RESUMEN
BACKGROUND: Asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous diseases. The phenotypes that have clinical features of both asthma and COPD are still incompletely understood. OBJECTIVE: To clarify the best discriminators of the asthma-COPD overlap phenotype from asthma and COPD subgroups using a clustering approach. METHODS: This study assessed pathophysiological parameters, including mRNA expression levels of T helper cell-related transcription factors, namely TBX21 (Th1), GATA3 (Th2), RORC (Th17) and FOXP3 (Treg), in peripheral blood mononuclear cells in asthma patients (n=152) and in COPD patients (n=50). Clusters were determined using k-means clustering. Exacerbations of asthma and COPD were recorded during the 1-year follow-up period. RESULTS: The cluster analysis revealed four biological clusters: cluster 1, predominantly patients with COPD; cluster 2, patients with an asthma-COPD overlap phenotype; cluster 3, patients with non-atopic and late-onset asthma; and cluster 4, patients with early-onset atopic asthma. Hazard ratios for exacerbation were 2.5 (95% confidence interval [CI], 1.1-5.6) in cluster 1 and 2.3 (95% CI, 1.0-5.0) in cluster 2 compared with patients in other clusters. Cluster 2 was discriminated from other clusters by total serum IgE level ≥310 IU/mL, blood eosinophil counts ≥280 cells/µL, a higher ratio of TBX21/GATA3, FEV1 /FVC ratio <0.67 and smoking ≥10 pack-years with an area under the curve of 0.94 (95% CI, 0.90-0.98) in the receiver operating characteristic analysis. CONCLUSIONS AND CLINICAL RELEVANCE: The asthma-COPD overlap phenotype was characterized by peripheral blood eosinophilia and higher levels of IgE despite the Th2-low endotype.
Asunto(s)
Asma/diagnóstico , Análisis por Conglomerados , Fenotipo , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Anciano , Asma/etiología , Asma/metabolismo , Biomarcadores , Comorbilidad , Diagnóstico Diferencial , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Calidad de Vida , Curva ROC , Pruebas de Función Respiratoria , Factores de Riesgo , Evaluación de SíntomasRESUMEN
BACKGROUND: Clinical application of the forced oscillation technique (FOT) has progressed with the spread of commercially available FOT devices. The correlation between respiratory impedance and spirometry has been reported; however, the association with airway inflammation and pulmonary function, in the lung periphery in particular, is unclear. OBJECTIVE: To assess whether respiratory impedance is associated with peripheral airway inflammation and dysfunction in asthma. METHODS: Subjects included 78 patients with overall controlled asthma. We measured whole-breath or within-breath respiratory system resistance (Rrs) and reactance (Xrs) using a commercially available multi-frequency FOT device (MostGraph-01), and assessed the correlation with the fraction of exhaled nitric oxide (FeNO), alveolar nitric oxide concentration (CANO), maximal NO flux in the conductive airways (J'awNO), and the N2 phase III slope of single breath N2 washout (delta N2 ). RESULTS: The differences between inspiratory and expiratory phases of Xrs at 5 Hz (X5), resonant frequency (Fres), and a low-frequency reactance area (ALX) were significantly correlated with CANO; however, there was no correlation between respiratory impedance and FeNO or J'awNO. The delta N2 values were significantly correlated with whole-breath, inspiratory, and expiratory Rrs and Xrs, except for R20. CONCLUSIONS AND CLINICAL RELEVANCE: We conclude that respiratory impedance reflects peripheral airway inflammation and ventilation inhomogeneity.
Asunto(s)
Asma/fisiopatología , Mecánica Respiratoria , Adulto , Anciano , Estudios de Casos y Controles , Espiración , Femenino , Humanos , Masculino , Persona de Mediana Edad , Óxido Nítrico , Pruebas de Función Respiratoria , Factores de RiesgoRESUMEN
Methylviologen (MV) induces oxidative damages in leaves. In order to understand its mechanism we studied initial biochemical events under light in MV-fed spinach leaves. When isolated chloroplasts were illuminated in the presence of MV, both stromal and thylakoid-bound ascorbate peroxidases (APX) were inactivated rapidly at the same rates, and their inactivation was retarded by ascorbate (AsA) at higher concentrations. Since MV accelerates the photoproduction of O2- in Photosystem (PS) I and simultaneously inhibits the photoreduction of monodehydroascorbate (MDA) to AsA, the inactivation of APX was attributed to the loss of AsA and accumulation of H2O2 in the stroma. Following APX, superoxide dismutase and NADP(+)-glyceraldehyde 3-phosphate dehydrogenase, both of which are vulnerable to H2O2, were inactivated by MV plus light. Dehydroascorbate reductase, monodehydroascorbate reductase, PS II, PS I and ferredoxin-NADP(+) reductase were far less sensitive to the treatment. In the treated leaves, cytosolic APX and guaiacol-specific peroxidase were also inactivated, but slower than chloroplastic APXs were. Catalase was not inactivated. Thus the MV-induced photooxidative damages of leaves are initiated with the inactivation of chloroplastic APXs and develop via the inactivation of other H2O2-sensitive targets. The decay half-life of the MDA signal after a short illumination in the leaves, as determined by in vivo electron spin resonance spectrometry (ESR), was prolonged when the H2O2-scavenging capacity of the leaf cells was abolished by the inactivation of chloroplastic and cytosolic APXs. The measurement of MDA in leaves by ESR, therefore, allows to estimate in vivo cellular capacity to scavenge the photoproduced H2O2.
Asunto(s)
Ácido Deshidroascórbico/análogos & derivados , Paraquat/farmacología , Peroxidasas/antagonistas & inhibidores , Spinacia oleracea/efectos de los fármacos , Ascorbato Peroxidasas , Cloroplastos/efectos de los fármacos , Cloroplastos/enzimología , Ácido Deshidroascórbico/química , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Peróxido de Hidrógeno/metabolismo , Luz , Oxidación-Reducción , Estrés Oxidativo , Peroxidasas/química , Peroxidasas/metabolismo , Spinacia oleracea/química , Spinacia oleracea/enzimología , Tilacoides/enzimologíaRESUMEN
Arabidopsis thaliana P1 protein was crystallized using the hanging drop vapor-diffusion method in 0.1 M piperazine-1, 4-bis(2-ethanesulfonic acid) buffer, containing 14% polyethylene glycol 6000 and 0.2 M magnesium acetate at pH 6.5 and 20 degrees C. The crystals are orthorhombic and belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a=49.8, b=122.4 and c=149. 9 A. The diffraction data up to 2.9 A were collected by a multiwire area detector.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/enzimología , Cristalinas/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Ácidos Alcanesulfónicos , Tampones (Química) , Cristalización , Cristalografía por Rayos X , PiperazinasRESUMEN
BACKGROUND: Vascular tissues of humans and dogs contain chymase as an angiotensin II-forming enzyme. In this study, we investigated whether chymase-dependent angiotensin II formation plays a crucial role in the development of vascular proliferation in dog grafted veins. METHODS AND RESULTS: The right external jugular vein of dogs was grafted to the ipsilateral carotid artery. As a control group, the right external jugular veins in dogs that had not received grafts were used. In the chymase inhibitor-treated group, the vein was infiltrated with 10 micromol/L Suc-Val-Pro-Phe(P)(OPh)(2) and was grafted to the carotid artery. In the placebo-treated group, ACE activity in the grafted veins was significantly lower than that in the control veins up to 7 days after the operation, whereas chymase activity was increased significantly. After 7 days, the mRNA levels of collagen I, collagen III, and fibronectin, all of which are induced by an increase of angiotensin II action, were significantly increased in the grafted veins, and the intima-media ratio of the grafted veins was also increased. In the chymase inhibitor-treated group, the chymase activity in the grafted veins 7 days after the operation was suppressed to 12.1%. The elevated mRNA levels of fibronectin, collagen I, and collagen III in the grafted veins were significantly suppressed by treatment with the chymase inhibitor, and the intima-media ratio was also decreased significantly. CONCLUSIONS: We demonstrate for the first time that chymase-dependent angiotensin II formation plays an important role in the development of vascular proliferation in the grafted veins.
Asunto(s)
Angiotensina II/metabolismo , Vasos Sanguíneos/crecimiento & desarrollo , Serina Endopeptidasas/metabolismo , Angiotensina II/efectos de los fármacos , Animales , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/trasplante , Quimasas , Colágeno/genética , Perros , Factores de Crecimiento Endotelial/genética , Fibronectinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Venas Yugulares/crecimiento & desarrollo , Venas Yugulares/metabolismo , Venas Yugulares/trasplante , Linfocinas/genética , Oligopéptidos/farmacología , Peptidil-Dipeptidasa A/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina Endopeptidasas/efectos de los fármacos , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1 , Túnica Íntima/efectos de los fármacos , Túnica Íntima/crecimiento & desarrollo , Túnica Íntima/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
BACKGROUND: Few quantitative studies have been conducted on the relationship between society and its languages. Individuals with autistic spectrum disorder (ASD) are known to experience social hardships, and a wide range of clinical information about their quality of life has been provided through numerous narrative analyses. However, the narratives of ASD patients have thus far been examined mainly through qualitative approaches. OBJECTIVES: In this study, we analyzed adults with ASD to quantitatively examine the relationship between language abilities and ASD severity scores. METHODS: We generated phonetic transcriptions of speeches by 16 ASD adults at an ASD workshop, and divided the participants into 2 groups according to their Social Responsiveness Scale(TM), 2nd Edition (SRS(TM)-2) scores (where higher scores represent more severe ASD): Group A comprised high-scoring ASD adults (SRS(TM)-2 score: ≥â 76) and Group B comprised low- and intermediate-scoring ASD adults (SRS(TM)-2 score: <â 76). Using natural language processing (NLP)-based analytical methods, the narratives were converted into numerical data according to four language ability indicators, and the relationships between the language ability scores and ASD severity scores were compared. RESULTS AND DISCUSSION: Group A showed a marginally negative correlation with the level of Japanese word difficulty (p <â .10), while the "social cognition" subscale of the SRS(TM)-2 score showed a significantly negative correlation (p <â .05) with word difficulty. When comparing only male participants, Group A demonstrated a significantly lower correlation with word difficulty level than Group B (p <â .10). CONCLUSION: Social communication was found to be strongly associated with the level of word difficulty in speech. The clinical applications of these findings may be available in the near future, and there is a need for further detailed study on language metrics designed for ASD adults.
Asunto(s)
Trastorno del Espectro Autista/diagnóstico , Trastorno del Espectro Autista/fisiopatología , Cognición , Lenguaje , Conducta Social , Vocabulario , Adulto , Anciano , Femenino , Humanos , Japón , Masculino , Persona de Mediana EdadRESUMEN
We developed a modified nonradioactive method for the detection of DNA. This method makes use of the polymerase chain reaction for preparation of probes; that is, a DNA fragment inserted in the polylinker region of an M13 or pUC vector is amplified with primers that have a modified cytosine tail at the 5' terminus (C-tailed primers). By this method, large amounts of labeled probes can be obtained easily. After hybridization, modified cytosine tails can be detected immunologically. DNA labeled by this method could be used in plaque hybridization. We could detect 0.05 pg of dot-blotted labeled DNA in 30 min with an enzyme-catalyzed chemiluminescence reaction.
Asunto(s)
Citosina/análogos & derivados , Sondas de ADN/metabolismo , ADN/análisis , Inmunoensayo/métodos , Secuencia de Bases , Vectores Genéticos/genética , Mediciones Luminiscentes , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Oligodesoxirribonucleótidos/metabolismo , Plásmidos/genética , Poli C/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
The COOH-termini of the 32 kDa D1 and 44 kDa CPa-2 were determined by protein sequencing of peptides from trypsinized photosystem II core complexes. COOH-terminal fragments were isolated by affinity chromatography using anhydrotrypsin-agarose. One peptide had a sequence corresponding to the segment from Asn at position 335 to Ala at position 344 of the sequence deduced from the psbA gene coding for D1. Nine amino acids may be cleaved from the COOH-terminus of pre-D1 during maturation. In contrast, CPa-2 was not modified at its COOH-terminus.
Asunto(s)
Clorofila , Proteínas de Plantas , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Complejos de Proteína Captadores de Luz , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/análisis , Proteínas del Complejo del Centro de Reacción Fotosintética , Complejo de Proteína del Fotosistema II , PlantasRESUMEN
Molecular cloning and nucleotide sequence analysis were performed for the identification of the regulator genes of methicillin resistance in the genome of a MRSA strain N315. Two open reading frames (orfs) were identified in the 5'-flanking region of the mecA gene. Predicted amino acid sequences of these orfs showed extensive homology to the co-inducer and the repressor protein of the penicillinase (PCase) production in Staphylococcus aureus as well as in Bacillus licheniformis. These orfs are considered to encode putative co-inducer and repressor proteins specific for the regulation of methicillin resistance in MRSA.
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Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Genes Bacterianos , Genes Reguladores , Hexosiltransferasas , Resistencia a la Meticilina/genética , Muramoilpentapéptido Carboxipeptidasa/genética , Peptidil Transferasas , Staphylococcus aureus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas de Unión a las Penicilinas , Mapeo Restrictivo , Alineación de SecuenciaRESUMEN
We have found that a boxA-like sequence is conserved in the 16 S and 23 S rRNA intergenic spacer regions of mycoplasmas, and that it always locates on loop regions of the hypothetical secondary stem-loop structures. A nucleotide sequence similar to the '-10' box of prokaryotic promoters was identified at upstream sites of the boxA-like sequence in the 16 S/23 S spacer regions. These structures may represent an internal promoter between the 16 S and 23 S rRNA genes in mycoplasmas.
Asunto(s)
Mycoplasma/genética , Operón , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Bases , ADN Bacteriano , ADN Ribosómico/química , ADN Ribosómico/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Bacteriano/genéticaRESUMEN
After a brief exposure to supra-saturating light, leaves of a tobacco transformant, in which chloroplastic NAD(P)H dehydrogenase (NDH) was defective, showed more severe photoinhibition than the wild-type, when judged by the parameter of chlorophyll fluorescence Fv/Fm. Repeated application of supra-saturating light eventually resulted in chlorosis in the NDH-defective mutant, while the wild-type sustained less photodamage and was able to recover from it. The mechanism of the phenomena is discussed with respect to the potential role of NDH in photosynthesis.
Asunto(s)
Cloroplastos/enzimología , Luz/efectos adversos , NADPH Deshidrogenasa/fisiología , Ferredoxina-NADP Reductasa/metabolismo , Complejos de Proteína Captadores de Luz , Mutagénesis Insercional , Oxidación-Reducción , Fotosíntesis/fisiología , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteínas de Plantas/fisiología , Plantas Tóxicas , Factores de Tiempo , Nicotiana/enzimología , Nicotiana/efectos de la radiación , Transformación GenéticaRESUMEN
A simple and reliable method is described for direct sequencing of material generated by the polymerase chain reaction. The protocol is based on the purification of the amplified double-stranded product by polyethylene glycol precipitation, annealing of primer with template by a "snap-cooling" procedure and sequencing by the dideoxy chain termination method with the use of Klenow fragment or Taq polymerase. The limit of the size of PCR products that can be sequenced is also discussed.
Asunto(s)
ADN/genética , Amplificación de Genes , Mapeo Nucleótido/métodos , Reacción en Cadena de la Polimerasa , Secuencia de Bases , ADN/síntesis química , ADN Polimerasa Dirigida por ADN , Datos de Secuencia Molecular , Polimerasa Taq , Moldes GenéticosRESUMEN
Although most humans have been exposed to wild-type adenoviruses in their childhood, titers of neutralizing antibodies against viruses decrease with the passage of time. In the present study, we infused adenoviruses carrying the lacZ gene into the tail vein of rats, and re-infused the same adenoviruses long after the initial administration. However, development of neutralizing antibodies against adenovirus and proliferation of adenovirus-specific T cells were elicited profoundly by adenoviral readministration, and transgene expression was not induced in rats. Our results may have important implications for efficacy considerations when adenoviral vectors are employed in clinical settings for the treatment of cancer.
Asunto(s)
Adenoviridae/genética , Vectores Genéticos/administración & dosificación , Operón Lac , Hígado/metabolismo , Animales , Formación de Anticuerpos/inmunología , Femenino , Expresión Génica , Terapia Genética/métodos , Inyecciones Intravenosas , Operón Lac/fisiología , Vena Porta , Ratas , Ratas Sprague-Dawley , Linfocitos T/inmunologíaRESUMEN
OBJECTIVES: The great saphenous vein graft is known to be less patent than the internal thoracic artery graft. Recently, we reported that chymase-dependent angiotensin II formation plays an important role in the development of intimal hyperplasia in dog grafted veins. In this study we investigated the levels of angiotensin II-forming enzymes, angiotensin-converting enzyme, and chymase in human saphenous veins and internal thoracic arteries. METHODS: The saphenous vein and internal thoracic artery specimens were obtained from coronary artery bypass grafts of patients during surgical procedures (saphenous vein, n = 16; internal thoracic artery, n = 16). Activities of angiotensin-converting enzyme and chymase were determined by using the extract from the saphenous vein or internal thoracic artery. Sections of the saphenous vein or internal thoracic artery were stained with van Gieson's elastin stain and were immunostained with anti-human chymase antibody. RESULTS: The activities of angiotensin-converting enzyme in the saphenous vein and internal thoracic artery were 0.34 +/- 0.12 and 0.32 +/- 0.17 mU/mg protein, respectively, and the difference was not significant. The chymase activity in the saphenous vein was significantly higher than that in the internal thoracic artery (saphenous vein, 10.1 +/- 0.81 mU/mg protein; internal thoracic artery, 6.21 +/- 1.86 mU/mg protein). Chymase-positive cells in the saphenous vein were located in both the media and adventitia, and those in the internal thoracic artery were located only in the adventitia. The number of chymase-positive cells in the saphenous vein was about 2.6 times that in the internal thoracic artery. CONCLUSION: The chymase activity, but not the angiotensin-converting enzyme activity, was significantly higher in the saphenous vein, suggesting that the high levels of chymase activity may be related to the poorer performance of the saphenous vein for use as a bypass conduit.
Asunto(s)
Angiotensina II/metabolismo , Puente de Arteria Coronaria , Arterias Mamarias/enzimología , Vena Safena/enzimología , Serina Endopeptidasas/metabolismo , Anciano , Anciano de 80 o más Años , Angiotensina II/inmunología , Anticuerpos Monoclonales/análisis , Quimasas , Puente de Arteria Coronaria/métodos , Enfermedad Coronaria/cirugía , Femenino , Humanos , Inmunoglobulina G/inmunología , Masculino , Arterias Mamarias/citología , Arterias Mamarias/trasplante , Persona de Mediana Edad , Peptidil-Dipeptidasa A/metabolismo , Vena Safena/citología , Vena Safena/trasplante , Serina Endopeptidasas/inmunologíaRESUMEN
Pulse-generation of O2- by a flash was used to determine the reactivity of O2-, O2- was produced within 10 ms by a flash of light through the excitation of FMN in the presence of N,N,N',N'-tetramethylethylenediamine and oxygen. Kinetic analysis of cytochrome c reduction by O2- generated by flash yielded the reaction rate constant between cytochrome c and O2- and the spontaneous disproportionation rate constant of O2-. We applied it for superoxide dismutase assay using a linear relation between superoxide dismutase concentration and the apparent rate constant of cytochrome c reduction by O2-. The catalytic rate constant and activation energy at pH 7.3 of bovine liver Cu,Zn-superoxide dismutase were found to be 1.75 x 10(9) M-1 . s-1 at 25 degrees C and 26.9 kJ . M-1, respectively. The kinetics of O2- decay can be also monitored at 240 nm in this flash-photometric system and gave the spontaneous disproportionation rate constant of O2- and the catalytic rate constant of superoxide dismutase.
Asunto(s)
Oxígeno/análisis , Superóxido Dismutasa/metabolismo , Superóxidos/análisis , Animales , Bovinos , Grupo Citocromo c/metabolismo , Mononucleótido de Flavina , Caballos , Cinética , Luz , Hígado/enzimología , Miocardio , Fotólisis , Espectrofotometría/métodosRESUMEN
Ketoacid oxidation in rat liver mitochondria was very sensitive to t-butyl hydroperoxide (t-BuOOH). Furthermore, 2-oxoglutarate and pyruvate each enhanced t-BuOOH-induced oxidative stresses of mitochondria, such as oxidation of pyridine nucleotides and GSH, inhibition of respiration with the other NAD-linked substrates, and peroxidation of mitochondrial lipids. We provide evidence that the t-BuOOH and ketoacid-induced effects are due to the failure of supply of NADH by 2-oxoglutarate dehydrogenase, and report the inactivation of the dehydrogenase in mitochondria by simultaneous addition of 2-oxoglutarate and t-BuOOH. Using the purified enzyme, we confirmed that t-BuOOH-induced inactivation of 2-oxoglutarate dehydrogenase was enhanced by its substrate and thiamine pyrophosphate protected the dehydrogenase from the inactivation. In contrast, succinate-dependent oxidation of mitochondria was not only scarcely affected by t-BuOOH, but also succinate protected against inactivation of 2-oxoglutarate dehydrogenase by t-BuOOH in mitochondria.
Asunto(s)
Complejo Cetoglutarato Deshidrogenasa/antagonistas & inhibidores , Ácidos Cetoglutáricos/farmacología , Cetona Oxidorreductasas/antagonistas & inhibidores , Mitocondrias Hepáticas/enzimología , Peróxidos/farmacología , Animales , Femenino , Glutatión/metabolismo , Peróxidos Lipídicos/biosíntesis , NAD/metabolismo , NADP/metabolismo , Oxidación-Reducción , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Endogámicas , terc-ButilhidroperóxidoRESUMEN
After Tris(hydroxymethyl)aminomethane[Tris buffer]-treatment cross-linking of the chloroplast thylakoid peptides of 11, 13, 18, 43, 55, and 87 kdaltons was observed on SDS-polyacrylamide gel electrophoresis. Tris-induced disulfide formation was suggested by the decrease of thiol groups of the chloroplast thylakoids in Tris medium at pHs above 8. In addition to the finding that Tris coordinates to Cu2+ in the forms of Tris-Cu2+ and Tris2-Cu2+ whose successive stability constants are 5.78 x 10(3) M-1 and 4.46 x 10(6) M-2, respectively, it was observed that Tris-Cu2+ complexes catalyze cysteine oxidation. Sinc release of copper from the chloroplast thylakoids is enhanced by increasing the concentration of Tris, oxidation of thiols by Tris-Cu2+ complexes formed in chloroplast thylakoids is a possible mechanism for the cross-linking of chloroplast thylakoid peptides.
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Cobre , Reactivos de Enlaces Cruzados , Proteínas de la Membrana , Trometamina , Cloroplastos , Disulfuros , Peso Molecular , PlantasRESUMEN
To reveal the molecular orientation of plastocyanin (PC) on spinach thylakoid membranes, the position of Lys residues modified by acetic anhydride was compared between thylakoid-bound PC and the isolated one. Digestion of the isolated PC by a trypsin yielded a peptide map with seven spots prior to the acetylation of the protein; none of the spots appeared after the isolated PC was acetylated. On the other hand, there were two spots on the peptide map of the PC acetylated when it was bound to the thylakoids. Those spots were revealed by their amino acid compositions to correspond to the peptide fragments Phe 82-Lys 95 and Val 96-Asn 99. Thus, the Lys residues 81 and 95 of the thylakoid-bound PC were not acetylated. These results suggest that the PC molecule binds to the thylakoids with a specific region including the Lys's 81 and 95 in contact with the membranes. The Lys's 81 and 95 are located near Tyr 83, which has been thought to be the delivery site of electrons from the Cu2+ center.
Asunto(s)
Lisina/análisis , Proteínas de la Membrana/análisis , Proteínas de Plantas/análisis , Plastocianina/análisis , Acetilación , Secuencia de Aminoácidos , Membranas/análisis , Plantas/análisis , Plantas/ultraestructura , Unión Proteica , Conformación Proteica , Propiedades de SuperficieRESUMEN
The pol gene of the thermophilic eubacterium Bacillus caldotenax was cloned in a plasmid and expressed in Escherichia coli. The PCR method was used to clone the gene with no previous knowledge of the gene or protein sequence. The 3,329-bp DNA fragment containing the structural gene for DNA polymerase was sequenced. DNA polymerase, as deduced from the DNA sequence, consisted of 877 amino acids, had a molecular weight of 99,452, and was structurally homologous to the DNA polymerases of the Pol I family (family A), which includes E. coli DNA polymerase I and T7 DNA polymerase. B. caldotenax DNA polymerase (Bca polymerase) purified from the recombinant E. coli strain was characterized. Like E. coli Pol I, Bca polymerase had 5'-->3' exonuclease activity. The degraded product with the molecular weight of 65,000 was also purified and found to have polymerase activity. To overproduce this Klenow-type fragment of Bca polymerase, a recombinant expression plasmid pUI205 with a deletion in the 5'-region of the pol structural gene was constructed. The DNA polymerase produced by pUI205 is more suitable for use in the dideoxy sequencing method than the other DNA polymerases that have been used for sequencing.