RESUMO
Localized two-dimensional constant-time correlation spectroscopy (CT-COSY) was used to resolve glutamate (Glu), gamma-aminobutyric acid (GABA), and glutamine (Gln) in the human brain at 4.7 T. In this method, three-dimensional localization was achieved using three radio frequency pulses of the CT-COSY module for slice selection. As this sequence could decouple JHH along the F1 direction, peak resolution of metabolites was improved even on a magnitude-mode display. In experiments on a phantom containing N-acetylaspartate, creatine, Glu, Gln, and GABA with a constant time delay (Tct) of 110 ms, cross peaks of Glu, Gln, and GABA were obtained on a spectrum processed with standard sine-bell windows, which emphasize sine-dependent signals along the t2 direction. In contrast, diagonal peaks of Glu C4H at 2.35 ppm, GABA C2H at 2.28 ppm, and Gln C4H at 2.44 ppm were resolved on a spectrum processed with Gaussian windows, which emphasize cosine-dependent signals along t2. Human brain spectra were obtained from a 27 mL voxel within the parieto-occipital region using a volume transverse electromagnetic (TEM) coil for both transmission and reception. Tct was 110 ms; the total scan time was 30 min. Diagonal peaks of Glu C4H, GABA C2H, and Gln C4H were also resolved on the spectrum processed with Gaussian windows. These results show that the localized two-dimensional CT-COSY method featuring 1H decoupling along the F1 direction could resolve Glu, GABA, and Gln signals in the human brain.
Assuntos
Química Encefálica , Ácido Glutâmico/análise , Glutamina/análise , Espectroscopia de Ressonância Magnética/métodos , Processamento de Sinais Assistido por Computador , Ácido gama-Aminobutírico/análise , Ácido Aspártico/análogos & derivados , Ácido Aspártico/análise , Ácido Aspártico/química , Encéfalo/metabolismo , Química Encefálica/efeitos da radiação , Creatina/análise , Creatina/química , Interpretação Estatística de Dados , Transferência de Energia/efeitos da radiação , Ácido Glutâmico/química , Glutamina/química , Humanos , Aumento da Imagem/métodos , Magnetismo , Imagens de Fantasmas , Prótons , Ondas de Rádio , Sensibilidade e Especificidade , Ácido gama-Aminobutírico/químicaRESUMO
OBJECTIVE: Ethnicity-based research may identify new clues to the pathogenesis of atherosclerotic disease. Therefore, we sought to determine whether carotid lesions differ between 20 Chinese and 20 Caucasian Americans by MRI. METHODS AND RESULTS: Inclusion criteria were >50% stenosis as measured by duplex ultrasound and recent symptoms attributed to carotid artery disease. The patients were imaged in 2 centers (Beijing, China and Seattle, Wash) using a standardized protocol. Both carotid arteries were reviewed quantitatively (lumen, wall, outer wall, tissue components) and morphologically (lesion types, fibrous cap status). Significant differences between the Chinese and Americans were found for the mean size of the lipid/necrotic core (13.6 versus 7.8 mm2; P=0.002), percentage of slices with calcified type VII lesions (1.6 versus 12.4%; P=0.03), and percentage of slices with early type III lesions (19.3 versus 9.3%; P=0.02). Furthermore, the mean outer wall area in the common carotid artery was larger in the Chinese population (P=0.007). CONCLUSIONS: This pilot study suggests that composition and morphology of atherosclerotic lesions in symptomatic carotid disease differ between ethno-racial groups. Quantitative MRI-based review of carotid atherosclerosis comparing plaque morphology and composition between ethno-racial groups is feasible, and future MRI studies may improve our understanding of the pathophysiology of this disease.
Assuntos
Povo Asiático/estatística & dados numéricos , Doenças das Artérias Carótidas/etnologia , Doenças das Artérias Carótidas/patologia , Imageamento por Ressonância Magnética , População Branca/estatística & dados numéricos , Idoso , Idoso de 80 Anos ou mais , Artéria Carótida Primitiva/patologia , China/epidemiologia , Feminino , Humanos , Imageamento por Ressonância Magnética/normas , Masculino , Pessoa de Meia-Idade , Imagens de Fantasmas , Projetos Piloto , Reprodutibilidade dos Testes , Fatores de Risco , Estados Unidos/epidemiologiaRESUMO
OBJECTIVE: This study evaluates the ability of MRI to quantify all major carotid atherosclerotic plaque components in vivo. METHODS AND RESULTS: Thirty-one subjects scheduled for carotid endarterectomy were imaged with a 1.5T scanner using time-of-flight-, T1-, proton density-, and T2-weighted images. A total of 214 MR imaging locations were matched to corresponding histology sections. For MRI and histology, area measurements of the major plaque components such as lipid-rich/necrotic core (LR/NC), calcification, loose matrix, and dense (fibrous) tissue were recorded as percentages of the total wall area. Intraclass correlation coefficients (ICCs) were computed to determine intrareader and inter-reader reproducibility. MRI measurements of plaque composition were statistically equivalent to those of histology for the LR/NC (23.7 versus 20.3%; P=0.1), loose matrix (5.1 versus 6.3%; P=0.1), and dense (fibrous) tissue (66.3% versus 64%; P=0.4). Calcification differed significantly when measured as a percentage of wall area (9.4 versus 5%; P<0.001). Intrareader and inter-reader reproducibility was good to excellent for all tissue components, with ICCs ranging from 0.73 to 0.95. CONCLUSIONS: MRI-based tissue quantification is accurate and reproducible. This application can be used in therapeutic clinical trials and in prospective longitudinal studies to examine carotid atherosclerotic plaque progression and regression.
Assuntos
Doenças das Artérias Carótidas/patologia , Imageamento por Ressonância Magnética/métodos , Idoso , Feminino , Humanos , Masculino , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The mechanism of the heme-thiolate-dependent NADH-NO reductase (P450(NOR)) from Fusarium oxysporum was investigated by kinetic isotope effects including protio, [4S-2H]-, [4R-2H]-, [4,4(2)H(2)]-NADH and stopped-flow measurements. The respective kinetic isotope effects were measured at high NO concentrations and were found to be 1.7, 2.3 and 3.8 indicating a rate-limitation at the reduction step and a moderate stereoselectivity in binding of the cofactor NADH. In a different approach the kinetic isotope effects were determined directly for the reaction of the Fe(III)-NO complex with [4R-2H]- and [4S-2H]-NADH by stopped-flow spectroscopy. The resulting isotope effects were 2.7+/-0.4 for the R-form and 1.1+/-0.1 for the S-form. In addition the 444 nm intermediate could be chemically generated by addition of an ethanolic borohydride solution to the ferric-NO complex at -10 degrees C. In pulse radiolysis experiments a similar absorbing species could be observed when hydroxylamine radicals were generated in the presence of Fe (III) P450(NOR). Based on these results we postulate hydride transfer from NADH to the ferric P450-NO complex resulting in a ferric hydroxylamine-radical or ferryl hydroxylamine-complex and this step, as indicated by the kinetic isotope effects, to be rate-limiting at high concentrations of NO. However, at low concentrations of NO the decay of the 444 nm species becomes the rate-limiting step as envisaged by stopped-flow and optical kinetic measurements in a system in which NO was continuously generated. The last step in the catalytic cycle may proceed by a direct addition of the NO radical to the Fe-hydroxylamine complex or by electron transfer from the NO radical to the ferric-thiyl moiety in analogy to the postulated mechanisms of prostacyclin and thromboxane biosynthesis by the corresponding P450 enzymes. The latter process of electron transfer could then constitute a common step in all heme-thiolate catalyzed reactions.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Óxido Nítrico/química , Oxirredutases/química , Sistema Enzimático do Citocromo P-450/metabolismo , Deutério , Espectroscopia de Ressonância de Spin Eletrônica , Fusarium/enzimologia , Cinética , NAD/química , NAD/metabolismo , Óxido Nítrico/metabolismo , Oxirredução , Oxirredutases/metabolismoRESUMO
This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 01 meeting in Orlando, FL. The presentations addressed the mechanisms of inhibition and regulation of cytochrome P450 and flavin monooxygenase enzymes by nitric oxide. They also highlighted the consequences of these effects on metabolism of drugs and volatile amines as well as on important physiological parameters, such as control of blood pressure, renal ion transport, and steroidogenesis. This is achieved via regulation of P450-dependent prostacyclin, hydroxyeicosatetraenoic acid, and epoxyeicosatrienoic acid formation. Conversely, the mechanisms and relative importance of nitric oxide synthases and P450 enzymes in NO production from endogenous and synthetic substrates were also addressed.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Óxido Nítrico/metabolismo , Oxigenases/metabolismo , Animais , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Hepatite/enzimologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , NG-Nitroarginina Metil Éster/farmacologia , Nitratos/metabolismo , Nitratos/farmacologia , Óxido Nítrico Sintase/metabolismo , Oxigenases/antagonistas & inibidores , Ratos , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/enzimologiaRESUMO
A novel denitrifier Alcaligenes sp. STC1 was identified. The strain efficiently denitrifies under an atmosphere of 10% oxygen (O2) where Paracoccus denitrificans, one of the most studied aerobic denitrifiers, had less denitrifying activity, indicating that the strain has an O2-torelant denitrifying system. It denitrified by using C1-carbon sources such as formate and methanol as well as glucose, glycerol, and succinate. The genes for the copper-containing nitrite reductase and azurin of this C1-using denitrifier were cloned. Their predicted products of them were similar to those of their counterparts and the maximum similarities were 90% and 92%, respectively.
Assuntos
Alcaligenes/metabolismo , Azurina/genética , Cloretos/metabolismo , Cobre/metabolismo , Genes Bacterianos , Nitrito Redutases/genética , Alcaligenes/genética , Sequência de Aminoácidos , Azurina/química , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , Nitrito Redutases/química , Homologia de Sequência de AminoácidosRESUMO
Cytochrome P450nor is involved in fungal denitrification as nitric oxide (NO) reductase. Although the heme protein has been known to occur in restricted species of fungi that belong to ascomycotina, we have previously suggested that it would also occur in the yeast Trichosporon cutaneum, which is phylogenetically far from those P450nor-producing ascomycetous fungi. Here we isolated and characterized the heme protein from the basidiomycetous yeast T. cutaneum. P450nor of the yeast (TcP450nor) exhibited properties in terms of catalysis, absorption spectrum and molecular mass that are almost identical to those of its counterparts in ascomycetous fungi. We also isolated and sequenced its cDNA. The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicating that they belong to the same family. TcP450nor protein cofractionated with cytochrome c oxidase by subcellular fractionation and its predicted primary structure contained an extension on its amino terminus that is characteristic of a mitochondrial-targeting signal, indicating that it is a mitochondrial protein like some of the isoforms of other fungi. On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells. The occurrence of P450nor across the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought.
Assuntos
Sistema Enzimático do Citocromo P-450/isolamento & purificação , Oxirredutases/isolamento & purificação , Trichosporon/enzimologia , Sequência de Aminoácidos , Western Blotting , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar/química , DNA Complementar/metabolismo , Dados de Sequência Molecular , Oxirredutases/biossíntese , Oxirredutases/genética , Alinhamento de Sequência , Espectrofotometria , Frações Subcelulares/enzimologiaRESUMO
The effects of dioxygen (O2) on the denitrification activity of the fungus Fusarium oxysporum MT-811 in fed-batch culture in a stirred jar fermentor were examined. The results revealed that fungal denitrifying activity requires a minimal amount of O2 for induction, which is repressed by excess O2. The optimal O2 supply differed between the denitrification substrates : 690 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrate (NO3-) and about 250 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrite (NO2-). The reduction of NO3- required more O2 than that of NO2- . With an optimal O2 supply, 80% and 52% of nitrogen atoms in NO3- and NO2-, respectively, were recovered as the denitrification product N2O. These features of F. oxysporum differ from those of bacterial denitrifiers that work exclusively under anoxic conditions. The denitrification activity of F. oxysporum MT-811 mutants with impaired NO3- assimilation was about double that of the wild-type strain, suggesting competition for the substrate between assimilatory and dissimilatory types of NO3- reduction. These results showed that denitrification by F. oxysporum has unique features, namely, a minimal O2 requirement and competition with assimilatory NO3-.
Assuntos
Fusarium/metabolismo , Oxigênio/análise , Aerobiose , Fusarium/genética , Mutação , Nitratos/metabolismo , Nitritos/metabolismo , Oxirredução , Oxigênio/farmacologiaRESUMO
Nitric oxide (NO) plays an important role in vascular regulation through its vasodilatory, antiatherogenic, and antithrombotic properties. NO inhibits platelet adhesion and aggregation and modulates smooth muscle cell (SMC) proliferation and migration. In animals with experimentally induced vascular injury, ec-NOS gene transfection not only restored NO production to normal levels but also increased vascular reactivity of the injured vessels. However, it is unclear whether NO regulates smooth-muscle-specific gene expression. We report here that addition of PDGF-BB to vascular smooth muscle cells suppressed SM-MHC expression but treatment with the NO donors FK409 and SNAP restored SM-MHC mRNA/protein expression. In vitro transfection and subsequent CAT assays demonstrated that exogenous NO can restore PDGF-BB-induced suppression of SM-MHC promoter activity. Promoter deletion analysis revealed that a CArG-3 box located at -1276 bp in the SM-MHC promoter was important for NO-dependent promoter regulation and as well as high level promoter activity. Gel mobility shift assays showed that CArG-3 contained the SRF binding site and a binding site for YY1, a nuclear factor which acts as a negative regulator on muscle-specific promoters. Interestingly, NO donor FK409 reduced YY1 binding to the CArG-3 element but increased SRF binding, suggesting that these two factors bind competitively to the overlapping sites. We also found that mutation to the YY1 binding site in the CArG-3 element resulted in a loss of PDGF-BB-induced suppression of the SM-MHC promoter activity. These findings indicate that NO regulates SM-MHC gene expression at the transcriptional level at least partially through the regulation of transcription factor binding activities on the CArG element. Thus we propose that NO plays a positive role in maintaining the differentiated state of VSMCs.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Cadeias Pesadas de Miosina/genética , Óxido Nítrico/farmacologia , Proteínas Nucleares/fisiologia , Penicilamina/análogos & derivados , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/efeitos dos fármacos , Animais , Aorta Torácica/citologia , Becaplermina , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Genes Reporter , Músculo Liso Vascular/metabolismo , Mutagênese Sítio-Dirigida , Cadeias Pesadas de Miosina/biossíntese , Óxido Nítrico/biossíntese , Óxido Nítrico/fisiologia , Doadores de Óxido Nítrico/farmacologia , Nitrocompostos/farmacologia , Penicilamina/farmacologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-sis , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Sequências Reguladoras de Ácido Nucleico , Deleção de Sequência , Fator de Resposta Sérica , Transfecção , Fator de Transcrição YY1RESUMO
Arg and Lys residues are concentrated on the distal side of cytochrome P450nor (P450nor) to form a positively charged cluster facing from the outside to the inside of the distal heme pocket. We constructed mutant proteins in which the Arg and Lys residues were replaced with Glu, Gln, or Ala. The results showed that this cluster plays crucial roles in NADH interaction. We also showed that some anions such as bromide (Br(-)) perturbed the heme environment along with the reduction step in P450nor-catalyzed reactions, which was similar to the effects caused by the mutations. We determined by x-ray crystallography that a Br(-), termed an anion hole, occupies a key region neighboring heme, which is the terminus of the positively charged cluster and the terminus of the hydrogen bond network that acts as a proton delivery system. A comparison of the predicted mechanisms between the perturbations caused by Br(-) and the mutations suggested that Arg(174) and Arg(64) play a crucial role in binding NADH to the protein. These results indicated that the positively charged cluster is the unique structure of P450nor that responds to direct interaction with NADH.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Heme/química , NAD/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Sequência de Aminoácidos , Arginina/fisiologia , Sítios de Ligação , Brometos/metabolismo , Cânfora 5-Mono-Oxigenase/química , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Cinética , Lisina/fisiologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Óxido Nítrico/metabolismo , Oxirredutases/genética , Fosfatos/metabolismo , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , EspectrofotometriaRESUMO
The gene of a fatty-acid hydroxylase of the fungus Fusarium oxysporum (P450foxy) was cloned and expressed in yeast. The putative primary structure revealed the close relationship of P450foxy to the bacterial cytochrome P450BM3, a fused protein of cytochrome P450 and its reductase from Bacillus megaterium. The amino acid sequence identities of the P450 and P450 reductase domains of P450foxy were highest (40.6 and 35.3%, respectively) to the corresponding domains of P450BM3. Recombinant P450foxy expressed in yeast was catalytically and spectrally indistinguishable from the native protein, except most of the recombinant P450foxy was recovered in the soluble fraction of the yeast cells, in marked contrast to native P450foxy, which was exclusively recovered in the membrane fraction of the fungal cells. This difference implies that a post (or co)-translational mechanism functions in the fungal cells to target and bind the protein to the membrane. These results provide conclusive evidence that P450foxy is the eukaryotic counterpart of bacterial P450BM3, which evokes interest in the evolutionary aspects concerning the P450 superfamily along with its reducing systems. P450foxy was classified in the new family, CYP505.
Assuntos
Bacillus megaterium/enzimologia , Proteínas de Bactérias , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Graxos/metabolismo , Proteínas Fúngicas , Fusarium/enzimologia , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Western Blotting , Sistema Enzimático do Citocromo P-450/química , Eletroforese em Gel de Poliacrilamida , Evolução Molecular , Cinética , Dados de Sequência Molecular , NADPH-Ferri-Hemoproteína Redutase , Filogenia , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The involvement of cytochrome P450nor (P450nor) is the most striking feature of the fungal denitrifying system, and has never been shown in bacterial systems. To establish the physiological significance of the P450nor, we constructed and investigated mutants of Fusarium oxysporum that lacked the gene for P450nor. We mutated the gene by targeted integration of a disrupted gene into the chromosome of F. oxysporum. The mutants were shown to contain neither P450nor protein nor nitric oxide (NO) reductase (Nor) activity, implying that they are indeed deficient in P450nor. These mutants had apparently lost the denitrifying activity and failed to evolve nitrous oxide (N2O) upon incubation under oxygen-limiting conditions in the presence of nitrate. Their mycelia exhibited normal levels of dissimilatory nitrite reductase (Nir) activity and were able to evolve NO under these conditions. The promoter region of the P450nor gene was fused to lacZ and introduced into the wild-type strain of F. oxysporum. The transformed strain produced beta-galactosidase under denitrifying conditions as efficiently as the wild type does P450nor. These results represent unequivocal genetic evidence that P450nor is essential for the reduction of NO to N2O, the last step in denitrification by F. oxysporum.
Assuntos
Sistema Enzimático do Citocromo P-450/fisiologia , Fusarium/metabolismo , Óxido Nítrico/metabolismo , Oxirredutases/fisiologia , Southern Blotting , Western Blotting , Sistema Enzimático do Citocromo P-450/genética , Fusarium/genética , Modelos Genéticos , Mutação , Oxirredutases/genética , Plasmídeos , Fatores de Tempo , Transformação GenéticaRESUMO
Fusarium oxysporum, an imperfect filamentous fungus performs nitrate respiration under limited oxygen. In the respiratory system, Cytochrome P450nor (P450nor) is thought to catalyze the last step; reduction of nitric oxide to nitrous oxide. We examined its intracellular localization using enzymatic, spectroscopic, and immunological analyses to show that P450nor is found in both the mitochondria and the cytosol. Translational fusions between the putative mitochondrial targeting signal on the amino terminus of P450nor and Escherichia coli beta-galactosidase resulted in significant beta-galactosidase activity in the mitochondrial fraction of nitrate-respiring cells, suggesting that one of the isoforms of P450nor (P450norA) is in anaerobic mitochondrion of F. oxysporum and acts as nitric oxide reductase. Furthermore, these findings suggest the involvement of P450nor in nitrate respiration in mitochondria.
Assuntos
Respiração Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Fusarium/enzimologia , Fusarium/metabolismo , Mitocôndrias/enzimologia , Nitratos/metabolismo , Oxirredutases/metabolismo , Aerobiose , Anaerobiose , Transporte Biológico , Códon de Iniciação/genética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/imunologia , Citosol/enzimologia , Citosol/metabolismo , Citosol/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Fusarium/citologia , Fusarium/ultraestrutura , Isoenzimas/química , Isoenzimas/imunologia , Isoenzimas/metabolismo , Microscopia Eletrônica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Óxido Nítrico/metabolismo , Óxido Nitroso/metabolismo , Oxirredutases/química , Oxirredutases/imunologia , Oxigênio/metabolismo , Biossíntese de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , EspectrofotometriaRESUMO
A patient with Cowden disease and multiple arteriovenous malformations (AVMs) that resulted in high output heart failure is described. Cowden disease is a familial syndrome characterized by endodermal, mesodermal and ectodermal dysplasia causing benign and malignant tumors of the skin, breast, gastrointestinal tract, and thyroid gland. Our patient had gastrointestinal polyposis, a right renal tumor, a left lung tumor, an adenomatous goiter, and typical dermatologic findings such as facial papules, acral keratosis, gingival papillomatosis and hemangiomas. AVMs were observed in the pelvis, cervical vertebra, liver, and right supraclavicular area. Transcatheter embolization was performed 7 times for the pelvic AVMs, but the effect decreased with repetition and the patient died of heart failure 2 years after the first embolization. The serum levels of tissue plasminogen activator (t-PA), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta1 were high, suggesting that these angiogenic molecules may play a role in the pathogenesis of AVMs in Cowden disease.
Assuntos
Malformações Arteriovenosas/terapia , Embolização Terapêutica , Síndrome do Hamartoma Múltiplo , Adulto , Malformações Arteriovenosas/patologia , Malformações Arteriovenosas/fisiopatologia , Humanos , Masculino , Pelve/irrigação sanguínea , Pelve/patologiaRESUMO
Yeasts of various genera were screened for denitrifying activity, and several yeast strains such as Trichosporon cutaneum, Fellomyces fuzhouensis, and Candida sp. were found to exhibit distinct activities to convert nitrite to nitrous oxide. Dissimilatory nitrite reductase (Nir) or nitric oxide reductase (Nor) activities were detected in the cell-free extracts of these yeasts. Spectrophotometric as well as Western blot analyses showed that T. cutaneum contains Nor of the cytochrome P450 type. This is the first report that shows that denitrification is also distributed among yeasts although their systems are incomplete, only capable of reducing nitrite to nitrous oxide.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Nitritos/metabolismo , Oxirredutases/metabolismo , Trichosporon/enzimologia , Leveduras/enzimologia , Anaerobiose , Western Blotting , Indução Enzimática , Nitrito Redutases/metabolismo , Óxido Nitroso/metabolismo , Oxirredução , Espectrofotometria , Trichosporon/classificação , Trichosporon/crescimento & desenvolvimento , Leveduras/classificação , Leveduras/metabolismoRESUMO
Many actinomycete strains are able to convert nitrate or nitrite to nitrous oxide (N2O). As a representative of actinomycete denitrification systems, the system of Streptomyces thioluteus was investigated in detail. S. thioluteus attained distinct cell growth upon anaerobic incubation with nitrate or nitrite with concomitant and stoichiometric conversion of nitrate or nitrite to N2O, suggesting that the denitrification acts as anaerobic respiration. Furthermore, a copper-containing, dissimilatory nitrite reductase (CuNir) and its physiological electron donor, azurin, were isolated. This is the first report to show that denitrification generally occurs among actinomycetes.
Assuntos
Ascomicetos/metabolismo , Azurina/metabolismo , Nitrito Redutases/metabolismo , Streptomyces/metabolismo , Azurina/isolamento & purificação , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Nitrito Redutases/isolamento & purificação , Streptomyces/enzimologiaRESUMO
A 62-year-old woman with IgA-lambda type monoclonal gammopathy had been followed up since January 1988. In March 1991, multiple myeloma (IgA-lambda) was diagnosed on the basis of bone marrow biopsy findings and increased serum IgA levels. She was treated intermittently with melphalan and prednisolone over a perioa of about 6 years, but was eventually admitted due to renal dysfunction, hypercalcemia, increased serum IgA and the formation of subcutaneous masses. During chemotherapy she underwent emergency surgery for obturative ileus. Histological examination of the resected tissues revealed invasion of myeloma cells into the small intestine and peritoneum. Despite continued chemotherapy, the patient's soft tissue masses enlarged, and new lesions appeared in other organs. In the terminal stage, lower serum IgA levels were observed despite an increase in Bence-Jones protein levels in urine. The patient died five months after admission. An autopsy found infiltration by atypical myeloma cells in multiple organs. An immunohistochemical examination revealed and increase in lambda-light chain positive cells relative to the number of alpha-heavy chain positive cells. The terminal course was considered to be representative of aggressive phase multiple myeloma. The case was rare in that the patient's ileus was caused by invasion of myeloma cells into the small intestine.
Assuntos
Neoplasias Intestinais/patologia , Obstrução Intestinal/etiologia , Mieloma Múltiplo/patologia , Progressão da Doença , Feminino , Humanos , Obstrução Intestinal/cirurgia , Intestino Delgado , Pessoa de Meia-Idade , Mieloma Múltiplo/complicações , Invasividade NeoplásicaRESUMO
We cloned a chitinase-encoding gene from Aspergillus nidulans by polymerase chain reaction using degenerated oligonucleotide primers designed from the conserved amino acid sequences among chitinases from yeasts and Rhizopus spp. The cloned gene, named chiA, encoded a polypeptide consisting of 660 amino acids. Disruption of chiA had no effect on hyphal or conidiophore morphology, but germination frequency and hyphal growth rate decreased substantially. Expression of chiA was investigated using Escherichia coli beta-galactosidase as a reporter enzyme. The beta-galactosidase activity was present during hyphal growth and increased twice as the conidiophores developed. In situ staining of beta-galactosidase activity found high expression in metulae, phialides, and conidia during conidiophore development, indicating that the expression of chiA is developmentally regulated. This is the first report to isolate a chitinase gene from A. nidulans and investigate its functions using the gene disruption technique and gene fusion methods in filamentous fungi.
Assuntos
Aspergillus nidulans/enzimologia , Quitinases/genética , Proteínas Fúngicas/genética , Sequência de Aminoácidos , Aspergillus nidulans/genética , Aspergillus nidulans/crescimento & desenvolvimento , Sequência de Bases , Quitinases/fisiologia , Clonagem Molecular , DNA Fúngico , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
A flavohemoprotein was purified to homogeneity from the denitrifying fungus Fusarium oxysporum. The purified protein existed as a monomer with a molecular weight of 44 kDa. It was purified in an oxidized form and exhibited the absorption maxima at 401, 540 and 643 nm in its resting form, and at 434 and 555 nm upon reduction with dithionite, respectively. The protein contained 0.5 mol protoheme/mol and 1.1 mol FAD/mol, respectively. When the resting flavohemoprotein was aerobically incubated with NAD(P)H, it was converted to a spectral species that is spectrally very similar to oxyhemoglobins. These properties are characteristics of flavohemoglobins (FHb) of Alcaligenes eutrophus, Escherichia coli, and baker's yeast. Further the amino terminal amino acid sequence of the protein of F. oxysporum was similar to those of these FHbs. These results suggest that the isolated flavohemoprotein of F. oxysporum would be a counterpart of the proteins in the FHb family.