RESUMO
AIM: To examine whether proton pump inhibitors modify the production of oxygen-derived free radicals and related cytokines in the human gastric mucosa infected with H. pylori. METHODS: Thirty-four H. pylori-positive peptic ulcer patients (23 gastric ulcer, 11 duodenal ulcer) were enrolled. Biopsy tissue samples were obtained endoscopically from the antrum and corpus. Tissue content of neutrophil myeloperoxidase (myeloperoxidase) and IL-8 was measured by ELISA. Mucosal production of oxygen-derived free radical was measured using luminol-dependent chemiluminescence (ChL). A proton pump inhibitor (either lansoprazole 30 mg, omeprazole 20 mg, or rabeprazole 10 mg) was administered daily by mouth to all patients for 8 weeks. Endoscopic examination was then repeated, and biochemical analysis was performed. RESULTS: Antral myeloperoxidase decreased significantly after proton pump inhibitor treatment (5.23 +/- 7.00-2.76 +/- 5.11 ng/mg, P < 0.02), but corpus myeloperoxidase was unchanged. IL-8 was also modified by proton pump inhibitors and these changes were parallel to those of myeloperoxidase. Corpus ChL was significantly increased from 88.5 +/- 69.8-159 +/- 172 counts/10 s/mg after proton pump inhibitor treatment, whereas antrum ChL was not altered. H. pylori infection rate was decreased in the antrum as well as the corpus. CONCLUSIONS: Proton pump inhibitor treatment stimulated oxygen-derived free radical production in the corpus mucosa.
Assuntos
Antiulcerosos/uso terapêutico , Úlcera Duodenal/complicações , Infecções por Helicobacter/complicações , Helicobacter pylori/isolamento & purificação , Inibidores da Bomba de Prótons , Úlcera Gástrica/complicações , Adulto , Idoso , Úlcera Duodenal/tratamento farmacológico , Úlcera Duodenal/metabolismo , Úlcera Duodenal/microbiologia , Feminino , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/enzimologia , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Masculino , Pessoa de Meia-Idade , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Úlcera Gástrica/tratamento farmacológico , Úlcera Gástrica/metabolismo , Úlcera Gástrica/microbiologiaRESUMO
BACKGROUND: Proton pump inhibitors have been reported to modify the level of Helicobacter pylori gastritis. AIM: To quantitatively investigate the effect of a proton pump inhibitor on the mucosal neutrophil reaction. METHODS: Forty-six H. pylori-infected patients (17 duodenal ulcer, 29 gastric ulcer) were enrolled. During endoscopic examination, biopsy samples were obtained from the antrum and the corpus. The tissue content of neutrophil myeloperoxidase was measured by enzyme-linked immunoabsorbent assay, and H. pylori infection was histologically assessed. A proton pump inhibitor was administered orally for 8 weeks. RESULTS: In the patients as a whole, antral myeloperoxidase decreased significantly after proton pump inhibitor treatment, but corpus myeloperoxidase remained largely unchanged. In duodenal ulcer patients, myeloperoxidase significantly decreased in the antrum, but increased in the corpus. In gastric ulcer patients, a significant reduction was observed in antral myeloperoxidase, but corpus myeloperoxidase remained unchanged. In the antral myeloperoxidase > corpus myeloperoxidase subgroup (n=24), antral myeloperoxidase significantly decreased, whereas corpus myeloperoxidase increased. No changes were observed at either site in the corpus myeloperoxidase > antral myeloperoxidase subgroup. Histology showed that the antral bacterial load of H. pylori decreased in all subgroups, but that it was mostly unchanged in the corpus. CONCLUSIONS: Proton pump inhibitor treatment stimulated the neutrophil reaction in the corpus mucosa of duodenal ulcer patients and of patients in whom antral neutrophil accumulation was more predominant than that of the corpus. This phenomenon may not be caused by increased bacterial density.
Assuntos
Inibidores Enzimáticos/farmacologia , Gastrite/etiologia , Infecções por Helicobacter/tratamento farmacológico , Omeprazol/análogos & derivados , Peroxidase/metabolismo , Inibidores da Bomba de Prótons , 2-Piridinilmetilsulfinilbenzimidazóis , Administração Oral , Adulto , Idoso , Benzimidazóis/farmacologia , Biópsia , Úlcera Duodenal/tratamento farmacológico , Úlcera Duodenal/microbiologia , Endoscopia Gastrointestinal , Feminino , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/imunologia , Mucosa Gástrica/microbiologia , Gastrite/microbiologia , Infecções por Helicobacter/complicações , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/patogenicidade , Humanos , Lansoprazol , Masculino , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Omeprazol/farmacologia , Antro Pilórico/microbiologia , Antro Pilórico/patologia , Rabeprazol , Úlcera Gástrica/tratamento farmacológico , Úlcera Gástrica/microbiologiaRESUMO
Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformis luciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement of N-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.
Assuntos
Candida albicans/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas , Tetraciclina/farmacologia , Animais , Doxiciclina/farmacologia , Masculino , Camundongos , Transativadores/fisiologiaRESUMO
BACKGROUND: CXC chemokines such as interleukin (IL)-8 are neutrophil chemoattractants, the levels of which increase in Helicobacter pylori-infected gastric mucosa. Many investigators have focused on the chemotactic aspects of IL-8: however, CXC chemokines are also reported to have angiogenic activity and to serve as remodelling factors. Rat GRO/CINC-1 is a rodent counterpart of human GROalpha, a member of the family of CXC chemokines. Gastric mucosa infected with H. pylori is in a state of hyperproliferation, with increases in the amounts of growth factors such as hepatocyte growth factor (HGF). AIM: To investigate whether rat GRO/CINC-1 had growth-stimulating activity for gastric epithelial cells. METHODS: The rat gastric epithelial cell line RGM-1 was incubated in serum-free medium for 12 h to adjust the cell cycle to the G0 phase, and GRO/CINC-1 was then added for 24 h. The total cell number was determined by fluorogenic analysis after propidium iodide staining, and cell proliferation was assessed by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation. The activity of p42/p44 mitogen-activated protein kinase (MAPK) was measured 5-20 min after the start of GRO/CINC-1 exposure. RESULTS: Cultures treated with GRO/CINC-1 showed a significant increase in cell number and BrdU incorporation in a concentration-dependent fashion. The MAPK activity increased within 5 min after GRO/CINC-1 application and returned to the control level at 20 min. CONCLUSION: The growth-stimulatory effect of GRO/CINC-1 on rat gastric epithelial cells suggests a dual function of this chemokine: proinflammatory action and induction of epithelial proliferation.
Assuntos
Quimiocinas CXC/farmacologia , Células Epiteliais/fisiologia , Mucosa Gástrica/citologia , Animais , Autorradiografia , Bromodesoxiuridina , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Mucosa Gástrica/efeitos dos fármacos , Inflamação , RatosRESUMO
BACKGROUND: Mongolian gerbils have been reported to be a suitable model for Helicobacter pylori-associated gastric mucosal injury, including gastric cancer. Although ethanol is known to be one of the harmful substances in the gastric mucosa, the relationship between ethanol and H. pylori infection remains unknown. The aim of the present study is to investigate the effect of ethanol treatment prior to H. pylori inoculation on associated gastric mucosal injury. METHODS: Male Mongolian gerbils were used for the study. Helicobacter pylori was orally inoculated after 15 h fasting (Hp group). Thirty minutes prior to H. pylori inoculation, a group of gerbils was orally treated with 40% ethanol (20 mL/kg; E + Hp group). Another group of animals was treated either with H. pylori culture media alone (controls) or with 40% ethanol plus culture media (E group). Gerbils were killed 2, 4 or 12 weeks after H. pylori inoculation. Helicobacter pylori infection was confirmed by both histological examination and serological tests. Mucosal damage was evaluated histologically according to the modified Sydney system. RESULTS: Although in the controls and E group no significant change to the gastric mucose was observed, persistent H. pylori infection was seen in the mucosa and mucosal leucocyte infiltration and severe epithelial damage was observed in the Hp and E + Hp groups after 4 weeks. The histological scores for polymorphonuclear cell infiltration and myeloperoxidase activity were higher in the E + Hp group at 4 weeks than in the Hp group (P < 0.05). CONCLUSIONS: Ethanol intake preceding H. pylori inoculation could promote the progression of gastric mucosal inflammation in Mongolian gerbils.
Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Gastrite/patologia , Infecções por Helicobacter/patologia , Helicobacter pylori , Consumo de Bebidas Alcoólicas/patologia , Animais , Modelos Animais de Doenças , Mucosa Gástrica/patologia , Gerbillinae , Masculino , Neutrófilos/patologiaRESUMO
BACKGROUND: Spontaneously hypertensive rats (SHR) are a representative animal model for disturbance of the pituitary-adrenal axis, as well as disturbance of the autonomic nervous system. METHODS AND RESULTS: In this study, we showed that adrenalectomy in SHR-induced spontaneous gastric ulcer formation. We further investigated how abnormal adrenal secretion is related to the attenuation of gastric ulcerogenesis, in terms of leucocyte infiltration and nitric oxide (NO) formation. Bilateral adrenalectomy, as well as a sham-operation, were carried out at 12 weeks in hypertensive SHR and Wistar-Kyoto rats (WKY) and observations were made three weeks later. The number of myeloperoxidase (MPO) positive cells, NADPH diaphorase histochemistry and NO synthase (NOS) activity were determined in gastric specimens. Only in adrenalectomized, but not sham-operated SHR, WKY and adrenalectomized WKY, could gastric ulcers be observed. Although the number of cells positive for MPO was significantly lower in hypertensive SHR than those in WKY, such cells were increased after adrenalectomy in SHR. In contrast, adrenalectomized WKY developed no increase in MPO-positive numbers. The number of NADPH diaphorase-positive cells increased after adrenalectomy in both strains, the extent of which was much greater in SHR than in WKY. Although NOS activity in SHR was lower than that in WKY, it was significantly increased after adrenalectomy. CONCLUSIONS: Our data show that the development of a significant gastric ulceration may be associated with entrapment of activated leucocytes in the gastric mucosa, as well as with an excessive production of NO in adrenalectomized SHR. An enhanced adrenal glucocorticoid may be a key factor for protecting the gastric mucosa in SHR.
Assuntos
Córtex Suprarrenal/fisiologia , Mucosa Gástrica/metabolismo , Glucocorticoides/fisiologia , Úlcera Gástrica/prevenção & controle , Adrenalectomia , Animais , Dexametasona/análogos & derivados , Dexametasona/uso terapêutico , Mucosa Gástrica/patologia , Leucócitos/fisiologia , NADPH Desidrogenase/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Peroxidase/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Úlcera Gástrica/patologiaRESUMO
We previously reported that NH2Cl induced extensive DNA fragmentation in gastric cells. Polaprezinc, a zinc-carnosine chelate compound, is reported to be a potent antioxidant in gastric mucosa. The present study was designed to examine whether polaprezinc could attenuate the NH3Cl-induced DNA damage. Gastric cell lines, MKN45, were exposed to NH2Cl in Ca(2+)-containing Hanks' balanced salt solution. DNA fragmentation was evaluated by photometric enzyme immunoassay for in vitro determination of cytoplasmic mono- and oligonucleosomes. Polaprezinc, L-carnosine, and zinc sulfate (ZnSO4) were added to the cell incubation medium to evaluate the inhibitory effect on the formation of cytoplasmic mono- and oligonucleosomes. Separately, the bleaching level of beta-carotene with the addition of each test solution was evaluated to confirm the inhibitory effect against hypochlorous acid. Polaprezinc or L-carnosine, but not ZnSO4, at a concentration of 0.001 mM, significantly attenuated the increased levels of cytoplasmic mono- and oligonucleosomes evoked by 0.001 mM NH2Cl. Polaprezinc and L-carnosine, but not ZnSO4, also inhibited NH2Cl-induced beta-carotene bleaching in the cell-free system. In conclusion, polaprezinc, especially its subportion L-carnosine, inhibited NH2Cl-evoked gastric epithelial DNA fragmentation, suggesting a role for this agent in preventing the progression of gastric epithelial injury induced by NH2Cl.
Assuntos
Antiulcerosos/farmacologia , Carnosina/análogos & derivados , Fragmentação do DNA/efeitos dos fármacos , Mucosa Gástrica/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Análise de Variância , Antiulcerosos/uso terapêutico , Carnosina/farmacologia , Carnosina/uso terapêutico , Linhagem Celular , Cloraminas , Dano ao DNA , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori , Humanos , Compostos Organometálicos/uso terapêutico , Taurina/farmacologia , Compostos de Zinco , Sulfato de Zinco/farmacologiaRESUMO
The Candida glabrata KRE9 (CgKRE9) and KNH1 (CgKNH1) genes have been isolated as multicopy suppressors of the tetracycline-sensitive growth of a Saccharomyces cerevisiae mutant with the disrupted KNH1 locus and the KRE9 gene placed under the control of a tetracycline-responsive promoter. Although a cgknh1Delta mutant showed no phenotype beyond slightly increased sensitivity to the K1 killer toxin, disruption of CgKRE9 resulted in several phenotypes similar to those of the S. cerevisiae kre9Delta null mutant: a severe growth defect on glucose medium, resistance to the K1 killer toxin, a 50% reduction of beta-1,6-glucan, and the presence of aggregates of cells with abnormal morphology on glucose medium. Replacement in C. glabrata of the cognate CgKRE9 promoter with the tetracycline-responsive promoter in a cgknh1Delta background rendered cell growth tetracycline sensitive on media containing glucose or galactose. cgkre9Delta cells were shown to be sensitive to calcofluor white specifically on glucose medium. In cgkre9 mutants grown on glucose medium, cellular chitin levels were massively increased.
Assuntos
Candida/genética , Proteínas Fúngicas/genética , Glucanos/análise , Glicoproteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , beta-Glucanas , Sequência de Aminoácidos , Sequência de Bases , Benzenossulfonatos/farmacologia , Candida/citologia , Candida/efeitos dos fármacos , Parede Celular/química , Quitina/análise , Clonagem Molecular , Resistência Microbiana a Medicamentos , Genes Fúngicos/genética , Teste de Complementação Genética , Fatores Matadores de Levedura , Dados de Sequência Molecular , Mutação , Micotoxinas/farmacologia , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão , Mapeamento por Restrição , Análise de Sequência de DNARESUMO
Eradication therapy for Helicobacter pylori (H. pylori) has been established. However, the physiological factors influencing the success of treatment remain unclear. The aim of this study was to analyze these factors and to evaluate the efficacy of sofalcone on H. pylori eradication therapy. Forty-four H. pylori-infected and peptic ulcer patients were enrolled in this study. Twenty-seven patients were treated with lansoprazole (LPZ, 30 mg o.d. for 1-8 weeks) and amoxicillin (AMPC, 500 mg q.i.d, 1-2 weeks), followed by 8 weeks of treatment with famotidine (FAM, 20 mg o.d.). Moreover, sofalcone (SOF, 100 mg t.i.d) was administered to 17 patients throughout the therapeutic period. Endoscopic and serologic evaluations and the urea breath test (UBT) were performed before therapy. At the endoscopic examination, mucosal samples were biopsied and then tissue myeloperoxidase (MPO) content, an index of neutrophil infiltration was measured. Cure of H. pylori infection was determined 8 weeks after the cessation of LPZ. This eradication regimen afforded an overall cure rate of 63.0% (17/27) without SOF and 76.5% (13/17) with SOF. In the control group, treatment success was inversely associated with pre-UBT value (gastric urease activity), whereas this association was not observed in the SOF group. Furthermore, in the patients exhibiting a high preUBT value (>40%), a twofold higher eradication rate was obtained by the administration of SOF. In patients who were successfully eradicated, mucosal MPO level was slightly higher than those of unsuccessful cases, whereas there was no significant association with serum pepsinogen (PG I, PG II) concentration and its ratio (PG I/PG II). These results suggest that a low UBT value is a factor predicting treatment success. SOF administration may improve the eradication rate, especially in the high-UBT subgroup.
Assuntos
Antiulcerosos/uso terapêutico , Chalcona/análogos & derivados , Úlcera Duodenal/tratamento farmacológico , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori , Úlcera Gástrica/tratamento farmacológico , 2-Piridinilmetilsulfinilbenzimidazóis , Adulto , Idoso , Amoxicilina/uso terapêutico , Testes Respiratórios/métodos , Isótopos de Carbono , Chalcona/uso terapêutico , Chalconas , Quimioterapia Combinada , Úlcera Duodenal/complicações , Úlcera Duodenal/enzimologia , Endoscopia Gastrointestinal , Feminino , Mucosa Gástrica/enzimologia , Infecções por Helicobacter/complicações , Infecções por Helicobacter/enzimologia , Humanos , Lansoprazol , Masculino , Pessoa de Meia-Idade , Omeprazol/análogos & derivados , Omeprazol/uso terapêutico , Penicilinas/uso terapêutico , Inibidores da Bomba de Prótons , Úlcera Gástrica/complicações , Úlcera Gástrica/enzimologia , Resultado do Tratamento , Ureia , Urease/metabolismoRESUMO
A convenient system for the control of gene expression in Saccharomyces cerevisiae was developed. Tetracycline-responsive promoters were constructed by fusing the tetracycline operator (tetO) to the S. cerevisiae HOP1 promoter. When fused to the tetracycline repressor (tetR), trans-activation domains of both GAL4 and HAP4 were capable of promoting transcription from the tetO-HOP1 chimeric promoter, but the tetR-HAP4 fusion activator was the more efficient transcriptional activator. Addition of tetracycline nearly completely repressed activator-dependent transcription from the tetO-HOP1 promoter. Moreover, tetracycline-dependent repression of YEF3, CDC28 and RAM2 expression impaired cell growth. Thus, this system is useful for the elucidation of gene function in S. cerevisiae.
Assuntos
Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Engenharia Genética/métodos , Saccharomyces cerevisiae/genética , Tetraciclina/farmacologia , Regiões Operadoras Genéticas , Regiões Promotoras Genéticas , Proteínas Repressoras/genéticaRESUMO
A canCHS1A gene encoding the chitin synthase of Candida albicans was cloned. DNA sequencing and comparison with another canCHS1 gene described elsewhere indicated that the canCHS1A gene encoded a polypeptide with 775 amino acid residues, a protein with one less amino acid than that encoded by the canCHS1 gene. A six-base alteration was observed between the two genes, suggesting that the canCHS1A gene is a variant gene of the canCHS1. The pH profile/activity relationship of canChs1A in permeabilized cells was identical to that of canChs1. The canChs1A enzyme was competitively inhibited by polyoxin D (5.2 microM) and nikkomycin Z (12 microM). When the cloned gene was expressed in a Saccharomyces cerevisiae chs2 mutant that exhibited aberrant morphology, the normal structure was restored. We conclude that the function of the canCHS1A gene is similar to that of sacCHS2 in S. cerevisiae.
Assuntos
Aminoglicosídeos , Candida albicans/genética , Quitina Sintase/genética , Genes Fúngicos , Variação Genética , Antibacterianos/farmacologia , Sequência de Bases , Candida albicans/enzimologia , Clonagem Molecular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos , Teste de Complementação Genética , Dados de Sequência Molecular , Nucleosídeos de Pirimidina/farmacologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade da EspécieRESUMO
Chitin synthase 2 of Saccharomyces cerevisiae was characterized by means of site-directed mutagenesis and subsequent expression of the mutant enzymes in yeast cells. Chitin synthase 2 shares a region whose sequence is highly conserved in all chitin synthases. Substitutions of conserved amino acids in this region with alanine (alanine scanning) identified two domains in which any conserved amino acid could not be replaced by alanine to retain enzyme activity. These two domains contained unique sequences, Glu561-Asp562-Arg563 and Gln601-Arg602-Arg603-Arg604-Trp605, that were conserved in all types of chitin synthases. Glu561 or arginine at 563, 602, and 603 could be substituted by glutamic acid and lysine, respectively, without significant loss of enzyme activity. However, even conservative substitutions of Asp562 with glutamic acid, Gln601 with asparagine, Arg604 with lysine, or Trp605 with tyrosine drastically decreased the activity, but did not affect apparent Km values for the substrate significantly. In addition to these amino acids, Asp441 was also found in all chitin synthase. The mutant harboring a glutamic acid substitution for Asp441 severely lost activity, but it showed a similar apparent Km value for the substrate. Amounts of the mutant enzymes in total membranes were more or less the same as found in the wild type. Furthermore, Asp441, Asp562, Gln601, Arg604, and Trp605 are completely conserved in other proteins possessing N-acetylglucosaminyltransferase activity such as NodC proteins of Rhizobium bacterias. These results suggest that Asp441, Asp562, Gln601, Arg604, and Trp605 are located in the active pocket and that they function as the catalytic residues of the enzyme.
Assuntos
Quitina Sintase/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Relação Estrutura-AtividadeRESUMO
The chitin synthase 3 gene (CACHS3) has been cloned from Candida albicans. The yeast CAL1 gene encoding the chitin synthase 3 of Saccharomyces cerevisiae was used as a probe for the isolation of the gene from C. albicans. The CAL1 homolog was identified in Southern blots of C. albicans genomic DNA and cloned from a C. albicans genomic DNA library. The nucleotide sequences of two partial clones were determined and combined giving a total length of 4610 bp. A continuous open reading frame of 3525 bp encoding a predicted protein of 1175 amino acids and molecular mass of 131 850 daltons was identified. A comparison of the deduced amino acid sequences of CAL1 and the Candida chitin synthase 3 protein showed 59.3% identity. Southern blot analysis indicates that the CACHS3 gene is present in a single copy in the genome and maps to chromosome I. Northern blot analysis shows that expression of chitin synthase 3 gene is dramatically increased during the transition from the yeast to the hyphal form of C. albicans. This change in transcription level strongly suggests that CACHS3 may play a role in Candida morphogenesis.
Assuntos
Candida albicans/genética , Quitina Sintase/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Quitina Sintase/metabolismo , Clonagem Molecular , DNA Fúngico , Expressão Gênica , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
A previous report from this laboratory showed that in vitro transcription of total genomic DNA of the newt Cynopus pyrrhogaster resulted in a discrete sized 8 S RNA, which represented highly repetitive and transcribable sequences with a glutamic acid tRNA-like structure in the newt genome. We isolated four independent clones from a newt genomic library and determined the complete sequences of three 2000 to 2400 base-pair PstI fragments spanning the 8 S RNA gene. The glutamic acid tRNA-related segment in the 8 S RNA gene contains the CCA sequence expected as the 3' terminus of a tRNA molecule. Further, the 11 nucleotides located 13 nucleotides upstream from one of the two transcription initiation sites of the 8 S RNA were found to be repeated in the region upstream from the termination site, suggesting that the original unit, which is shorter than the 8 S RNA, was retrotransposed via cDNA intermediates from the PolIII transcript. In the upstream region of the 8 S RNA gene, a 360 nucleotide unit containing the glutamic acid tRNA-related segment was found to be duplicated (clones NE1 and NE10) or triplicated (clone NE3). Except for the difference in the number of the 360 nucleotide unit, the three sequences of the 2000 to 2400 base-pair PstI fragment were essentially the same with only a few mutations and minor deletions. Inverse polymerase chain reaction and sequence determination of the products, together with a Southern hybridization experiment, demonstrated that the family consists of a tandemly repeated unit of 3300, 3700 or 4100 base-pairs. Thus during evolution, this family in the newt was created by retroposition via cDNA intermediates, followed by duplication or triplication of the 360 nucleotide unit and multiplication of the 3300 to 4100 base-pair region at the DNA level.
Assuntos
Elementos de DNA Transponíveis , RNA de Transferência de Ácido Glutâmico/genética , Sequências Repetitivas de Ácido Nucleico , Salamandridae/genética , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , Genes , Ligação de Hidrogênio , Dados de Sequência Molecular , Estrutura Molecular , Mapeamento por Restrição , Transcrição GênicaRESUMO
On in vitro transcription of total genomic DNA of the tortoise (Geoclemys reevessi), a discrete-sized RNA of 6.5S was obtained that represented a highly repetitive and transcribable sequence in the tortoise genome. Three sequences of the 6.5S RNA gene were sequenced, and a consensus sequence was deduced from these three sequences and one reported previously [Endoh, H & Okada, N. (1986) Proc. Natl Acad. Sci. USA 83, 251-255]. The 5' part of the gene showed close similaries to lysine (rabbit) and threonine (mouse) tRNAs (overall similarity 68-70%), so this tortoise sequence may have evolved from one of these tRNAs. The consensus sequence retained the expected CCA triplet at the 3' end of tRNA, but not at the 3' end of tDNA, supporting the idea that the tRNA-related region of the gene was generated via an RNA intermediate. The 5' and 3' flanking sequences of the four genes were found to be completely different from each other. Fingerprint analysis and S1 nuclease mapping analysis also showed that sequence boundaries of tortoise repetitive units exactly corresponded to RNA species. These results, together with data obtained by Southern blot hybridization, indicated that the 6.5S RNA genes are dispersed in the tortoise genome. Therefore, generation of the tRNA-related region of the gene and amplification of the whole unit of the gene are both RNA-mediated events. The existence of this tortoise sequence suggests that short interspersed sequences are more common in eukaryotic genomes than had previously been thought.
Assuntos
Elementos de DNA Transponíveis , Biblioteca Genômica , RNA/genética , Transcrição Gênica , Tartarugas/genética , Animais , Sequência de Bases , Evolução Biológica , DNA/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Mapeamento de Nucleotídeos , Plasmídeos , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
A 25 year old with vitiligo por 6 years now. For one year has being presenting hyperthyroidism, myasthenia gravis and periodic paralysis. The normalization of the thyroid function was followed by a significant improvement of the myasthenic syndrome that did not interfere with the paralytical attacks.
Assuntos
Hipertireoidismo/complicações , Miastenia Gravis/complicações , Quadriplegia/complicações , Vitiligo/complicações , Adulto , Humanos , Hipertireoidismo/tratamento farmacológico , Masculino , Propiltiouracila/uso terapêuticoRESUMO
O diagnostico anatomopatologico das lesoes encefalicas de natureza expansiva e necessario para a instituicao do tratamento adequado. Muitas lesoes localizadas nas estruturas cerebrais profundas ou em centros nervosos de grande importancia funcional nao podem ser abordadas por procedimentos neurocirurgicos habituais, sem riscos para os doentes. Nesses casos, a biopsia percutanea, estereotaxica ou assistida pela tomografia computadorizada deve ser empregada para esclarecimento diagnostico. Os autores apresentam o resultado da bipsia por trepanacao de lesoes encefalicas inabordaveis pelos metodos neurocirurgicos tradicionais, em 63 doentes. O procedimento foi realizado a mao livre em 18 casos, por estereotaxia em 28 e por estereotomografia em 17. O indice de mortalidade e de morbidade foi baixo. Cinco doentes foram, durante o mesmo ato operatorio, submetidos a radioterapia intersticial por tecnica de "after loading". Os criterios de selecao, a avaliacao pre-operatoria e as vantagens da biopsia por trepanacao sao discutidos