RESUMO
Mutations in the CDH23 gene are known to be responsible for both Usher syndrome type ID (USH1D) and non-syndromic hearing loss (DFNB12), and the molecular confirmation of the CDH23 gene has become important in the diagnosis of these conditions. The present study was performed to find whether the CDH23 mutations are also responsible for non-syndromic hearing loss in patients in the Japanese population. A total of 51 sequence variants were found in 64 Japanese probands with non-syndromic sensorineural hearing impairment from autosomal recessive families. Among them, at least four missense mutations in six patients from five families were confirmed to be responsible for deafness by segregation study. All mutations detected were missense mutations, corroborating the previous reports regarding DFNB12. The present data confirmed that CDH23 mutations are frequently found and significantly responsible in Japanese. Interestingly, the CDH23 mutation spectrum in Japanese is very different from that found in Caucasians. This Japanese spectrum may be representative of those in Eastern Asian populations and its elucidation is expected to facilitate the molecular diagnosis of DFNB12 and USH1D.
Assuntos
Caderinas/genética , Perda Auditiva Neurossensorial/genética , Mutação , Proteínas Relacionadas a Caderinas , Estudos de Casos e Controles , Análise Mutacional de DNA , Éxons , Feminino , Regulação da Expressão Gênica , Genótipo , Perda Auditiva Neurossensorial/etnologia , Humanos , Japão , Masculino , Modelos Genéticos , Linhagem , FenótipoRESUMO
The NH2-terminal domain of sonic hedgehog (residue 25-198) was expressed in both yeast and animal cells. The yeast-derived NH2-terminal domain of sonic hedgehog was less active by far than the animal cell-derived counterpart. The yeast-derived NH2-terminal domain of sonic hedgehog lacked 10 amino acids from the NH2-terminus. This cleavage of the yeast-derived NH2-terminal domain of sonic hedgehog might due to Kex 2. In contrast, a mutant yeast-derived NH2-terminal domain of sonic hedgehog (Lys-33 to Thr) retained its NH2-terminus and its activity was comparable to that of the animal cell-derived NH2-terminal domain of sonic hedgehog. The NH2-terminal deleted NH2-terminal domain of sonic hedgehog completely lost its activity, nevertheless it inhibited the alkaline phosphatase activity induced by the animal cell-derived NH2-terminal domain of sonic hedgehog in a dose-dependent manner. These data suggest that the NH2-terminal deleted NH2-terminal domain of sonic hedgehog retains a receptor-binding ability and that the NH2-terminal peptide of the NH2-terminal domain of sonic hedgehog is necessary for its signal transduction.
Assuntos
Proteínas/química , Proteínas/metabolismo , Transativadores , Fosfatase Alcalina/antagonistas & inibidores , Substituição de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Primers do DNA/genética , Expressão Gênica , Proteínas Hedgehog , Técnicas In Vitro , Células L , Camundongos , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Deleção de Sequência , Transdução de SinaisRESUMO
In order to deduce which cellular molecules react with the sera from patients with rheumatoid arthritis (RA), human and mouse cellular extracts were fractionated stepwise, by ethanol precipitation and their reactivity analysed by Western blotting. It was found that three cytoplasmic molecules with molecular weights of 80,000, 81,000 and 77,000 were immunoreactive and they were identified as ezrin (E), radixin (R), and moesin (M), respectively, by partial amino acid sequencing. Using cDNA clones of these human molecules, recombinant proteins were produced in Escherichia coli and used to enable the antigens to detect the antibodies in the sera of patients with RA. Of 71 sera tested, 24 sera (33.8%) reacted with at least one of three recombinant antigens, although there was no significant correlation between the presence of the antibodies and clinical manifestations, such as disease duration or stage. There was also no discernible relationship to other auto-antibodies such as antinuclear antibodies (ANA) and rheumatoid factor. The results suggest that ERM proteins are possible novel auto-immune target antigens for RA.
Assuntos
Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Proteínas Sanguíneas/imunologia , Proteínas do Citoesqueleto , Proteínas de Membrana/imunologia , Proteínas dos Microfilamentos , Fosfoproteínas/imunologia , Proteínas/imunologia , Adulto , Idoso , Sequência de Aminoácidos , Reações Antígeno-Anticorpo , Autoantígenos/sangue , Proteínas Sanguíneas/química , Western Blotting , Citoplasma/imunologia , Feminino , Humanos , Masculino , Proteínas de Membrana/química , Pessoa de Meia-Idade , Fosfoproteínas/química , Proteínas/química , Proteínas Recombinantes/imunologiaRESUMO
The cDNAs encoding the porcine 65- and 67-kDa glutamic acid decarboxylases (GAD65 and GAD67, respectively) were cloned by the PCR method. The 2246-nucleotide (nt) GAD65 cDNA contained an open reading frame (ORF) coding for a protein of 585 amino acids (aa), and the 3262-nt GAD67 cDNA contained an ORF coding for a protein of 594 aa. A remarkable conservation was shown when the deduced aa sequences of porcine GAD65 and GAD67 were compared with those of other mammalian species (human, cat and rat). Porcine GAD65 is 96% identical to human and rat GAD65, and porcine GAD67 is more than 95% identical to human, cat and rat GAD67 at the aa level.
Assuntos
Glutamato Descarboxilase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Gatos , Clonagem Molecular , DNA Complementar , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , SuínosRESUMO
Abnormal antithrombin III (AT III) was found in a 30-year-old woman who suffered from recurrent thrombosis during pregnancy and the postpartum period. Among her family members, only her father had recurrent episodes of deep vein thrombosis of the lower extremities, from his youth. The antithrombin and antifactor Xa heparin cofactor activities of the proposita's plasma were 61% and 42% of normal, respectively. The progressive antithrombin and antifactor Xa activities were also decreased to 55% and 58% of normal, respectively. The immunoreactive level of AT III was within the normal range (23.1 mg/dl). Analysis of the proposita's plasma by crossed immunoelectrophoresis in the presence or absence of heparin and by affinity chromatography on heparin-Sepharose revealed that the proposita's AT III had apparently normal affinity for heparin. Nucleotide sequencing of 7 exons of the proposita's AT III gene amplified by polymerase chain reaction (PCR) disclosed that the second base of codon 393 comprised both G and A, indicating Arg393-His conversion. The base sequences of exons 1, 2, 3a, 3b, 4, and 5 were normal, excluding any other mutation. These findings indicated that the proposita's AT III was a variant of AT III at the thrombin binding site and that the proposita was a heterozygote for the abnormality. Heparin affinity of purified abnormal AT III from the proposita's plasma was demonstrated to be increased upon affinity chromatography using heparin-Sepharose, suggesting that the mutation (Arg393-His) per se could possibly increase the affinity of antithrombin III for heparin. For this variant AT III (Arg393-His), the name AT III Kumamoto II is proposed.
Assuntos
Antitrombina III/genética , Mutação , Tromboflebite/genética , Adulto , Antitrombina III/metabolismo , Arginina , Sequência de Bases , Cromatografia de Afinidade , Fator X/antagonistas & inibidores , Feminino , Heparina/metabolismo , Histidina , Humanos , Imunoeletroforese Bidimensional , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Gravidez , Complicações Hematológicas na Gravidez/sangue , Transtornos Puerperais/genética , Recidiva , Trombina/antagonistas & inibidores , Tromboflebite/sangueRESUMO
Five human nuclear antigens, RNP 70 kD, SS-A, SS-B, Sm-B and Sm-D, were produced in E. coli using the expression vector pSEM. cDNAs encoding these antigens were ligated to a truncated lacZ' gene of the vector and the beta-galactosidase fusion proteins were efficiently expressed as intracellular inclusion bodies after isopropyl-beta-thiogalactopyranoside induction. The antibody reactivities of these fusion proteins were evaluated by Western blot and by ELISA employing panel sera from patients with autoimmune diseases such as systemic lupus erythematosus, Sjögren's syndrome or mixed connective tissue disease. The three fusion proteins, RNP 70 kD, SS-B, and Sm-B, showed good reactivities in both systems, whereas the other two fusion proteins, SS-A and Sm-D, showed poor and no reactivity in both systems, respectively. It can be concluded that RNP 70 kD, SS-B and Sm-B recombinant antigens are useful reagents for the differential diagnosis of the autoimmune diseases.
Assuntos
Autoantígenos/imunologia , Soros Imunes/imunologia , RNA Citoplasmático Pequeno , Ribonucleoproteínas Nucleares Pequenas/imunologia , Ribonucleoproteínas/imunologia , Núcleo Celular/imunologia , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Proteínas Recombinantes/imunologia , Proteínas Centrais de snRNP , Antígeno SS-BRESUMO
A Japanese patient with congenital antithrombin III (AT-III) deficiency, named AT-III Kyoto, is associated with reduced levels (60% of normal) of AT-III antigen, progressive activity and heparin cofactor activity. The antithrombin III gene of this patient was investigated by polymerase chain reaction (PCR) method followed by direct DNA sequencing analysis, which revealed a G to T transitional mutation resulting in the conversion of arginine-406 to methionine in exon 6. Arginine-406 is located at the 12th amino acid residue from the reactive site on the C-terminal side of AT-III in a core region of the molecule which has been highly conserved during evolution of serine protease inhibitor (serpin) family. It is concluded that AT-III Kyoto is a newly described mutation which is similar to AT-III Utah and lends support to the idea that the conserved region near the reactive site is important in maintaining biological function of the AT-III molecule.
Assuntos
Deficiência de Antitrombina III , Arginina/genética , Metionina/genética , Adulto , Sequência de Aminoácidos , Antitrombina III/genética , Sequência de Bases , Sítios de Ligação/fisiologia , Amplificação de Genes/genética , Humanos , Focalização Isoelétrica , Masculino , Dados de Sequência Molecular , Mutação/genética , Oligonucleotídeos/síntese química , Homologia de Sequência do Ácido NucleicoRESUMO
We have isolated four clones of integrated human papillomavirus type 16 (HPV-16) DNA from four different primary cervical cancer specimens. All clones were found to be monomeric or dimeric forms of HPV-16 DNA with cellular flanking sequences at both ends. Analysis of the viral sequences in these clones showed that E6/E7 open reading frames and the long control region were conserved and that no region specific for the integration was detected. Analysis of the cellular flanking sequences revealed no significant homology with any known human DNA sequences, except Alu sequences, and no homology among the clones, indicating no cellular sequence specific for the integration. By probing with single-copy cellular flanking sequences from the clones, it was demonstrated that the integrated HPV-16 DNAs, with different sizes in the same specimens, shared the same cellular flanking sequences at the ends. Furthermore, it was shown that the viral sequences together with cellular flanking sequences were amplified. The possible process of viral integration into cell chromosomes in cervical cancer is discussed.
Assuntos
DNA de Neoplasias/isolamento & purificação , DNA Viral/isolamento & purificação , Genes Virais , Papillomaviridae/isolamento & purificação , Neoplasias do Colo do Útero/microbiologia , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA de Neoplasias/genética , DNA Viral/genética , Feminino , Amplificação de Genes , Genes Reguladores , Humanos , Lisogenia , Dados de Sequência Molecular , Papillomaviridae/genéticaRESUMO
Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) fractions were isolated from Friend erythroleukemia cells and separated by 15-45% sucrose gradient centrifugation. The distribution of small nuclear RNAs (snRNAs) in hnRNP fractions indicated that the snRNAs are associated with hnRNP particles. HnRNP fractions were incubated with normal IgG or anti-U1 RNP IgG, and the resulting immunocomplexes were isolated by binding to a protein A-Sepharose column. HnRNP was found in bound fractions only when anti-U1 RNP IgG was used. By Northern hybridization of RNA extracted from the immunocomplexes with a beta-globin genomic DNA probe, 15S beta-globin mRNA precursors and 10S mature mRNA were detected. These findings suggest the existence of a complex of U1 RNP particles and hnRNP particles containing beta-globin pre-mRNA.
Assuntos
Globinas/genética , Precursores de Ácido Nucleico/análise , Splicing de RNA , RNA Mensageiro/análise , Ribonucleoproteínas/análise , Animais , Especificidade de Anticorpos , Autoanticorpos/isolamento & purificação , Linhagem Celular , Núcleo Celular/análise , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Vírus da Leucemia Murina de Friend , Ribonucleoproteínas Nucleares Heterogêneas , Imunoglobulina G/isolamento & purificação , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Experimental/metabolismo , Camundongos , Hibridização de Ácido Nucleico , Precursores de RNA , Ribonucleoproteínas/imunologiaRESUMO
In order to improve the antibacterial activity of aminobenzylpenicillin, penicillin derivatives having an asparagine moiety in the 6-acyl side chain (11a approximately g, 12a, b, f, g) were synthesized. The structure-activity relationship of new penicillins, N4-alkyl-asparaginylaminobenzylpenicillins, was investigated. N4-Methyl-D-asparaginylamoxicillin (11a), TA-058, was found to possess a broad spectrum of antibacterial activity against Gram-positive and Gram-negative bacteria. In acute toxicity, TA-058 showed good tolerance in mice (LD50 greater than 10 g/kg, i.v.).
Assuntos
Amoxicilina/análogos & derivados , Bactérias/efeitos dos fármacos , Amoxicilina/síntese química , Amoxicilina/farmacologia , Animais , Camundongos , Relação Estrutura-AtividadeRESUMO
Linear, small and large circular forms of unintegrated viral DNAs were detected in Hirt supernatant fraction of human cultured cells infected with baboon endogenous virus M7. The circular M7 DNAs were cloned in bacteriophage lambda, Charon 28. Seventeen independent clones were isolated and analyzed by restriction endonuclease mapping. Nine clones were carrying a viral sequence of 8.6 kilobase pairs (kb) with two tandem repeats of 0.6 kb, which correspond to the large circular form of the unintegrated M7 DNA. Eight other clones had the viral insert of 8.0 kb, i. e., the small circular form, and were deleted one of the repeated sequences. The repeated sequences correspond to the long terminal repeats of 0.6 kb, located at both ends of the linear M7 DNA of 8.6 kb. One of the recombinants of the large circular M7 DNA had an inversion of 2.5 kb. One end of the inverted sequence was near the terminus of the long terminal repeats and the other in the gag gene region. The inversion seems to be occurred by integration of a viral DNA within itself during early periods of infection. The mechanism of the processes leading to integration is discussed from the structure of these unintegrated M7 DNAs as the precursors.
Assuntos
Clonagem Molecular , DNA Circular/genética , DNA Viral/genética , Retroviridae/genética , Bacteriófago lambda/genética , Sequência de Bases , Centrifugação com Gradiente de Concentração , Enzimas de Restrição do DNA , DNA Recombinante , HumanosRESUMO
Infection of BHK cells with western equine encephalitis (WEE) virus resulted in rapid inhibition of cellular DNA synthesis. The rate of inhibition of DNA synthesis depended on the multiplicity of infection, and was closely related to virus replication. Cellular DNA synthesis was not inhibited in infected BHK cells that had been irradiated with ultraviolet radiation. These results indicated that a functional viral genome was required for the inhibition of DNA synthesis by WEE virus. The sharp decrease in thymidine incorporation into the acid-insoluble fraction was not due to a change in the intracellular pool of the acid-soluble fraction. Sedimentation analysis in alkaline sucrose gradients was used to show that cellular DNA was not degraded during WEE viirus infection. DNA polymerase activity in infected cells was not significantly reduced.