Streptomyces thermocerradoensis I3 secretes a novel bifunctional xylanase/endoglucanase under solid-state fermentation.

Lignocellulosic wastes can be potentially converted into several bioproducts such as glucose, xylo-oligosaccharides, and bioethanol. Certain processes, such as enzymatic hydrolysis, are generally needed to convert biomass into bioproducts. The present study investigated the production of xylanases and cellulases by Streptomyces thermocerradoensis I3 under solid-state fermentation (SSF), using wheat bran as a low-cost medium. The activities of xylanase and carboxymethyl cellulase (CMCase) were evaluated until 96 hr of incubation. The highest enzyme activity was observed after 72 hr of incubation. The crude enzyme extract was sequentially filtered, first using a 50 kDa filter, followed by a 30 kDa filter. Fraction 3 (F3) exhibited activities of both xylanase and CMCase. Xylanase and CMCase showed optimum activity at 70°C and pH 6.0 and 55°C and pH 6.0, respectively. The zymogram analysis showed a single activity band with a molecular mass of approximately 17 kDa. These findings provide strong evidence that the enzyme is a bifunctional xylanase/endoglucanase. This enzyme improved the saccharification of sugarcane bagasse by 1.76 times that of commercial cellulase. This enzyme has potential applications in various biotechnological procedures.

Cultivable microbiome and its resistance to antimicrobials isolated from Zaprionus indianus.

The objective of this study was to identify Z. indianus in PEJC and PESCAN. Listed are the following methods: to isolate bacteria from the integument of the Z. indianus species collected and to check the resistance of microorganisms to antibiotics. Collections of Z. indianus were performed in four seasons of the two parks. The results obtained suggest that the low amount of Z. indianus collected can be justified by environmental factors such as high average temperature and low average humidity. It is noted that there is a predominance of bacteria of the Enterobacteriaceae family found in both the PEJC and the PESCAN. The antibiogram performed for the isolated PEJC bacteria shows statistical significance when comparing the edge and inside values of the park. Studies with fungi were also carried out, and it was evidenced that Trichophyton spp. was the genus that most inhabited the two environments studied. The ability of fluconazole and ketoconazole to inhibit fungal growth was also investigated, and considering the concentration tested may suggest that they have good action spectra. Plasmid profile data show that 60% of antibiotic-resistant bacteria have plasmids. The values found show that Z. indianus can act as vectors of microorganisms that affect the healthy animals and humans and that these organisms may be influenced by seasons.

Development of a nanogold slot blot inhibition assay for the detection of antibodies against bovine herpesvirus type 1.

Bovine herpesvirus type 1 (BoHV-1) is recognized as an important pathogen causing respiratory, reproductive, and neurological disorders in cattle and is associated with economic losses to animal industry. Accurate diagnostic methods are needed for prevention of disease transmission. While the virus neutralization test is considered the gold standard method, it requires maintenance of the virus and cell cultures, which is time consuming and expensive. Serological techniques such as enzyme-linked immunosorbent assay (ELISA) are widely applied, as these are easy to perform and provide quick results. In the present study, a nanogold slot blot inhibition assay was developed for the serological diagnosis of BoHV-1 and compared with standard ELISA and horseradish peroxidase (HRP) slot blot assays. Of 42 serum samples tested by ELISA, 32 (76.2%) were positive and 10 (23.8%), were negative. The sensitivity and specificity of the nanogold slot blot inhibition assay was similar to that observed for ELISA and HRP slot blot assays, and a strong correlation was observed between the tests. Thus, the nanogold slot blot inhibition assay may serve as an efficient and rapid alternative to ELISA in settings, where plate-reading equipment is lacking.

Improvement of bread making quality by supplementation with a recombinant xylanase produced by Pichia pastoris.

Xylanases (EC 3.2.1.8) are hydrolytic enzymes, which randomly cleave the ß-1,4-linked xylose residues from xylan. The synthetic gene xynBS27 from Streptomyces sp. S27 was successfully cloned and expressed in Pichia pastoris. The full-length gene consists of 729 bp and encodes 243 amino acids including 51 residues of a putative signal peptide. This enzyme was purified in two steps and was shown to have a molecular weight of 20 kDa. The purified r-XynBS27 was active against beechwood xylan and oat spelt xylan as expected for GH 11 family. The optimum pH and temperature values for the enzyme were 6.0 and 75 °C, respectively. The Km and Vmax were 12.38 mg/mL and 13.68 µmol min/mg, respectively. The r-XynBS27 showed high xylose tolerance and was inhibited by some metal ions and by SDS. r-XynBS27 was employed as an additive in the bread making process. A decrease in firmness, stiffness and consistency, and improvements in specific volume and reducing sugar content were recorded.

Sequence analysis and characterization of plasmid pLK39 isolated from endophytic Salmonella sp. isolated from Solanum lycocarpum.

The complete nucleotide sequence of a small cryptic plasmid pLK39 isolated from endophytic Salmonella sp. was determined. This plasmid is 4,029 bp long with an overall GC content of 55.4 %. Sequence analyses of pLK39 revealed extensive homology to several plasmids: pRK10, pK, pSW200, pBERT, pST728/06-2, pSW100, pEC3, and pUCD5000. Using the ORF Finder program, 35 putative ORFs was identified, 30 showed more than 35 residues. After performing a search for homologous sequences to the pLK39 at BLASTn software on NCBI, it was ascertained that the plasmid has a ColE1-like replication origin and also a region of mobilization proteins from relaxase family (mobCABD). Besides these mobilization proteins, the pLK39 codes a putative DUF903 protein family, which is characterized as assumed external cytoplasmic membrane lipoprotein. A recombinant form of pLK39 carrying a kanamycin resistance gene is stably maintained in Escherichia coli cells grown in the absence of selection pressure. pLK39 was compatible with pUC18, pBR322, and pACYC184.

A1 noradrenergic neurons lesions reduce natriuresis and hypertensive responses to hypernatremia in rats.

Noradrenergic neurons in the caudal ventrolateral medulla (CVLM; A1 group) contribute to cardiovascular regulation. The present study assessed whether specific lesions in the A1 group altered the cardiovascular responses that were evoked by hypertonic saline (HS) infusion in non-anesthetized rats. Male Wistar rats (280-340 g) received nanoinjections of antidopamine-ß-hydroxylase-saporin (A1 lesion, 0.105 ng.nL(-1)) or free saporin (sham, 0.021 ng.nL(-1)) into their CVLMs. Two weeks later, the rats were anesthetized (2% halothane in O2) and their femoral artery and vein were catheterized and led to exit subcutaneously between the scapulae. On the following day, the animals were submitted to HS infusion (3 M NaCl, 1.8 ml • kg(-1), b.wt., for longer than 1 min). In the sham-group (n = 8), HS induced a sustained pressor response (ΔMAP: 35±3.6 and 11±1.8 mmHg, for 10 and 90 min after HS infusion, respectively; P<0.05 vs. baseline). Ten min after HS infusion, the pressor responses of the anti-DßH-saporin-treated rats (n = 11)were significantly smaller(ΔMAP: 18±1.4 mmHg; P<0.05 vs. baseline and vs. sham group), and at 90 min, their blood pressures reached baseline values (2±1.6 mmHg). Compared to the sham group, the natriuresis that was induced by HS was reduced in the lesioned group 60 min after the challenge (196±5.5 mM vs. 262±7.6 mM, respectively; P<0.05). In addition, A1-lesioned rats excreted only 47% of their sodium 90 min after HS infusion, while sham animals excreted 80% of their sodium. Immunohistochemical analysis confirmed a substantial destruction of the A1 cell group in the CVLM of rats that had been nanoinjected withanti-DßH-saporin. These results suggest that medullary noradrenergic A1 neurons are involved in the excitatory neural pathway that regulates hypertensive and natriuretic responses to acute changes in the composition of body fluid.

Screening and xylanase production by Streptomyces sp. grown on lignocellulosic wastes.

Xylanases have raised interest because of their potential applications in various industrial fields, including the pulp and paper industries, bioethanol production, and the feed industry. In bioethanol production from lignocellulosic compounds, xylanase can improve the hydrolysis of cellulose into fermentable sugars, since the xylan restricts the cellulases from acting efficiently. In this work, a new thermophilic Streptomyces sp. was selected for its ability to produce xylanase. Carbon source selection is an important factor in the production of hemicellulases. The highest activity was obtained when Streptomyces sp. I3 was grown in the presence of wheat bran. Xylanase activity was partially characterized concerning the effect of pH and temperature on activity and thermostability, and the effects of different metal ions were also tested. The pH and temperature profile showed optimal activity at pH 6.0/70 °C. Zymogram analysis showed multiple xylanases (39, 21, 18, and 17 kDa). Xylanases studied in this work are thermophilic, thermostable, and active in a wide pH range; they have potential to be used in the development of new processes of biotechnological interest.

Dificuldades na identificação laboratorial da talassemia alfa / Difficulty on laboratory identification of alpha thalassemia

Introdução: Talassemia alfa é uma síndrome associada à redução da síntese de cadeias de globina do tipo alfa. A gravidade das manifestações clínicas está relacionada com a quantidade de globinas produzida e a estabilidade das cadeias beta presentes em excesso. A talassemia alfa mínima resulta da deleção de apenas um dos quatro genes a (-α/αα). Clinicamente apresenta anemia leve com microcitose ou ausência de anemia, sendo o diagnóstico realizado por meio de visualização da hemoglobina (Hb) H por eletroforese alcalina em acetato de celulose ou por identificação de inclusões celulares de Hb H coradas pelo azul de crezil brilhante. Objetivo: Avaliar portadores de talassemia alfa e seus respectivos progenitores, correlacionando perfil hematológico e presença de Hb H, utilizando procedimentos laboratoriais clássicos em três diferentes amostragens. Discussão e conclusão: Os dados obtidos mostram que a presença de Hb H, indicativo de talassemia alfa, pode não ser confirmada em uma análise posterior. Entre os fatores que podem influenciar no não aparecimento de Hb H em pessoa comprovadamente com talassemia alfa está a deficiência de ferro. A talassemia alfa está associada a defeitos envolvendo os genes codificadores da cadeia alfa, mas também pode estar relacionada com desbalanciamento temporário na expressão dos genes globina, diminuição de alfa ou aumento de beta, o que poderia explicar o aparecimento de tetrâmeros de cadeia beta (Hb H), sugerindo diagnóstico de talassemia alfa mínima.

Introduction: Alpha thalassemia is a syndrome with associated with the reduction of alpha globin chain synthesis. The severity of clinical manifestations is related to the amount of globins produced and the stability of beta chains that are present in excess. Alpha thalassemia minor is caused by the deletion of one of the four genes a (-α/αα). Clinically, it presents mild anemia with microcytosis or absence of anemia. The diagnosis is made by the visualization of Hb H through alkaline electrophoresis on cellulose acetate or by the identification of inclusion bodies stained with brilliant cresyl blue. Objective: Evaluate alpha thalassemia carriers and their respective progenitors, correlating their hematology profile and the presence of Hb H by means of standard laboratory procedures in three different samplings. Discussion and conclusion: The results show that the presence of Hb H, which is indicative of alpha thalassemia, may not be confirmed in a subsequent analysis. Iron deficiency in Hb H carriers is among the factors that may influence on the absence of Hb H in alpha thalassemia proven patients. Alpha thalassemia is associated with genetic defects involving alpha chain encoding genes, but may be also associated with a temporary imbalance of globin gene expression, alpha chain reduction or beta increase, which could explain the presence of beta chain tetramer (Hb H) leading to the diagnosis of alpha thalassemia minor.

Estudo comparativo da eficiência da eletroforese alcalina em acetato de celulose na identificação de hemoglobinas utilizando diferentes tampões / Comparative study of the alkaline electrophoresis on cellulose acetate efficiency to identify hemoglobins by using different buffers

As hemoglobinas podem apresentar alterações moleculares denominadas hemoglobinopatias caracterizadas pela ineficiência da produção das cadeias de globinas (alfa ou beta) ou por alterações estruturais na síntese destas cadeias. Tais alterações estruturais promovem a formação de hemoglobinas variantes (anômalas) e o desbalanceamento na síntese destas cadeias tem como consequência a talassemia. O diagnóstico das hemoglobinopatias é realizado essencialmente por avaliação eletroforética. Este trabalho teve como objetivo avaliar a influência de diferentes tampões de eletroforese na caracterização de hemoglobinas variantes por meio de técnica de eletroforese alcalina em acetato de celulose. Considerando as características de separação e nitidez das bandas obtidas concluímos que o tampão TEB (Tris base 0,090 M, Ácido bórico 0,090 M, EDTA0,002 M, pH 8,2) na concentração de 0,5X foi o mais eficiente na caracterização das hemoglobinas testadas. Este tampão além de permitir uma melhor separação eletroforética entre as diferentes hemoglobinas, também permitiu uma maior estabilidade da hemoglobina H, favorecendo a identificação mais eficaz dessa hemoglobina instável.

Purification and properties of an N-acetylglucosaminidase from Streptomyces cerradoensis.

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The Km and Vmax values for the enzyme using p-nitrophenyl-beta-N-acetylglucosaminide as substrate were of 0.13 mM: and 1.95 U mg(-1) protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 degrees C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu2+ and Hg2+ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-beta-N-acetylglucosaminide and p-nitrophenyl-beta-D-N,N'-diacetylchitobiose.

Chemotaxis and flagellar genes of Chromobacterium violaceum.

The availability of the complete genome of the Gram-negative beta-proteobacterium Chromobacterium violaceum has increasingly impacted our understanding of this microorganism. This review focuses on the genomic organization and structural analysis of the deduced proteins of the chemosensory adaptation system of C. violaceum. C. violaceum has multiple homologues of most chemotaxis genes, organized mostly in clusters in the bacterial genome. We found at least 67 genes, distributed in 10 gene clusters, involved in the chemotaxis of C. violaceum. A close examination of the chemoreceptors methyl-accepting chemotaxis proteins (MCPs), and the deduced sequences of the members of the two-component signaling system revealed canonical motifs, described as essential for the function of the deduced proteins. The chemoreceptors found in C. violaceum include the complete repertoire of such genes described in bacteria, designated as tsr, tar, trg, and tap; 41 MCP loci were found in the C. violaceum genome. Also, the C. violaceum genome includes a large repertoire of the proteins of the chemosensory transducer system. Multiple homologues of bacterial chemotaxis genes, including CheA, CheB, CheD, CheR, CheV, CheY, CheZ, and CheW, were found in the C. violaceum genome.

Hypothesis testing of genetic similarity based on RAPD data using Mantel tests and model matrices

Clustering and ordination procedures in multivariate analyses have been widely used to describe patterns of genetic distances. However, in some cases, such as when dealing with Jaccard coefficients based on RAPD data, these techniques may fail to represent genetic distances because of the high dimensionality of the genetic distances caused by stochastic variation in DNA fragments among the units analyzed (species or populations). In this note, we show how Mantel tests can be used to test hypotheses about genetic distances and avoid problems with multivariate data representation. The procedure is illustrated with RAPD data comparing 20 anuran species from the cerrado, in which Jaccard genetic distances were compared to three model matrices linking the species at distinct taxonomic levels