RESUMO
The fungus Thermothelomyces thermophilus is a thermotolerant microorganism that has been explored as a reservoir for enzymes (hydrolytic enzymes and oxidoreductases). The functional analysis of a recombinant cellobiose dehydrogenase (MtCDHB) from T. thermophilus demonstrated a thermophilic behavior, an optimal pH in alkaline conditions for inter-domain electron transfer, and catalytic activity on cellooligosaccharides with different degree of polymerization. Its applicability was evaluated to the sustainable production of cellobionic acid (CBA), a potential pharmaceutical and cosmetic ingredient rarely commercialized. Dissolving pulp was used as a disaccharide source for MtCDHB. Initially, recombinant exoglucanases (MtCBHI and MtCBHII) from T. thermophilus hydrolyzed the dissolving pulp, resulting in 87% cellobiose yield, which was subsequently converted into CBA by MtCDHB, achieving a 66% CBA yield after 24 h. These findings highlight the potential of MtCDHB as a novel approach to obtaining CBA through the bioconversion of a plant-based source.
Assuntos
Desidrogenases de Carboidrato , Proteínas Recombinantes , Desidrogenases de Carboidrato/metabolismo , Proteínas Recombinantes/metabolismo , Concentração de Íons de Hidrogênio , Dissacarídeos/biossíntese , Dissacarídeos/metabolismo , Temperatura , Celobiose/metabolismo , Sordariales/enzimologia , Hidrólise , Eurotiales/enzimologiaRESUMO
A new species of Synthesium from the bottlenose dolphin Tursiops truncatus in South Brazilian waters is described. Morphological and molecular identification was performed, and phylogenetic analyses were carried out using the ribosomal small subunit and internal transcribed spacer 1 and the mitochondrial NDH dehydrogenase subunit 3 and cytochrome c oxidase subunit 1 genes. The main characteristics of the new species are the subterminal round-shaped oral sucker, the anterior distribution of vitellaria reaching the level of the ovary and the oval-shaped testes. The results obtained with the molecular markers supported the inclusion of the specimens into the genus Synthesium. The nucleotide divergence detected for the mitochondrial genes among the new species and others of the same genus supported the erection of a new species. This is the ninth species assigned to the genus and the third Synthesium species recorded in the South Atlantic Ocean.
Assuntos
Golfinho Nariz-de-Garrafa/parasitologia , Trematódeos/classificação , Trematódeos/isolamento & purificação , Infecções por Trematódeos/veterinária , Animais , Oceano Atlântico , Brasil , Desidrogenases de Carboidrato/genética , DNA Intergênico/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Masculino , Filogenia , Subunidades Ribossômicas Menores/genética , Trematódeos/genética , Infecções por Trematódeos/parasitologiaRESUMO
Background: Surgery is an effective method for the management of renal hyperparathyroidism. Aim: To report the clinical presentation and results of surgical treatment of renal hyperparathyroidism. Material and Methods: Retrospective analysis of 58 patients aged 46 ± 11 years with secondary hyperparathyroidism (HPT2) and 13 patients aged 53 ± 11 years with tertiary hyperparathyroidism (HPT3), operated at a clinical hospital. Results: In 55 cases (77.4%) the indications for surgery were complications of excess parathyroid hormone (PTH) and in 16 patients (22.6%) a failure of medical treatment. Total parathyroidectomy with intraoperative measurement of PTH (PTHop) plus a forearm parathyroid autograft was performed in 54 (93.1%) patients with HPT2 and in all patients with HPT3. PTHop decreased ≥ 75% in 51 patients (88%) with HPT2 and in 9 patients (69.2%) with HPT3, respectively. Cure of the disease was achieved in 52 (89.7%) and 11 (84.6%) patients with HPT2 and 3, respectively. Median follow-up was 41 months. Five (9.6%) patients with HPT2 and two patients (18.2%) with HPT3 had a recurrence of the disease. Conclusions: In patients with renal hyperparathyroidism, the primary indication for surgery was the presence of complications of PTH excess. A drop in PTHop ≥ 75% from baseline predicts healing in 98% and 100% of cases with secondary or tertiary HPT respectively. Surgery was a safe and effective treatment in both groups.
Assuntos
Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Desidrogenases de Carboidrato/genética , Espessura Intima-Media Carotídea , Polimorfismo de Nucleotídeo Único , Sequência de Aminoácidos , Aterosclerose/genética , Saúde da Família , Frequência do Gene , Predisposição Genética para Doença/genética , Genótipo , Modelos Lineares , Dados de Sequência Molecular , Fatores de Risco , Homologia de Sequência de AminoácidosRESUMO
The effect of inoculation of Aspergillus flavus, Fusarium verticillioides, and Penicillium sp. in Dystrophic Red Latosol (DRL) and Eutroferric Red Latosol (ERL) soils with or without glucose on the total carbohydrate content and the dehydrogenase and amylase activities was studied. The fungal growth and spore production in culture medium with and without glucose were also evaluated. A completely randomized design with factorial arrangement was used. The addition of glucose in the culture medium increased the growth rate of A. flavus and Penicillium sp. but not of F. verticillioides. The number of spores increased 1.2 for F. verticillioides and 8.2 times for A. flavus in the medium with glucose, but was reduced 3.5 times for Penicillium sp. The total carbohydrates contents reduced significantly according to first and second degree equations. The consumption of total carbohydrates by A. flavus and Penicillium sp. was higher than the control or soil inoculated with F. verticillioides. The addition of glucose to soils benefited the use of carbohydrates, probably due to the stimulation of fungal growth. Dehydrogenase activity increased between 1.5 to 1.8 times (p <0.05) in soils with glucose and inoculated with the fungi (except F. verticillioides), in relation to soil without glucose. Amylase activity increased 1.3 to 1.5 times due to the addition of glucose in the soil. Increased amylase activity was observed in the DRL soil with glucose and inoculated with A. flavus and Penicillium sp. when compared to control.
Assuntos
Amilases/análise , Desidrogenases de Carboidrato/análise , Esporos Fúngicos/crescimento & desenvolvimento , Fungos Mitospóricos/crescimento & desenvolvimento , Ativação Enzimática , Métodos , MétodosRESUMO
AIMS: To isolate and characterize phosphate-solubilizing strains from a constrained environment such as the Salado River Basin and to assess their phosphate-solubilizing mechanisms, to further selection of the most promising strains to inoculate and improve the implantation and persistence of Lotus tenuis in the most important area devoted to meat-cow production in Argentina. METHODS AND RESULTS: Fifty isolates were obtained and through BOX-PCR analysis, 17 non-redundant strains were identified. Subsequently, they were found to be related to Pantoea, Erwinia, Pseudomonas, Rhizobium and Enterobacter genera, via 16S rRNA gene sequence analysis. This was in agreement with the clusters obtained by antibiotic resistance analysis. All isolates were tested for their phosphate-solubilizing activity and selected strains were inoculated onto L. tenuis plants. The most efficient isolate, was identified as Pantoea eucalypti, a novel species in terms of plant growth-promoting rhizobacteria. CONCLUSIONS: The isolates obtained in this study showed a significant in vitro plant-growth promoting activity onto Lotus tenuis and the best of them solubilizes phosphate mainly via induction of the metabolism through secretion and oxidation of gluconic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of these bacteria as bioinoculants, alone or in combination with nitrogen-fixing micro-organisms, could be a sustainable practice to facilitate the nutrient supply to Lotus tenuis plants and preventing negative side-effects such as eutrophication.
Assuntos
Lotus/crescimento & desenvolvimento , Lotus/microbiologia , Pantoea/fisiologia , Fosfatos/metabolismo , Rizosfera , Microbiologia do Solo , Argentina , Desidrogenases de Carboidrato/metabolismo , Enterobacter/genética , Erwinia/genética , Eucalyptus/metabolismo , Gluconatos/metabolismo , Glucose Desidrogenase/metabolismo , Oxirredução , Pantoea/genética , Pantoea/isolamento & purificação , Filogenia , Pseudomonas/genética , RNA Ribossômico 16S/genética , Rhizobium/genética , RiosRESUMO
White rot fungi (Ascomycota and Basidiomycota) were collected on fallen trunks with different decay stages, in a subandean forest (La Montaña del Ocaso nature reserve), and it was evaluated their ligninolitic activity. They were cultured on malt extract agar. Then it was performed semiquantitative tests for laccase and cellobiose dehydrogenase (CDH) activity using ABTS and DCPIP as enzymatic inducers. Based on the results of these tests, the fungi with higher activities from trunks with different decay stages were selected: Cookeina sulcipes (for stage 1), a fungus from the family Corticiaceae (for stage 2), Xylaria polymorpha (for stage 3) and Earliella sp. (for stage 4). A fermentation was performed at 28 degrees C, during 11 days, in a rotatory shaker at 150 rpm. Biomass, glucose, proteins and enzyme activities measurements were performed daily. The fungi that were in the trunks with decay states from 1 to 3, showed higher laccase activity as the state of decay increased. A higher DCH activity was also associated with a higher. Also, there was a positive relationship between both enzymes' activities. Erliella was the fungus which presented the highest biomass production (1140,19 g/l), laccase activity (157 UL(-1)) and CDH activity (43,50 UL(-1)). This work is the first report of laccase and CDH activity for Cookeina sulcipes and Earliella sp. Moreover, it gives basis for the use of these native fungi in biotechnological applications and the acknowledgment of their function in the wood decay process in native forest.
Assuntos
Desidrogenases de Carboidrato/análise , Proteínas Fúngicas/análise , Fungos/isolamento & purificação , Lacase/análise , Madeira/microbiologia , Ascomicetos/enzimologia , Ascomicetos/isolamento & purificação , Basidiomycota/enzimologia , Basidiomycota/isolamento & purificação , Biomassa , Colômbia , Fungos/enzimologia , Especificidade da EspécieRESUMO
Pfaffia glomerata (Spreng.) Pedersen, conhecida como ginseng brasileiro, é uma planta extensivamente usada na medicina popular em decorrência de possuir propriedades fitoterápicas. Este trabalho teve como objetivo determinar a melhor combinação de concentração da sacarose (30, 45 e 60g L-1) e do nitrogênio (50, 75, 100 e 125 por cento da concentração padrão do meio MS) para a multiplicação in vitro de P. glomerata. Aos 30 dias de cultivo, verificou-se que concentrações entre 40 e 45g L-1 de sacarose e 50 por cento de N propiciaram maior crescimento em altura e número de segmentos nodais por plântula. O número de brotações foi maior na concentração de 55g L-1 de sacarose combinada com 70 por cento de N. A matéria seca de raízes, da parte aérea e do total da plântula foi maior na concentração de 45g L-1 de sacarose associada com 50 por cento de N. No geral, a redução da concentração de N para 50 por cento daquela padrão do meio MS, associada a um incremento na dose de sacarose para 45g L-1, favorece o crescimento em altura, número de segmentos nodais e brotações, bem como a produção de biomassa de P. glomerata cultivada in vitro, devido ao estímulo ao uso do carbono.
Assuntos
Amaranthaceae , Desidrogenases de Carboidrato , Nitrogênio/administração & dosagem , Panax , SacaroseRESUMO
Aberrant expression and mutation of E-cadherin is frequent in gastric carcinoma (GC) especially of the diffuse type. The frequency of CDH1 (gene encoding E-cadherin) mutation in populations with high incidence of diffuse GC and its prognostic significance is unknown. One hundred seventy-seven gastrectomies from Mexican mestizo patients with intestinal (53), mixed (55), or diffuse (69) GC were included. In addition, 101 endoscopic biopsies from patients with GC not subjected to surgery were analyzed. Immunohistochemistry against wild-type E-cadherin (clone 36) and against 2 mutation-specific antibodies (MSA) recognizing mutant CDH1 lacking exon-8 (del 8) or exon-9 (del 9) were performed. Staining was correlated with histotype, tumor node metastasis stage, and follow-up. Abnormal or absent E-cadherin expression (clone 36) was identified in 84% GC, predominantly in diffuse or mixed tumors (P = 0.004) in advanced stages (P = 0.003). No survival differences at 1 and 2 years were observed among patients showing normal, abnormal, or absent wild type E-cadherin expression. Overall reactivity with the MSA was observed in 10 (5.6%) patients who were treated with surgery. In 140 patients, dead from the disease or alive with the disease, the survival at 1 and 2 years was 37% versus 17% and 14% versus 0 for patients without and with del 8/9 positivity, respectively (log rank P = 0.01). Biopsies from patients with inoperable-GC (101) rendered 5 (4.95%) with del 8 or 9 immunoreactivity. Abnormal E-cadherin expression is frequent in GC. However, exon 8 or 9 deletions were observed in only 5.3% tumors in this series from Mexico, at a lower rate than previously published, but associated with a worse prognosis.
Assuntos
Caderinas/genética , Deleção de Genes , Neoplasias Gástricas/genética , Neoplasias Gástricas/mortalidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Caderinas/biossíntese , Desidrogenases de Carboidrato/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , México , Pessoa de Meia-Idade , Prognóstico , Análise de SobrevidaRESUMO
The fungus Phanerochaete chrysosporium was grown in a 10-l automatic fermenter using cellobiose as carbon source to monitor the induction of cellobiose dehydrogenase (CDH) and cellobiose quinone oxidoreductase (CBQ) enzymes, and to search for tentative cbq and cdh genes and their transcriptional products. After 24 h of induction, CDH was detected in the culture supernatant and a protein was recognized by a specific anti-CDH polyclonal antibody in the sonicated biomass. Northern blot experiments performed with several fungal RNA samples showed, after 24 h of induction, only one single species of an mRNA transcript corresponding in size to the cdh gene (2.5 kb) The relative amount of this transcript decreased as a function of time. Southern blot experiments done with genomic DNA and database search in the recently available genome information also ruled out the presence in this strain of a separate cbq gene distinct from the cdh gene. Taken together, these results demonstrated that CBQ originates from the cdh gene. Furthermore, it is not produced by differential splicing but by a posttranslational, predominantly intracellular, proteolytic cleavage.
Assuntos
Desidrogenases de Carboidrato/biossíntese , Proteínas Fúngicas/biossíntese , Phanerochaete/metabolismo , Alelos , Southern Blotting , Western Blotting , Desidrogenases de Carboidrato/química , Cromatografia Líquida de Alta Pressão , Proteínas Fúngicas/química , Phanerochaete/genética , Serina Endopeptidases , Transcrição GênicaRESUMO
Transcription of the Azotobacter vinelandii algD gene, which encodes GDP-mannose dehydrogenase (the rate-limiting enzyme of alginate synthesis), starts from three sites: p1, p2, and p3. The sensor kinase GacS, a member of the two-component regulatory system, is required for transcription of algD from its three sites during the stationary phase. Here we show that algD is expressed constitutively throughout the growth cycle from the p2 and p3 sites and that transcription from p1 started at the transition between the exponential growth phase and stationary phase. We constructed A. vinelandii strains that carried mutations in gacA encoding the cognate response regulator of GacS and in rpoS coding for the stationary-phase sigma(S) factor. The gacA mutation impaired alginate production and transcription of algD from its three promoters. Transcription of rpoS was also abolished by the gacA mutation. The rpoS mutation impaired transcription of algD from the p1 promoter and increased it from the p2 sigma(E) promoter. The results of this study provide evidence for the predominant role of GacA in a regulatory cascade controlling alginate production and gene expression during the stationary phase in A. vinelandii.
Assuntos
Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/fisiologia , Desidrogenases de Carboidrato/biossíntese , Fator sigma/fisiologia , Azotobacter vinelandii/genética , Azotobacter vinelandii/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Fator sigma/genéticaRESUMO
Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial species, the alternative sigma factor sigma(E) regulates the expression of functions related to the extracytoplasmic compartments. In A. vinelandii and Pseudomonas aeruginosa, the sigma(E) factor (AlgU) is essential for alginate production. In both bacteria, the activity of this sigma factor is regulated by the product of the mucA, mucB, mucC, and mucD genes. In this work, we studied the transcriptional regulation of the A. vinelandii algU-mucABCD gene cluster, as well as the role of the mucA and mucC gene products in alginate production. Our results show the existence of AlgU autoregulation and show that both MucA and MucC play a negative role in alginate production.
Assuntos
Alginatos/metabolismo , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Serina Endopeptidases , Fator sigma/genética , Fatores de Transcrição/genética , Azotobacter vinelandii/genética , Azotobacter vinelandii/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Desidrogenases de Carboidrato/genética , DNA Bacteriano , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ácido Glucurônico , Proteínas de Choque Térmico/genética , Ácidos Hexurônicos , Dados de Sequência Molecular , Família Multigênica , Mutagênese , Transcrição Gênica , Ativação TranscricionalRESUMO
The transcripts of structurally related cellobiohydrolase genes in Phanerochaete chrysosporium-colonized wood chips were quantified. The transcript patterns obtained were dramatically different from the transcript patterns obtained previously in defined media. Cellobiose dehydrogenase transcripts were also detected, which is consistent with the hypothesis that such transcripts play an important role in cellulose degradation.
Assuntos
Basidiomycota/enzimologia , Desidrogenases de Carboidrato/genética , Celulase/genética , RNA Mensageiro/análise , Madeira , Sequência de Bases , Celulose/metabolismo , Celulose 1,4-beta-Celobiosidase , Dados de Sequência MolecularRESUMO
The cDNA of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium has been cloned and sequenced. The 5' end was obtained by PCR amplification. The cDNA contains 2310 translated bases excluding the poly(A) tail. The deduced mature protein contains 770 amino acid residues and is preceded by a 18 residue long signal peptide. The regions of the amino acid sequence corresponding to the heme and FAD domains of CDH were identified as well as the nucleotide-binding motif, the disulfide pairing and a methionine residue chelating the heme iron. No homologous sequences were found for the heme domain, however, the FAD domain appears to be distantly related to the GMC oxidoreductase family.
Assuntos
Basidiomycota/genética , Desidrogenases de Carboidrato/genética , Genes Fúngicos/genética , Sequência de Aminoácidos , Aminoácidos/análise , Sequência de Bases , Basidiomycota/enzimologia , Desidrogenases de Carboidrato/química , Desidrogenases de Carboidrato/isolamento & purificação , Clonagem Molecular , DNA Complementar/genética , DNA Fúngico/genética , Flavina-Adenina Dinucleotídeo , Heme , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNAAssuntos
Hiperoxalúria Primária/complicações , Cálculos Renais/etiologia , Cálculos da Bexiga Urinária/etiologia , Adolescente , Adulto , Oxirredutases do Álcool/deficiência , Desidrogenases de Carboidrato/deficiência , Criança , Pré-Escolar , Feminino , Humanos , Hiperoxalúria Primária/classificação , Hiperoxalúria Primária/enzimologia , Lactente , MasculinoRESUMO
An inducible L-fucose dehydrogenase from the yeast-like fungus Pullularia pullulans was purified and studied. The enzyme catalyses the oxidation of L-fucose to L-fuconic acid possibly throught its unstable L-funcono-lactone. the enzyme was purified to hemogeneity by a sequence of protamine sulfate treatment, ammonium sulfate fractionation, gel filtration on Sephadex G-100, and DEAE-cellulose chromatography. This sequence resulted in an 87 fold purification. The apparent molecular weight determined by gel filtration and SDS polyacrilamide gel electrophoresis was 40 000. the dehydrogenase was NAD-especific and showed a high sugar substrate specificity. Of several D-and L-aldoses tested only L-fucose, L-galactose and-arabinose served as substrates. The maximum velocity for the reaction was at 33- and pH 10.5. Under these conditions, the Km values, and D-arabinose, respectively. The enzyme was strongly inhibited by thiol reagents, heavy metal ions, and was not particularly stable. At 4- it rapidly los activity, but remained active for two months when maintained at -20-. The enzyme has been used to quantativate L-fucose
Assuntos
Desidrogenases de Carboidrato/metabolismo , Fungos/enzimologiaRESUMO
En este estudio se comparó la técnica de la coaglutinación empleando un reactivo comercial (Phadebact Gonococcus Test) con la prueba de oxidación de carbohidratos para la identificación de N. gonorrhoeae. De un total de 148 cepas aisladas en medio de Thayer-Martin o gelosa chocolate, 145 se identificaron como N. gonorrhoeae, al haber cumplido con las siguientes características: ser diplococos gramnegativos, oxidasa positivos y capaces de oxidar solamente a la glucosa. Del total de gonococos se obtuvieron 143 cepas con resultado positivo para el reactivo de coaglutinación, es decir, 98.6% de concordancia. En las tres cepas identificadas bioquímicamente como N. meningitidis se obtuvieron resultados negativos. Al realizar la coaglutinación con anticuerpos monoclonales (Phadebact Monoclonal GC Test), 59 de las cepas se identificaron como pertenecientes al serogrupo WI y las 84 restantes, al serogrupo WII/III. Así, la coaglutinación constituye una prueba rápida, fácil de realizar y de alta especificidad, y una posible alternativa para la identificación del gonococo, en lugar de la prueba tradicional de oxidación de azúcares
Assuntos
Desidrogenases de Carboidrato , Neisseria gonorrhoeae/isolamento & purificação , Testes de Aglutinação/métodos , Anticorpos MonoclonaisRESUMO
An oxidative pathway by which L-arabinose is converted to alpha-ketoglutarate in crude extracts of Azospirillum brasiliense is demonstrated. Specific activities of enzymes involved in the pathway were determined, and several pathway intermediates were identified.