RESUMEN
Microbial lipids are sustainable feedstock for the production of oleochemicals and biodiesel. Oleaginous yeasts have recently been proposed as alternative lipid producers to plants and animals to promote sustainability in the chemical and fuel industries. The oleaginous yeast Lipomyces starkeyi has great industrial potential as an excellent lipid producer. However, improvement of its lipid productivity is essential for the cost-effective production of oleochemicals and fuels. Genetic and metabolic engineering of L. starkeyi via gene manipulation techniques may result in improvements in lipid production and our understanding of the mechanisms behind lipid biosynthesis pathways. We previously described an integrative transformation system using a drug-resistant marker for L. starkeyi. However, gene-targeting frequencies were very low because non-homologous recombination is probably predominant in L. starkeyi. Genetic engineering tools for L. starkeyi have not been sufficiently developed. In this study, we describe a new genetic tool and its application in L. starkeyi. To develop a highly efficient gene-targeting system for L. starkeyi, we constructed a series of mutants by disrupting genes for LsKu70p, LsKu80p, and/or LsLig4p, which share homology with other yeasts Ku70p, Ku80p, and Lig4p, respectively, being involved in non-homologous end-joining pathway. Deletion of the LsLIG4 gene dramatically improved the homologous recombination efficiency (80.0%) at the LsURA3 locus compared with that in the wild-type strain (1.4%), when 2000-bp homologous flanking regions were used. The homologous recombination efficiencies of the double mutant ∆l sku70∆lslig4 and the triple mutant ∆lsku70∆lsku80∆lslig4 were also markedly enhanced. Therefore, the L. starkeyi ∆lslig4 background strains have promise as efficient recipient strains for genetic and metabolic engineering approaches in this yeast.
Asunto(s)
Reparación del ADN por Unión de Extremidades/genética , Lípidos/biosíntesis , Lipomyces/genética , Ingeniería Metabólica , Biocombustibles/microbiología , Marcación de Gen , Lípidos/genética , Lipomyces/metabolismo , Mutación/genéticaRESUMEN
A 40-year-old woman was admitted to our hos- pital with cough and sputum production. A chest computed tomography (CT) scan revealed a diffuse nodular shadow in the upper lung. The patient was diagnosed with pulmonary tuberculosis, based on a positive T-SPOT®.TB test result of peripheral blood and a positive polymerase chain reaction (PCR) test result for Mycobacterium tuberculosis in gastric aspirates. M.tuberculosis was subsequently isolated from the gastric aspirate specimen. After 2 months of treatment with antituberculous medication, the patient developed a low grade fever and left-sided chest pain. A CT scan revealed a left pleural effusion and a right subphrenic abscess. Tuber- culous pleurisy with paradoxical response was diagnosed on the basis of an increased lymphocyte count and increased adenosine deaminase activity in the pleural fluid exudate. A percutaneous ultrasound-guided needle biopsy of the sub- phrenic abscess was performed. Histological analysis revealed epithelioid cell granulomas with necrosis and PCR for M. tuberculosis using puncture needle washing fluid returned positive results. Based on these findings, a diagnosis of subphrenic abscess with paradoxical response, caused by M. tuberculosis, was made. Subphrenic abscess caused by M. tuberculosis is an important consideration during antituber- culous therapy.
Asunto(s)
Mycobacterium tuberculosis/efectos de los fármacos , Absceso Subfrénico/etiología , Tuberculosis Pulmonar/tratamiento farmacológico , Adulto , Antituberculosos/uso terapéutico , Progresión de la Enfermedad , Combinación de Medicamentos , Femenino , Humanos , Tuberculosis Pulmonar/complicaciones , Tuberculosis Pulmonar/diagnósticoRESUMEN
A 78-year-old man had fatigue and appetite loss for 5 months. He had been receiving low-dose methotrexate for rheumatoid arthritis. Computed tomography revealed multiple pulmonary infiltrations and muddiness of the fatty tissue surrounding the right kidney, ureter wall thickening, and hydroureter/nephrosis, which were suspected retroperitoneal fibrosis. Lung biopsy revealed polymorphic/lymphoplasmacytic lymphoproliferative disorder. Methotrexate withdrawal resulted in spontaneous regression. Therefore, retroperitoneal lesion may account for the diagnosis as having retroperitoneal lymphoproliferative disorder, not retroperitoneal fibrosis.
Asunto(s)
Antirreumáticos/efectos adversos , Artritis Reumatoide/tratamiento farmacológico , Pulmón/patología , Trastornos Linfoproliferativos/inducido químicamente , Metotrexato/efectos adversos , Fibrosis Retroperitoneal/inducido químicamente , Anciano , Antirreumáticos/uso terapéutico , Artritis Reumatoide/patología , Biopsia , Humanos , Trastornos Linfoproliferativos/patología , Masculino , Metotrexato/uso terapéutico , Fibrosis Retroperitoneal/patología , Tomografía Computarizada por Rayos XRESUMEN
The oleaginous yeast, Lipomyces starkeyi, is an excellent lipid producer with great industrial potential. However, methods for molecular breeding have not been established for L. starkeyi. We describe the development of a system for targeted rDNA integration of multiple copies of a gene into L. starkeyi genome by spheroplast-polyethylene glycol transformation.
Asunto(s)
Ingeniería Genética/métodos , Lipomyces/genética , ADN Ribosómico/genética , Expresión Génica , Genoma Fúngico/genética , Polietilenglicoles/química , Transformación GenéticaRESUMEN
An antifungal defensin, AFP1, of Brassica juncea inhibits the growth of various microorganisms. The molecular details of this inhibition remain largely unknown. Herein, we reveal that a specific structure of fungal sphingolipid glucosylceramide (GlcCer) is critical for the sensitivity of Candida albicans cells to AFP1. Our results revealed that AFP1 induces plasma membrane permeabilization and the production of reactive oxygen species (ROS) in wild-type C. albicans cells, but not in cells lacking the ninth methyl residue of the GlcCer sphingoid base moiety, which is a characteristic feature of fungi. AFP1-induced ROS production is responsible for its antifungal activity, with a consequent loss of yeast cell viability. These findings suggest that AFP1 specifically recognizes the structural difference of GlcCer for targeting of the fungal pathogens.
Asunto(s)
Candida albicans/efectos de los fármacos , Defensinas/metabolismo , Planta de la Mostaza/crecimiento & desarrollo , Enfermedades de las Plantas/genética , Antifúngicos/metabolismo , Candida albicans/patogenicidad , Defensinas/genética , Glucosilceramidas/genética , Glucosilceramidas/metabolismo , Planta de la Mostaza/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
A 55-year-old man was admitted for coronavirus disease 2019 (COVID-19)-related respiratory failure. He was treated with corticosteroids and tocilizumab in the intensive care unit. Aspergillus fumigatus (A. fumigatus) was isolated from his sputum on admission. However, no radiological findings suggesting pulmonary aspergillosis were seen on chest computed tomography (CT). Since the fungus had merely colonized in airways, antifungal drugs were not administered immediately. On day 19 of hospitalization, a high (1â3)-ß-D-glucan (BDG) level was noted. A CT scan on day 22 revealed consolidations with a cavity in the right lung. A. fumigatus was isolated from his sputum again. Thus, we diagnosed the patient with COVID-19-associated pulmonary aspergillosis (CAPA) and started voriconazole. After the treatment, BDG levels and radiological findings were noted to improve. In this case, tocilizumab probably had a critical role in developing the disease. Although antifungal prophylaxis therapy for CAPA is not well established, this case shows that detecting Aspergillus in airway specimens before the disease onset possibly implies a high risk of developing CAPA and is an indicator of antifungal prophylaxis.
RESUMEN
We report a case with an atypical presentation of descending necrotizing mediastinitis (DNM). A 47-year-old woman with a medical history of untreated type 2 diabetes mellitus and influenza type A virus infection 2 weeks prior to admission was referred to our hospital complaining of right cervical pain and right upper limb swelling. A chest enhanced computed tomographic (CT) scan showed a ring-enhanced mass-like shadow extending from the right sternomastoid muscle down to the right upper mediastinum, compressing the right subclavicular vein. We diagnosed the patient as having DNM based on a physical examination and the CT findings. Because the abscess extended from deep in the neck to the upper mediastinum and right upper pleural space, emergent abscess debridement and drainage was required. After hospitalization, antibiotics (Ampicillin/Sulbactam 12 g/day) were also administered based on Gram-stain findings from the drainage fluid, which showed Gram-positive cocci resembling a string of beads. A culture of the drainage fluid identified Streptococcus agalactiae. Aggressive abscess drainage and early antibiotic therapy resulted in a favorable response. She was discharged without complications on the 33rd hospital day. DNM is well known as a rare but lethal disease. In this case, the presence of diabetes mellitus and post-influenza infection might have been risk factors for a serious S. agalactiae infection. Early aggressive therapy and adequate drainage are recommended for patients with DNM.
Asunto(s)
Complicaciones de la Diabetes , Mediastinitis/etiología , Infecciones Estreptocócicas/complicaciones , Streptococcus agalactiae , Femenino , Humanos , Persona de Mediana Edad , NecrosisRESUMEN
Mirtazapine (MTZ) is a noradrenergic and specific serotonergic antidepressant that has been associated with an increased risk of bleeding. However, there is insufficient evidence confirming this association. We hypothesised that 5-HT2A and α2 receptor-mediated inhibitory effects of MTZ on platelets suppress platelet aggregation and increase the risk of bleeding. In this study, we examined the antiplatelet effect of MTZ on human platelets to test our hypothesis. Blood samples for platelet aggregation tests were obtained from 14 healthy volunteers. The antiplatelet effect of MTZ was evaluated using light transmission aggregometry. MTZ significantly suppressed platelet aggregation mediated both by the synergistic interaction of serotonin (5-HT) and adrenaline and the synergistic interaction of ADP and 5-HT or adrenaline. In conclusion, MTZ exerts its antiplatelet effects by co-blocking the 5-HT2A and α2-adrenergic receptors on platelets and also suppresses platelet aggregation induced by ADP and 5-HT or adrenaline. Therefore, when MTZ is used, especially for patients with a high risk of bleeding, the significance of its use must be considered carefully. In addition, the platelet aggregation pattern by adrenaline + 5-HT, ADP + adrenaline, and ADP + 5-HT was similar between humans and mice; however, this study did not directly compare the effects of MTZ on human and murine platelets. Therefore, under the conditions for inducing platelet aggregation using adrenaline + 5-HT, ADP + adrenaline, and ADP + 5-HT, mouse platelets can be used in the evaluation of the efficacy of antiplatelet drugs in humans.
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PlaquetasRESUMEN
BACKGROUND: Anti-aquaporin-4 (AQP4) antibody is an autoantibody marker often observed in patients with neuromyelitis optica spectrum disorder (NMOSD). The pathological relevance of complicated pulmonary disorders in anti-AQP4 antibody-positive NMOSD remains unclear. We aimed to assess the clinical and histological relevance of complicated pulmonary disorders in anti-AQP4 antibody-positive NMOSD. METHODS: We retrospectively reviewed the medical records of 52 patients with anti-AQP4 antibody-positive NMOSD and conducted immunohistochemical evaluations of the lung biopsy specimens. RESULTS: Among 52 patients with anti-AQP4 antibody-positive NMOSD, 4 patients showed pulmonary involvement with a diagnosis of organizing pneumonia (OP). The proportion of males was larger (75% vs. 12.5%; p = 0.013) and creatine kinase levels were higher (458.3 U/L vs. 83.9 U/L; p = 0.003) in patients with OP than in those without OP. OP development preceded or coincided with the NMOSD symptoms. Chest computed tomography findings were consistent with OP in all four patients. Bronchoalveolar lavage fluid predominantly contained lymphocytes. Transbronchial lung biopsy revealed intraluminal plugs of inflammatory debris within the alveoli. Alveolar epithelial cells covering the OP lesions exhibited AQP4 loss, immunoglobulin G deposition, and complement activation. Corticosteroid treatment resulted in clinical improvement of OP. CONCLUSION: OP may be considered a pulmonary manifestation of anti-AQP4 antibody-positive NMOSD beyond the central nervous system. Complement-dependent cytotoxicity of the lung epithelial cells caused by anti-AQP4 antibody is at least partly involved in OP development. When diagnosing NMOSD, the possibility of OP should be carefully evaluated based on the detailed history and chest imaging findings.
Asunto(s)
Neuromielitis Óptica , Neumonía , Acuaporina 4/uso terapéutico , Autoanticuerpos , Humanos , Masculino , Neuromielitis Óptica/complicaciones , Neuromielitis Óptica/tratamiento farmacológico , Neumonía/complicaciones , Estudios RetrospectivosRESUMEN
Tuberculosis remains the leading cause of infectious disease related death worldwide with extrapulmonary tuberculosis being particularly difficult to diagnose. Here, we report a case of pancreatic tuberculosis (PTB) in an immunocompetent young female, which mimicked a malignant tumor diagnosed by endoscopic ultrasound-guided fine needle aspiration and biopsy (EUS-FNAB). A 19-year-old Japanese female with no prior medical history presented with abdominal epigastralgia and appetite loss lasting 2 months. A solid lobular mass was observed in the pancreatic head with enhanced abdominal computed tomography and magnetic resonance imaging suggested it was a malignant pancreatic tumor. Using EUS-FNAB, granulomas with caseous necrosis and acid-fast bacilli were observed. Polymerase chain reaction results were positive for Mycobacterium tuberculosis but negative for Mycobacterium avium complex. Therefore, the patient was diagnosed with PTB. Her symptoms and radiological findings improved with a standard antituberculosis therapy. PTB is difficult to differentiate from other pancreatic diseases with Magnetic resonance imaging (MRI) patterns of T1, T2 weighted, or diffusion-weighted image (DWI) images. To investigate novel radiological diagnostics for PTB, we focused on MRI apparent diffusion coefficient (ADC) values, which have not been investigated in this context. The present case showed 0.52 × 10-3 mm2/s; additionally, the mean value of other mass-forming pancreatic diseases, such as pancreatic cancer was 1.592 × 10-3 mm2/s (the range: 1.015-3.025 × 10-3 mm2/s). The range does not overlap with the present PTB case or other pancreatic diseases. Therefore, ADC values may be useful as a noninvasive radiological diagnostic method for PTB.
Asunto(s)
Enfermedades Pancreáticas , Neoplasias Pancreáticas , Tuberculosis , Adulto , Biopsia por Aspiración con Aguja Fina Guiada por Ultrasonido Endoscópico , Femenino , Humanos , Páncreas/diagnóstico por imagen , Enfermedades Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/diagnóstico por imagen , Tuberculosis/diagnóstico por imagen , Adulto JovenRESUMEN
A 73-year-old man developed diplopia after the administration of pembrolizumab for lung adenocarcinoma. He had ptosis and external ophthalmoplegia without general muscle weakness. Serum CK levels were elevated. Although autoantibodies to acetylcholine receptor and muscle-specific kinase, the edrophonium test, and the repetitive nerve stimulation test were all negative, anti-titin autoantibody was positive, leading to the diagnosis of myasthenia gravis (MG). Muscle pathology showed necrotizing myopathy with tubular aggregates. Unlike previously reported cases of pembrolizumab-associated MG, the present case showed ocular MG. This is the first case of pembrolizumab-associated MG with anti-titin antibody, as well as the first case with tubular aggregates.
Asunto(s)
Anticuerpos Monoclonales Humanizados/efectos adversos , Antineoplásicos Inmunológicos/efectos adversos , Autoanticuerpos/sangre , Conectina/inmunología , Miastenia Gravis/inducido químicamente , Anciano , Biomarcadores/sangre , Blefaroptosis/inducido químicamente , Diplopía/inducido químicamente , Humanos , Masculino , Enfermedades Musculares/inducido químicamente , Miastenia Gravis/diagnóstico , Miastenia Gravis/inmunología , Oftalmoplejía/inducido químicamenteRESUMEN
Thermotoga maritima tRNase Z cleaves pre-tRNAs containing the 74CCA76 sequence precisely after the A76 residue to create the mature 3' termini. Its crystal structure has revealed a four-layer alphabeta/betaalpha sandwich fold that is typically found in the metallo-beta-lactamase superfamily. The well-conserved six histidine and two aspartate residues together with metal ions are assumed to form the tRNase Z catalytic center. Here, we examined tRNase Z variants containing single amino acid substitutions in the catalytic center for pre-tRNA cleavage. Cleavage by each variant in the presence of Mg2+ was hardly detected, although it is bound to pre-tRNA. Surprisingly, however, Mn2+ ions restored the lost Mg2+-dependent activity with two exceptions of the Asp52Ala and His222Ala substitutions, which abolished the activity almost completely. These results provide a piece of evidence that Asp-52 and His-222 directly contribute the proton transfer for the catalysis.
Asunto(s)
Endorribonucleasas/química , Manganeso/química , Protones , Precursores del ARN/química , ARN de Transferencia/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Ácido Aspártico/química , Secuencia de Bases , Catálisis , Dominio Catalítico , Cationes Bivalentes , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Histidina/química , Metales/química , Datos de Secuencia Molecular , Precursores del ARN/metabolismo , ARN de Transferencia/metabolismo , Thermotoga maritima/enzimologíaRESUMEN
In the long-chain dicarboxylic acids (DCA)-hyperproducing mutant Candida maltosa strains, methyl-ends of n-alkanes and fatty acids are hydroxylated by n-alkane inducible cytochromes P450 (P450alk), presumably as an essential step in DCA production. A significantly higher production of P450alks was observed in response to n-alkane in the DCA-hyperproducing mutant strain M2030 than in the wild-type strain 1098. Northern analysis demonstrated that n-tetradecane induction levels of mRNAs of all four ALK genes encoding major P450alk isoforms involved in n-alkane assimilation were significantly higher in the DCA-hyperproducing mutant than in the wild-type strain. Among these four ALK genes, enhancement of the transcriptional induction level of ALK5, which prefers fatty acids as substrates, was prominent in the mutant. In agreement with Northern analysis, promoters of ALK genes, especially that of ALK5, more strongly responded to n-alkanes in the DCA-hyperproducing mutant than in the wild-type strain. These results suggest that the transcriptional control of ALK genes in the DCA-hyperproducing mutant strains was altered preferably to accelerate DCA production.
Asunto(s)
Candida/genética , Sistema Enzimático del Citocromo P-450/biosíntesis , Ácidos Dicarboxílicos/metabolismo , Proteínas Fúngicas/biosíntesis , Regulación Fúngica de la Expresión Génica , Oxigenasas de Función Mixta/biosíntesis , Mutación , Alcanos/farmacología , Fusión Artificial Génica , Northern Blotting , Western Blotting , Candida/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Proteínas Fúngicas/genética , Regulación de la Expresión Génica , Genes Reporteros , Oxigenasas de Función Mixta/genética , Regiones Promotoras Genéticas , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , ARN de Hongos/biosíntesis , ARN Mensajero/biosíntesis , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
2-Deoxy-scyllo-inosose (DOI) is a six-membered carbocycle formed from d-glucose-6-phosphate catalyzed by 2-deoxy-scyllo-inosose synthase (DOIS), a key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics. DOI is valuable as a starting material for the benzene-free synthesis of catechol and other benzenoids. We constructed a series of metabolically engineered Escherichia coli strains by introducing a DOIS gene (btrC) from Bacillus circulans and disrupting genes for phosphoglucose isomerase, d-glucose-6-phosphate dehydrogenase, and phosphoglucomutase (pgi, zwf and pgm, respectively). It was found that deletion of the pgi gene, pgi and zwf genes, pgi and pgm genes, or all pgi, zwf and pgm genes significantly improved DOI production by recombinant E. coli in 2YTG medium (3% glucose) up to 7.4, 6.1, 11.6, and 8.4 g l(-1), respectively, compared with that achieved by wild-type recombinant E. coli (1.5 g l(-1)). Moreover, E. coli mutants with disrupted pgi, zwf and pgm genes showed strongly enhanced DOI productivity of up to 29.5 g l(-1) (99% yield) in the presence of mannitol as a supplemental carbon source. These results demonstrated that DOI production by metabolically engineered recombinant E. coli may provide a novel, efficient approach to the production of benzenoids from renewable d-glucose.
Asunto(s)
Reactores Biológicos , Biotecnología/métodos , Escherichia coli/metabolismo , Inositol/análogos & derivados , Bacillus/genética , Escherichia coli/genética , Eliminación de Gen , Ingeniería Genética/métodos , Glucosa/metabolismo , Glucosa-6-Fosfato Isomerasa/genética , Glucosafosfato Deshidrogenasa/genética , Inositol/biosíntesis , Liasas/genética , Fosfoglucomutasa/genéticaRESUMEN
tRNA 3'-processing endoribonuclease (tRNase Z) is one of the enzymes involved in the 3'-end processing of precursor tRNAs and is a member of the metallo-beta-lactamase superfamily. tRNase Z crystal structures have revealed that the enzyme forms a dimer and has a characteristic domain, named a flexible arm or an exosite, which protrudes from the metallo-beta-lactamase core and is involved in tRNA binding. The refined structure of Thermotoga maritima tRNase Z has been determined at 1.97 A resolution, revealing the structure of the flexible arm and the zinc-bound active site. The structure of the flexible arm of T. maritima tRNase Z is distinct from those of the Bacillus subtilis and Escherichia coli tRNase Zs. A comparison of the three tRNase Z structures revealed differences in the dimer orientation, which may be related to the unique cleavage-site specificity of T. maritima tRNase Z.
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Endorribonucleasas/química , Pliegue de Proteína , Homología Estructural de Proteína , Thermotoga maritima/enzimología , Secuencia de Aminoácidos/fisiología , Sitios de Unión , Cristalografía por Rayos X , Datos de Secuencia MolecularRESUMEN
A 62-year-old woman, who had received immunosuppressive treatment for myasthenia gravis, was admitted to our hospital for the treatment of a right pulmonary tumor. In October 2003, a chest computed tomography showed a nodule-like lesion in the right lung. Two years later, the lesion was shown to have increased in size, and a right upper lobectomy was performed using video-assisted thoracic surgery. A tumor biopsy showed histological features of bronchioloalveolar carcinoma. It has been reported that malignant tumors occur more frequently in patients with myasthenia gravis with concurrent thymoma. Therefore, such patients need to be followed closely for a long period of time for any possible malignant tumor occurring in different organs. In addition, protection of the bronchial stump may be necessary to prevent a bronchial fistula, because wound healing can be delayed due to immunosuppressive treatment.
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Adenocarcinoma Bronquioloalveolar/cirugía , Neoplasias Pulmonares/cirugía , Miastenia Gravis/complicaciones , Neumonectomía/métodos , Timectomía , Adenocarcinoma Bronquioloalveolar/etiología , Femenino , Humanos , Inmunosupresores/uso terapéutico , Neoplasias Pulmonares/etiología , Persona de Mediana Edad , Miastenia Gravis/tratamiento farmacológico , Miastenia Gravis/cirugía , Complicaciones Posoperatorias , Cirugía Torácica Asistida por VideoRESUMEN
A 63-year-old man was referred to our hospital due to an abnormal shadow found on a chest CT scan. The CT scan showed a cystic tumor with a central high density in the right upper mediastinal area. The preoperative diagnosis was teratoma in the right upper mediastinal area. Video-assisted thoracic surgery was performed. The tumor was about 3cm in size and originated from the right vagal nerve. Microscopic findings of the resected specimen showed loose growth of spindle cells, in which a myxomatous framework was observed in the peripheral area as well as extended vessels, with thrombis, Verocay bodies, and a nuclear palisading pattern confirmed in the central area. The histological diagnosis was schwannoma originating from the mediastinal vagal nerve. This case was thought to be rare and difficult to diagnose by preoperative imaging findings. Postoperative hoarseness due to dysfunction of the right recurrent nerve occurred. Careful consideration should be given to the indication of video-assisted thoracic surgery for neurogenic mediastinal tumors since postoperative dysfunction may occur.
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Neoplasias de los Nervios Craneales/cirugía , Neurilemoma/cirugía , Cirugía Torácica Asistida por Video , Enfermedades del Nervio Vago/cirugía , Neoplasias de los Nervios Craneales/diagnóstico , Neoplasias de los Nervios Craneales/patología , Ronquera , Humanos , Imagen por Resonancia Magnética , Masculino , Mediastino/inervación , Persona de Mediana Edad , Neurilemoma/diagnóstico , Neurilemoma/patología , Complicaciones Posoperatorias , Tomografía Computarizada por Rayos X , Enfermedades del Nervio Vago/diagnóstico , Enfermedades del Nervio Vago/patología , Parálisis de los Pliegues VocalesRESUMEN
A Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterial strain was isolated from compost. This organism, identified as Bacillus megaterium N-18-25-9, produced a clearing zone on opaque NB-PHB agar, indicating the presence of extracellular PHB depolymerase. A PHB depolymerase gene, PhaZ(Bm), of B. megaterium N-18-25-9 was cloned and sequenced, and the recombinant gene product was purified from Escherichia coli. The N-terminal half region of PhaZ(Bm) shared significant homologies with a catalytic domain of other PHB depolymerases. Although the C-terminal half region of PhaZ(Bm) showed no significant similarity with those of other PHB depolymerases, that region was necessary for the PHB depolymerase activity. Therefore, this enzyme's domain structure is unique among extracellular PHB depolymerase domain structures. The addition of PHB to the medium led to a sixfold increase in PhaZ(Bm) mRNA, while the presence of glucose repressed PhaZ(Bm) expression. The maximum activity was observed at pH 9.0 at 65 degrees C.
Asunto(s)
Bacillus megaterium/enzimología , Bacillus megaterium/aislamiento & purificación , Hidrolasas de Éster Carboxílico/genética , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Microbiología del Suelo , Secuencia de Aminoácidos , Bacillus megaterium/clasificación , Bacillus megaterium/genética , Bacillus megaterium/crecimiento & desarrollo , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , SueloRESUMEN
tRNA 3' processing endoribonuclease (3' tRNase) is an enzyme responsible for the removal of a 3' trailer from precursor tRNA (pre-tRNA). We purified approximately 85 kDa 3' tRNase from pig liver and determined its partial sequences. BLAST search of them suggested that the enzyme was the product of a candidate human prostate cancer susceptibility gene, ELAC2, the biological function of which was totally unknown. We cloned a human ELAC2 cDNA and expressed the ELAC2 protein in Escherichia coli. The recombinant ELAC2 was able to cleave human pre-tRNA(Arg) efficiently. The 3' tRNase activity of the yeast ortholog YKR079C was also observed. The C-terminal half of human ELAC2 was able to remove a 3' trailer from pre-tRNA(Arg), while the N-terminal half failed to do so. In the human genome exists a gene, ELAC1, which seems to correspond to the C-terminal half of 3' tRNase from ELAC2. We showed that human ELAC1 also has 3'-tRNase activity. Furthermore, we examined eight ELAC2 variants that seem to be associated with the occurrence of prostate cancer for 3'-tRNase activity. Seven ELAC2 variants which contain one to three amino acid substitutions showed efficient 3'-tRNase activities, while one truncated variant, which lacked a C-terminal half region, had no activity.