RESUMO
PURPOSE: Antithrombotic therapy could trigger diffuse alveolar hemorrhage (DAH), and there are several case reports of DAH that occurred during antithrombotic therapy (DAH-AT). However, little is known about the clinical features and outcomes of DAH-AT. The purpose of this study was to clarify the features and mortality of DAH-AT. METHODS: 76 consecutive patients with DAH who were admitted to our hospital between January 2003 and April 2014 were retrospectively reviewed to identify the clinical features and outcomes of DAH-AT. The primary outcome was 90-day mortality. RESULTS: Of the 76 patients with DAH, 39 patients (51 %) had DAH-AT, and 37 patients (49 %) had DAH that occurred with no antithrombotic therapy (DAH-NAT). Of the patients with DAH-AT, 25 (64 %) were taking aspirin, 14 (36 %) were taking warfarin, 5 (13 %) were taking clopidogrel sulfate, and 4 (10 %) were taking cilostazol. Pre-existing cardiac disease was present in 23 (59 %) DAH-AT cases and 5 (14 %) DAH-NAT cases. Logistic regression analysis was used to assess the effect of antithrombotic therapy on the mortality of DAH patients, and no significant difference in survival was seen with antithrombotic therapy (OR 1.18, 95 % CI 0.38-3.78). CONCLUSIONS: Antithrombotic therapies had no effect on the 90-day mortality of DAH patients.
Assuntos
Fibrinolíticos/efeitos adversos , Hemorragia/etiologia , Pneumopatias/etiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Aspirina/efeitos adversos , Cilostazol , Clopidogrel , Doenças do Tecido Conjuntivo/complicações , Feminino , Insuficiência Cardíaca/complicações , Hemorragia/complicações , Humanos , Infecções/etiologia , Pneumopatias/complicações , Masculino , Pessoa de Meia-Idade , Neoplasias/complicações , Pneumonia/complicações , Alvéolos Pulmonares , Estudos Retrospectivos , Taxa de Sobrevida , Tetrazóis/efeitos adversos , Ticlopidina/efeitos adversos , Ticlopidina/análogos & derivados , Vasculite/complicações , Varfarina/efeitos adversosRESUMO
Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10(6) all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200,000) can leave the capsid and be replaced by water molecules from the outside in about 25 µs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.
Assuntos
Capsídeo/química , Simulação de Dinâmica Molecular , Poliovirus , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Pressão , Conformação Proteica , RNA Viral/metabolismo , Soluções , Água/químicaRESUMO
BACKGROUND: Hepatic ischaemia-reperfusion (IR) injury may lead to liver damage during liver surgery, and intrahepatic nitric oxide (NO) levels may play a role in this context. The aim of this study was to demonstrate real-time changes in intrahepatic NO concentration during IR and to correlate potential hepatic NO production with liver damage using a selective NO sensor. METHODS: Wistar rats were exposed to 15 min of hepatic ischaemia followed by reperfusion, after which changes in intrahepatic NO levels were measured using an NO sensor. Additionally, rats were exposed to five successive periods of IR, each consisting of 15 min ischaemia followed by 5 or 15 min reperfusion, and hepatic damage was evaluated by blood tests and histological examination. Hepatic expression of Akt, phosphorylated Akt, endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS was examined at different time points during and after IR by western blot and immunohistochemical analysis. RESULTS: During ischaemia, intrahepatic NO levels increased and reached a plateau at approximately 10 min. Repeated 15 min ischaemia-5 min reperfusion cycles reduced the maximum amount of NO produced during ischaemia gradually, and almost no NO production was observed during the fifth period of ischaemia. NO production following repeated ischaemia was proportional to the degree of hepatic viability. Phosphorylated eNOS was upregulated and correlated with the level of NO production during hepatic ischaemia. CONCLUSION: Intrahepatic NO levels decrease during repeated IR in rats. Real-time monitoring of intrahepatic NO levels is useful for the prediction of IR-related liver injury during experimental liver surgery.
Assuntos
Isquemia/metabolismo , Fígado/irrigação sanguínea , Óxido Nítrico Sintase Tipo III/metabolismo , Traumatismo por Reperfusão/diagnóstico , Animais , Western Blotting , Constrição , Inibidores Enzimáticos/farmacologia , Imuno-Histoquímica , Lisina/análogos & derivados , Lisina/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
Attached growth reactors were developed separately for solids retention time (SRT)-controlled partial nitrification and for anaerobic ammonia oxidation (Anammox) treatment, and a new nitrogen removal process is proposed for wastewater containing highly concentrated ammonia. For partial nitrification, an attached growth medium of polyurethane foam was used. Partial nitrification was achieved stably under a SRT of 4 days, and the abundance ratio of NO2(-)-N to the sum of NH4(+)-N, NO2(-)-N and NO3(-)-N was approximately 0.8 after 10 days. Under a SRT of4 days, the amoA gene concentrations of ammonia-oxidizing bacteria increased from 1 x 10(8) to 7 x 10(8) copies/l, whereas the 16S ribosomal RNA (16S rRNA) gene concentrations of nitrite-oxidizing bacteria did not increase. These results indicate that SRT-controlled operation is a promising technology for achieving partial nitrification. For the Anammox treatment, an attached growth medium of non-woven fabric was used. Inorganic nitrogen removal of approximately 80-90% was observed at an inorganic nitrogen loading rate of over 10 kgN/(m3-medium.d) and an influent nitrogen concentration of 400 mgN/l. Our non-woven fabric reactor showed similar or superior Anammox performance to that reported previously. By using a combination of these two rectors, we can develop a method that combines partial nitrification and Anammox treatment for effective and stable nitrogen removal.
Assuntos
Amônia/isolamento & purificação , Reatores Biológicos , Eliminação de Resíduos Líquidos/métodos , Bactérias/genética , Bactérias/metabolismo , Reatores Biológicos/microbiologia , Desenho de Equipamento , Nitratos/metabolismo , Nitrificação , Nitrogênio , Poliuretanos , RNA Ribossômico 16S , Eliminação de Resíduos Líquidos/instrumentaçãoRESUMO
Fluvoxamine, one of the oldest selective serotonin reuptaking inhibitors, is commonly prescribed to patients with major depression. Several studies have reviewed the efficacy and tolerability of fluvoxamine for the treatment of major depression. However, these reviews are outdated, have not been systematic and/or suffered from several methodological weaknesses. We conducted a systematic review to synthesize the best available evidence on the efficacy of fluvoxamine for adult patients suffering from major depression in comparison with other active antidepressive agents. Relevant randomized controlled trials were identified through a comprehensive search. The primary outcome was a relative risk of response, and the secondary outcome was a relative risk of remission. Tolerability and side-effect profile were also examined. Fifty-three trials were included. There were no large differences between fluvoxamine and any other antidepressants in terms of efficacy and tolerability. There is evidence of differing side effect profiles, especially when comparing gastrointestinal side effects between fluvoxamine and tricyclics. Clinicians should focus on practically or clinically relevant differences including those in side-effect profiles.
Assuntos
Antidepressivos de Segunda Geração/uso terapêutico , Transtorno Depressivo Maior/tratamento farmacológico , Fluvoxamina/uso terapêutico , Adulto , Antidepressivos de Segunda Geração/efeitos adversos , Fluvoxamina/efeitos adversos , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do TratamentoRESUMO
BACKGROUND: The basic and clinical implications of evaluating plasma atrial natriuretic peptide (ANP) concentration in calves are unknown. OBJECTIVE: To investigate the relationship between the plasma ANP concentration and left ventricular end-diastolic pressure (LVEDP) in healthy calves subjected to volume overload (Study 1), and to compare the plasma ANP concentration in calves with or without heart disease (Study 2). ANIMALS: Six healthy calves were used in Study 1; disease calves and sick calves with (n = 9) and without congenital heart disease (CHD) (n = 9) were used in Study 2. METHODS: In Study 1, LVEDP in anesthetized calves was manipulated by IV administration of acetated Ringer's solution (rate of 100 mL/kg/h for 20 minutes) and furosemide. In Study 2, disease calves were identified by blood examination and echocardiography or pathological examination. The plasma ANP concentration was determined by a chemiluminescence enzyme immunoassay for human alpha-ANP. RESULTS: In Study 1, preloading significantly increased the plasma ANP concentration (36 +/- 20-185 +/- 156, P < .01) and LVEDP (-11 +/- 7-2 +/- 12, P < .01) from the baseline. Furthermore, plasma ANP concentrations were strongly correlated with LVEDP (r= 0.61). In Study 2, the plasma ANP concentration was significantly higher in the calves with CHD than in the calves without heart disease (220 [67-970] versus 31 [10-86]; mean [range], P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Measurement of plasma ANP concentrations in calves can provide additional information useful for predicting hemodynamic abnormalities.
Assuntos
Fator Natriurético Atrial/sangue , Doenças dos Bovinos/congênito , Cardiopatias Congênitas/veterinária , Animais , Bovinos , Doenças dos Bovinos/sangue , Feminino , Cardiopatias Congênitas/sangue , MasculinoRESUMO
Neonatal hepatitis, a syndrome occurring in children, has various etiologies, such as viral infection, unidentified disorders of bile salt synthesis, and other poorly understood metabolic diseases. It is characterized by jaundice, giant cell hepatitis, and, rarely, liver failure necessitating liver transplantation. We experienced 3 cases of idiopathic neonatal hepatitis with unusual progressive fibrosis presenting with retrograde portal flow and portal-systemic shunt. Clinical manifestations were hyperammonemia, hyperbilirubinemia, and coagulopathy. Characteristic histological findings were giant cell transformation of hepatocytes and progressive severe fibrosis. Two patients underwent living donor liver transplantation. We consider that liver transplantation is indicated in cases of neonatal hepatitis with hepatofugal portal flow and collateral vein formation.
Assuntos
Circulação Colateral/fisiologia , Hepatite/fisiopatologia , Sistema Porta/fisiologia , Evolução Fatal , Humanos , Lactente , Transplante de Fígado , Masculino , Resultado do TratamentoRESUMO
Sagittal application of a titanium mini screw in the coronoid process at the time of coronoidotomy is a very efficient method for easy removal.
Assuntos
Parafusos Ósseos , Mandíbula/cirurgia , Osteotomia/métodos , Adulto , Fios Ortopédicos , Feminino , Humanos , Hiperplasia , Mandíbula/patologia , Músculo Masseter/patologia , Músculo Masseter/cirurgia , Osteotomia/instrumentação , Titânio , Trismo/cirurgiaRESUMO
After the chest wall resection, its reconstruction is often needed. A 45-year-old male lung adenocarcinoma patient with chest wall invasion underwent upper lobectomy of the right lung with partial resection of 4-6th ribs. The size of the removed chest wall was 11 x 6.5 cm. We reconstructed the chest wall with Bard Composix E/X Mesh. This prosthesis is consisted of a polypropylene mesh and an expanded polytetrafluoroethylene sheet This material is seems to be useful in the reconstruction of chest wall in both preventing pulmonary adhesion and enabling good wound healing.
Assuntos
Próteses e Implantes , Toracoplastia/instrumentação , Adenocarcinoma/cirurgia , Humanos , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Polipropilenos , PolitetrafluoretilenoRESUMO
BACKGROUND: Clinical testing to determine a suitable dose of linaclotide for Japanese patients with irritable bowel syndrome with constipation (IBS-C) was needed. METHODS: This was a randomized, double-blind, placebo-controlled, dose-finding trial. Japanese patients with IBS-C diagnosed using Rome III criteria (n = 559, men/women: 49/510) were randomly assigned to 1 of 4 linaclotide doses (0.0625, 0.125, 0.25, or 0.5 mg) or placebo for the 12-week treatment period. The primary endpoint was responder rate of global assessment of relief of IBS symptoms during 12 weeks. The secondary endpoints included responder rates of complete spontaneous bowel movement (CSBM), SBM and abdominal pain/discomfort relief and others. KEY RESULTS: The primary endpoint was 23.2%, 36.2%, 38.7%, 34.8%, and 38.3% in placebo (n = 112), 0.0625 (n = 116), 0.125 (n = 111), 0.25 (n = 112), and 0.5 (n = 107) mg of linaclotide groups with the difference from the placebo group in each linaclotide group (13.0%, 15.5%, 11.6%, 15.1%, P > .05). Monthly responder rate of global assessment of relief of IBS symptoms at month 3 (48.6%), responder rate of CSBM during 12 weeks (45.8%), and responder rate of abdominal pain/discomfort relief during 12 weeks (32.7%) in the 0.5 mg were significantly higher than those in placebo group (29.5%, P < .01; 25.9%, P < .01; and 18.8%, P < .05 respectively). The most frequent adverse event in the linaclotide groups was diarrhea. CONCLUSIONS & INFERENCES: This study suggests that a linaclotide dose of 0.5 mg may be appropriate in Japanese patients with IBS-C.
Assuntos
Dor Abdominal/tratamento farmacológico , Constipação Intestinal/tratamento farmacológico , Fármacos Gastrointestinais/administração & dosagem , Agonistas da Guanilil Ciclase C/administração & dosagem , Síndrome do Intestino Irritável/tratamento farmacológico , Peptídeos/administração & dosagem , Adulto , Defecação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Fármacos Gastrointestinais/uso terapêutico , Agonistas da Guanilil Ciclase C/uso terapêutico , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Peptídeos/uso terapêutico , Resultado do TratamentoRESUMO
Pod setting rate in soybean is an important trait that determines pod number, which is highly correlated with seed yield. Using two soybean cultivars with different pod setting rates, we examined the relationship between plant growth regulation by gibberellin (GA) and pod setting rate. Plant growth rate (PGR) after flowering was significantly higher in 'Fukuyutaka' (low pod setting rate) than in 'Kariyutaka' (high pod setting rate); this difference was caused by increasing of GA biosynthesis-related genes expression. Additionally, pod setting rate in 'Fukuyutaka' was lower than that in 'Kariyutaka'. Furthermore, when 'Kariyutaka' was treated with GA after flowering, the PGR increased and pod setting rate decreased. These results suggest that pod setting rate in soybean is regulated by vegetative growth after flowering through GA biosynthesis.
Assuntos
Giberelinas/biossíntese , Giberelinas/metabolismo , Glycine max/metabolismo , Reguladores de Crescimento de Plantas/biossíntese , Reguladores de Crescimento de Plantas/metabolismoRESUMO
BACKGROUND: The ribosome is a ribonucleoprotein complex which performs the crucial function of protein biosynthesis. Its role is to decode mRNAs within the cell and to synthesize the corresponding proteins. Ribosomal protein S7 is located at the head of the small (30S) subunit of the ribosome and faces into the decoding centre. S7 is one of the primary 16S rRNA-binding proteins responsible for initiating the assembly of the head of the 30S subunit. In addition, S7 has been shown to be the major protein component to cross-link with tRNA molecules bound at both the aminoacyl-tRNA (A) and peptidyl-tRNA (P) sites of the ribosome. The ribosomal protein S7 clearly plays an important role in ribosome function. It was hoped that an atomic-resolution structure of this protein would aid our understanding of ribosomal mechanisms. RESULTS: The structure of ribosomal protein S7 from Bacillus stearothermophilus has been solved at 2.5 A resolution using multiwavelength anomalous diffraction and selenomethionyl-substituted proteins. The molecule consists of a helical hydrophobic core domain and a beta-ribbon arm extending from the hydrophobic core. The helical core domain is composed of a pair of entangled helix-turn-helix motifs; the fold of the core is similar to that of a DNA architectural factor. Highly conserved basic and aromatic residues are clustered on one face of the S7 molecule and create a 16S rRNA contact surface. CONCLUSIONS: The molecular structure of S7, together with the results of previous cross-linking experiments, suggest how this ribosomal protein binds to the 3' major domain of 16S rRNA and mediates the folding of 16S rRNA to create the ribosome decoding centre.
Assuntos
Geobacillus stearothermophilus/química , Proteínas Ribossômicas/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Volta-Hélice , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Alinhamento de SequênciaRESUMO
BACKGROUND: The enzyme methylmalonyl-coenzyme A (CoA) mutase, an alphabeta heterodimer of 150 kDa, is a member of a class of enzymes that uses coenzyme B12 (adenosylcobalamin) as a cofactor. The enzyme induces the formation of an adenosyl radical from the cofactor. This radical then initiates a free-radical rearrangement of its substrate, succinyl-CoA, to methylmalonyl-CoA. RESULTS: Reported here is the crystal structure at 2 A resolution of methylmalonyl-CoA mutase from Propionibacterium shermanii in complex with coenzyme B12 and with the partial substrate desulpho-CoA (lacking the succinyl group and the sulphur atom of the substrate). The coenzyme is bound by a domain which shares a similar fold to those of flavodoxin and the B12-binding domain of methylcobalamin-dependent methionine synthase. The cobalt atom is coordinated, via a long bond, to a histidine from the protein. The partial substrate is bound along the axis of a (beta/alpha)8 TIM barrel domain. CONCLUSIONS: The histidine-cobalt distance is very long (2.5 A compared with 1.95-2.2 A in free cobalamins), suggesting that the enzyme positions the histidine in order to weaken the metal-carbon bond of the cofactor and favour the formation of the initial radical species. The active site is deeply buried, and the only access to it is through a narrow tunnel along the axis of the TIM barrel domain.
Assuntos
Cobamidas/metabolismo , Metilmalonil-CoA Mutase/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Radicais Livres/metabolismo , Ligantes , Modelos Moleculares , Propionibacterium/enzimologia , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
Background and aims: Conventional water jet devices have been used for injecting fluid to lift up lesions during endoscopic submucosal dissection or endoscopic mucosal resection procedures. However, these devices cannot dissect the submucosal layer effectively. Here we aim to elucidate the dissection capability of a laser-induced pulsed water jet and to clarify the mechanism of dissection with layer selectivity. Materials (Subjects) and methods: Pulsed water jets were ejected from a stainless nozzle by accelerating saline using the energy of a pulsed holmium: yttrium-aluminum-garnet laser. The impact force (strength) of the jet was evaluated using a force meter. Injection of the pulsed jet into the submucosal layer was documented by high-speed imaging. The physical properties of the swine esophagus were evaluated by measuring the breaking strength. Submucosal dissection of the swine esophagus was performed and the resection bed was evaluated histologically. Results: Submucosal dissection of the esophagus was accomplished at an impact force of 1.11-1.47 N/pulse (laser energy: 1.1-1.5 J/pulse; standoff distance: 60 mm). Histological specimens showed clear dissection at the submucosal layer without thermal injury. The mean static breaking strength of the submucosa (0.11 ± 0.04 MPa) was significantly lower than that of the mucosa (1.32 ± 0.18 MPa), and propria muscle (1.45 ± 0.16 MPa). Conclusions: The pulsed water jet device showed potential for achieving selective submucosal dissection. It could achieve mucosal, submucosal, and muscle layer selectivity owing to the varied breaking strengths.
RESUMO
The ribonuclease MC1 (RNase MC1) from seeds of bitter gourd (Momordica charantia) consists of 190 amino acid residues with four disulfide bridges and belongs to the RNase T(2) family, including fungal RNases typified by RNase Rh from Rhizopus niveus and RNase T(2) from Aspergillus oryzae. The crystal structure of RNase MC1 has been determined at 1.75 A resolution with an R-factor of 19.7% using the single isomorphous replacement method. RNase MC1 structurally belongs to the (alpha+beta) class of proteins, having ten helices (six alpha-helices and four 3(10)-helices) and eight beta-strands. When the structures of RNase MC1 and RNase Rh are superposed, the close agreement between the alpha-carbon positions for the total structure is obvious: the root mean square deviations calculated only for structurally related 151 alpha-carbon atoms of RNase MC1 and RNase Rh molecules was 1.76 A. Furthermore, the conformation of the catalytic residues His-46, Glu-105, and His-109 in RNase Rh can be easily superposed with that of the possible catalytic residues His-34, Glu-84, and His-88 in RNase MC1. This observation strongly indicates that RNase MC1 from a plant origin catalyzes RNA degradation in a similar manner as fungal RNases.
Assuntos
Cucurbitaceae/enzimologia , Ribonucleases/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia , Endorribonucleases/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ribonucleases/isolamento & purificação , Sementes/enzimologiaRESUMO
Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na+ + K+)-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45-80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles.
Assuntos
ATPase Trocadora de Sódio-Potássio/imunologia , 4-Nitrofenilfosfatase/metabolismo , Animais , Reações Antígeno-Anticorpo , Transporte Biológico Ativo , Membrana Eritrocítica/metabolismo , Cavalos , Humanos , Imunodifusão , Técnicas In Vitro , Rim/enzimologia , Ouabaína/farmacologia , Rubídio/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidoresRESUMO
Klebsiella pullulanase is a lipoprotein synthesized as a precursor with a signal peptide, which is processed by lipoprotein signal peptidase. To clarify the role of lipid modification of pullulanase, we purified lipid-modified wild-type and the unmodified (mutant) pullulanases and compared their properties. The Km and Vmax values of both pullulanases for pullulan were the same. The optimal pH and temperature, the stabilities over pH and temperature ranges, the specificity of substrates, and the patterns of inhibition of the lipid-modified and unmodified pullulanases were also the same. However, we found that the wild-type pullulanase formed trimers whereas the unmodified enzyme did not, and that the migrations of the two enzymes on sodium dodecyl sulphate/electrophoresis were different when the samples were applied on the gel without heating. The results presented in this paper and in previous work show that the correct processing and translocation of pullulanase in K. aerogenes require modification of lipid. However, the enzymatic properties and physical stabilities of pullulanase were not affected by the lipid modification.
RESUMO
The structures of deoxy (alpha(Mg(II))2 beta(Fe(II))2) and CO-liganded (alpha(Mg(II)2(Fe(II)-CO)2) forms of human hemoglobin were determined by X-ray crystallography to a resolution of 1.7 A and 1.9 A, respectively. The deoxy hybrid has virtually the same structure as that of the native deoxy HbA. Both the deoxy and CO-liganded hybrids assumed a T quaternary structure characteristic of native deoxy HbA. No significant structural difference was found between the alpha subunits of the CO-liganded hybrid and deoxy HbA, while in the beta subunit, significant tertiary structural changes were confined to the heme pocket. Baldwin showed by a comparison of COHbA and deoxy HbA that there is a 1.5 A shift of the beta subunit heme into its pocket. This shift was much reduced in alpha(Mg(II))2 beta(Fe(II)-CO)2. On the other hand, when the two structures are compared with superposition of hemes, the nearest neighbors of CO (Fe, E11 Val and E7 His) have shifted nearly to the same positions as those in COHbA. Thus the tertiary structure of the beta subunit of alpha(Mg(II))2 beta(Fe(II)-CO)2 is such that the CO molecule and the neighboring atoms assume nearly the same conformation as those of COHbA, while the block shift of these groups is impeded with respect to the structural invariant portions of the molecule.
Assuntos
Carboxihemoglobina/química , Hemoglobina A/química , Hemoglobinas/química , Conformação Proteica , Sequência de Aminoácidos , Cristalografia por Raios X , Hemoglobinas/isolamento & purificação , Humanos , Ligação de Hidrogênio , Ligantes , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase distinctly different in various properties from the family of pepsin-type aspartic proteinases, and so far it remains unknown which residues participate in the catalysis of the enzyme and how the mechanism operates. The acid proteinase A was crystallized from an ammonium sulfate solution by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1) with unit cell dimensions of a = 54.7 A, b = 70.4 A and c = 38.0 A. On the assumption that there is one enzyme molecule in the asymmetric unit, the calculated ratio of volume to unit protein mass (Vm) was 1.64 A3 per dalton. Diffraction data were collected up to a resolution higher than 1.5 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation. The crystal of proteinase A is, therefore, suitable for the structural analysis with a high resolution.