one, which differed from other regulatory mutants by overproduction of riboflavin and simultaneous upregulation of the rib C gene encoding flavokinase/FAD-synthase. Genetic and biochemical analysis showed that the ribU1 mutation determines a trans-acting factor that simultaneously regulates activity of riboflavin and truB-ribC-rpsO operons. Regulatory activity of the ribU1 mutation comprises about 10% of Rfn element activity on interaction with flavins. The ribUl mutation can be presumably ascribed to a gene of the transcriptional regulators family.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Mutação , Nucleotidiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Riboflavina/genética , Fatores de Transcrição/genética , Bacillus subtilis/enzimologia , Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/genética , Nucleotidiltransferases/biossíntese , Óperon/genética , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fármacos Fotossensibilizantes/farmacologia , Riboflavina/biossíntese , Riboflavina/farmacologia , Fatores de Transcrição/metabolismoRESUMO
Insertion and deletion mutagenesis of the leader region of the Bacillus subtilis rib operon encoding FMN-specific sensor RNA was conducted. Insertions of different structure and length in the conservative motif of the leader sequence (Rfn-element) were shown to cause partially constitutive expression of the operon resulted in an increased accumulation of riboflavin. At the same time, introducing into the genome of insertion mutants an additional ribC mutation blocking FMN synthesis leads to an increase in riboflavin production. Deletion of the main Rho-dependent transcription terminator gives rise to the same effect. These results indicate that some additional FMN-dependent regulation is involved in rib-operon control, which is, however, still connected with the primary and secondary structure of the leader region.
Assuntos
Bacillus subtilis/fisiologia , Óperon , Riboflavina/biossíntese , Bacillus subtilis/genética , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Riboflavina/genéticaRESUMO
Here we describe cloning, expression, and purification of the enzyme trehalose-6-phosphate hydrolase from thermoresistant strain Bacillus sp. GP16. Principal biochemical properties of the enzyme at different pH and temperature values were determined. Entropy and enthalpy of activation of the enzyme for substrates trehalose-6-phosphate and p-nitrophenyl glucoside were calculated, and the dependence of the kinetic parameters from ionic strength was established.
Assuntos
Bacillus/enzimologia , Bacillus/fisiologia , Dissacaridases/genética , Dissacaridases/isolamento & purificação , Dissacaridases/metabolismo , Trealose/análogos & derivados , Sequência de Aminoácidos , Bacillus/genética , Sequência de Bases , Catálise , Clonagem Molecular , Escherichia coli , Glucose/metabolismo , Glucose Oxidase/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Nitrofenilgalactosídeos/química , Nitrofenilgalactosídeos/metabolismo , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , Fosfatos Açúcares/metabolismo , Temperatura , Termodinâmica , Trealose/metabolismoRESUMO
This review highlights the main properties of mammalian, plant, and microbial alpha-glucosidases. Special attention is given to the classification of these enzymes, possible catalytic mechanisms, their tertiary structure, and the structure of major inhibitors. Experimental data on the elucidation of amino acid residues essential for catalysis are also discussed.
