Comprehensive transcriptome analysis based on RNA sequencing identifies critical genes for lipopolysaccharide-induced epididymitis in a rat model / 亚洲男科学杂志(英文版)

Epididymitis is a commonly diagnosed disease associated with male infertility. However, little is known about the molecules that are involved in its development. This study was to identify critical genes associated with lipopolysaccharide-induced epididymitis and analyze the molecular mechanism of epididymitis through RNA sequencing. Experimental epididymitis models were generated by administering male Sprague-Dawley rats' lipopolysaccharide. A total of 1378 differentially expressed genes, including 531 upregulated and 847 downregulated genes, were identified in the epididymitis model rats compared with those in sham-operated rats by RNA sequencing. Functional enrichment analyses suggested that the upregulated genes were markedly enriched in inflammation-related biological processes, as well as in the tumor necrosis factor (TNF) signaling pathway, cytokine-cytokine receptor interactions, complement and coagulation cascades, and in the chemokine signaling pathway. Four downregulated genes (collagen type XXVIII alpha 1 chain [Col28α1], cyclin-dependent kinase-like 1 [Cdkl1], phosphoserine phosphatase [Psph], and fatty acid desaturase 2 [Fads2]) and ten upregulated genes (CCAAT/enhancer-binding protein beta [Cebpβ], C-X-C motif chemokine receptor 2 [Cxcr2], interleukin 11 [Il11], C-C motif chemokine ligand 20 [Ccl20], nuclear factor-kappa-B inhibitor alpha [Nfkbiα], claudin 4 [Cldn4], matrix metallopeptidase 9 [Mmp9], heat shock 70 kDa protein 8 [Hspa8], intercellular cell adhesion molecule-1 [Icam1], and Jun) were successfully confirmed by real-time polymerase chain reaction. Western blot demonstrated that CDKL1 was decreased, while MMP9 and NFKBIA were increased in the experimental model group compared with those in the sham-operated group. Our study sheds new light on the understanding of the early response of the epididymis during bacterial epididymitis.

Selective intra-dinucleotide interactions and periodicities of bases separated by K sites: a new vision and tool for phylogeny analyses

Direct tests of the random or non-random distribution of nucleotides on genomes have been devised to test the hypothesis of neutral, nearly-neutral or selective evolution. These tests are based on the direct base distribution and are independent of the functional (coding or non-coding) or structural (repeated or unique sequences) properties of the DNA. The first approach described the longitudinal distribution of bases in tandem repeats under the Bose-Einstein statistics. A huge deviation from randomness was found. A second approach was the study of the base distribution within dinucleotides whose bases were separated by 0, 1, 2... K nucleotides. Again an enormous difference from the random distribution was found with significances out of tables and programs. These test values were periodical and included the 16 dinucleotides. For example a high ¨positive¨ (more observed than expected dinucleotides) value, found in dinucleotides whose bases were separated by (3K + 2) sites, was preceded by two smaller ¨negative¨ (less observed than expected dinucleotides) values, whose bases were separated by (3K) or (3K + 1) sites. We examined mtDNAs, prokaryote genomes and some eukaryote chromosomes and found that the significant non-random interactions and periodicities were present up to 1000 or more sites of base separation and in human chromosome 21 until separations of more than 10 millions sites. Each nucleotide has its own significant value of its distance to neutrality; this yields 16 hierarchical significances. A three dimensional table with the number of sites of separation between the bases and the 16 significances (the third dimension is the dinucleotide, individual or taxon involved) gives directly an evolutionary state of the analyzed genome that can be used to obtain phylogenies. An example is provided.

Draft genome sequence of Inquilinus limosus strain MP06, a multidrug-resistant clinical isolate

The bacterium, Inquilinus limosus, with its remarkable antimicrobial multiresistant profile, has increasingly been isolated in cystic fibrosis patients. We report draft genome sequence of a strain MP06, which is of considerable interest in elucidating the associated mechanisms of antibiotic resistance in this bacterium and for an insight about its persistence in airways of these patients.

Indole acetic acid production by fluorescent Pseudomonas spp. from the rhizosphere of Plectranthus amboinicus (Lour.) Spreng. and their variation in extragenic repetitive DNA sequences.

Fluorescent Pseudomonas (FP) is a heterogenous group of growth promoting rhizobacteria that regulate plant growth by releasing secondary metabolic compounds viz., indole acetic acid (IAA), siderophores, ammonia and hydrogen cyanide. In the present study, IAA producing FPs from the rhizosphere of Plectranthus amboinicus were characterized morphologically, biochemically and at the molecular level. Molecular identification of the isolates were carried out using Pseudomonas specific primers. The effect of varying time (24, 48, 72 and 96 h), Trp concentrations (100, 200, 300, 400 and 500 µg.ml-1), temperature (10, 26, 37 and 50±2 °C) and pH (6, 7 and 8) on IAA production by 10 best isolates were studied. Results showed higher IAA production at 72 h incubation, at 300 µg.ml-1 Trp concentration, temperature 26±2 °C and pH 7. TLC with acidified ethyl acetate extract showed that the IAA produced has a similar Rf value to that of the standard IAA. Results of TLC were confirmed by HPLC analysis. Genetic diversity of the isolates was also studied using 40 RAPD and 4 Rep primers. Genetic diversity parameters such as dominance, Shannon index and Simpson index were calculated. Out of 40 RAPD primers tested, 9 (2 OP-D series and 7 OP-E series) were shortlisted for further analysis. Studies using RAPD, ERIC, BOX, REP and GTG5 primers revealed that isolates exhibit significant diversity in repetitive DNA sequences irrespective of the rhizosphere.

Molecular detection of Mayaro virus during a dengue outbreak in the state of Mato Grosso, Central-West Brazil

Mayaro virus (MAYV) is frequently reported in Pan-Amazonia. The aim of this study was to investigate the circulation of alphaviruses during a dengue outbreak in the state of Mato Grosso, Brazil. Serum samples from dengue-suspected patients were subjected to multiplex semi-nested reverse transcriptase polymerase chain reaction for 11 flaviviruses and five alphaviruses, to nucleotide sequencing and to viral isolation. MAYV was detected in 15 (2.5%) of 604 patients. Twelve were co-infected with dengue virus 4, which was isolated from 10 patients. The molecular detection of MAYV in dengue-suspected patients suggests that other arboviruses may be silently circulating during dengue outbreaks in Brazil.

Limits and possibilities experienced by nurses in the treatment of women with chronic venous ulcers / Límites y posibilidades vividas por enfermeras en el tratamiento de mujeres con úlcera venosa crónica / Limites e possibilidades vivenciados por enfermeiras no tratamento de mulheres com úlcera venosa crônica

Objective To understand the experiences and expectations of nurses in the treatment of women with chronic venous ulcers. Method Phenomenological research was based on Alfred Schütz, whose statements were obtained in January, 2012, through semi-structured interviews with seven nurses. Results The nurse reveals the difficulties presented by the woman in performing self-care, the perceived limitations in the treatment anchored in motivation, and the values and beliefs of women. It showed professional frustration because venous leg ulcer recurrence, lack of inputs, interdisciplinary work and training of nursing staff. There was an expected adherence to the treatment of women, and it emphasized the need for ongoing care, supported self-care and standard practices in treatment. Conclusion That treatment of chronic venous leg ulcers constitutes a challenge that requires collective investment, involving women, professionals, managers and health institutions. .

Objetivo Comprender las experiencias y expectativas de enfermeras en el tratamiento de mujeres con úlcera venosa crónica. Método Investigación fenomenológica fundamentada en Alfred Schutz, que buscó Se realizó entrevista semiestructurada con siete enfermeras, en enero del 2012. Resultados La enfermera revela dificultades presentadas por la mujer para realizar el autocuidado, percibe limitaciones en el tratamiento relacionadas con la desmotivación, los valores y las creencias de las mujeres. Refiere frustración profesional debido a la recidiva de la lesión, a la falta de insumos, al deficiente trabajo interdisciplinar y a la limitada capacitación del equipo de enfermeras. Espera la adhesión de la mujer al tratamiento y resalta la necesidad del cuidado continuo, del autocuidado apoyado y de estandarizar conductas de tratamiento. Conclusión El tratamiento de la úlcera venosa crónica es un desafío que requiere contribución colectiva, involucrando a las mujeres, a los profesionales, a los gestores y a las instituciones de salud. .

Objetivo Compreender as experiências e expectativas de enfermeiras no tratamento de mulheres com úlcera venosa crônica na Atenção Primária à Saúde. Método Pesquisa fundamentada na fenomenologia social de Alfred Schütz, com depoimentos obtidos em janeiro de 2012, por meio de entrevista semiestruturada com sete enfermeiras. Resultados As enfermeiras revelam dificuldades apresentadas pelas mulheres com úlcera venosa crônica para realizar o autocuidado, percebem limitações na terapêutica ancoradas na desmotivação e nos valores e crenças das mulheres. Referem frustração profissional em razão da recidiva da lesão, falta de insumos e tecnologia, de trabalho interdisciplinar e da capacitação da equipe de enfermagem. Esperam a adesão das mulheres ao tratamento e ressaltam a necessidade do cuidado contínuo, do autocuidado apoiado e da padronização de condutas no tratamento. Conclusão O tratamento da úlcera venosa crônica constitui-se em um desafio que requer investimento coletivo, envolvendo a mulher, os profissionais, os gestores e as instituições de saúde. .

A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000 - 25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.

The structure of selective dinucleotide interactions and periodicities in D melanogaster mtDNA

BACKGROUND: We found a strong selective 3-sites periodicity of deviations from randomness of the dinucleotide (DN) distribution, where both bases of DN were separated by 1, 2, K sites in prokaryotes and mtDNA. Three main aspects are studied. I) the specific 3 K-sites periodic structure of the 16 DN. II) to discard the possibility that the periodicity was produced by the highly nonrandom interactive association of contiguous bases, by studying the interaction of non-contiguous bases, the first one chosen each I sites and the second chosen J sites downstream. III) the difference between this selective periodicity of association (distance to randomness) of the four bases with the described fixed periodicities of base sequences. RESULTS: I) The 16 pairs presented a consistent periodicity in the strength of association of both bases of the pairs; the most deviated pairs are those where G and C are involved and the least deviated ones are those where A and T are involved. II) we found significant non-random interactions when the first nucleotide is chosen every I sites and the second J sites downstream until I=J=76. III) we showed conclusive differences between these internucleotide association periodicities and sequence periodicities. CONCLUSIONS: This relational selective periodicity is different from sequence periodicities and indicates that any base strongly interacts with the bases of the residual genome; this interaction and periodicity is highly structured and systematic for every pair of bases. This interaction should be destroyed in few generations by recurrent mutation; it is only compatible with the Synthetic Theory of Evolution and agrees with the Wright's adaptive landscape conception and evolution by shifting balanced adaptive peaks.

Isolation and characterization of endophytic bacteria isolated from the leaves of the common bean (Phaseolus vulgaris)

The common bean is one of the most important legumes in the human diet, but little is known about the endophytic bacteria associated with the leaves of this plant. The objective of this study was to characterize the culturable endophytic bacteria of common bean (Phaseolus vulgaris. leaves from three different cultivars (Vermelhinho, Talismã, and Ouro Negro) grown under the same field conditions. The density of endophytic populations varied from 4.5 x 10² to 2.8 x 10³ CFU g-1 of fresh weight. Of the 158 total isolates, 36.7% belonged to the Proteobacteria, 32.9% to Firmicutes, 29.7% to Actinobacteria, and 0.6% to Bacteroidetes. The three P. vulgaris cultivars showed class distribution differences among Actinobacteria, Alphaproteobacteria and Bacilli. Based on 16S rDNA sequences, 23 different genera were isolated comprising bacteria commonly associated with soil and plants. The genera Bacillus, Delftia, Methylobacterium, Microbacterium, Paenibacillus, Staphylococcus and Stenotrophomonas were isolated from all three cultivars. To access and compare the community structure, diversity indices were calculated. The isolates from the Talismã cultivar were less diverse than the isolates derived from the other two cultivars. The results of this work indicate that the cultivar of the plant may contribute to the structure of the endophytic community associated with the common bean. This is the first report of endophytic bacteria from the leaves of P. vulgaris cultivars. Future studies will determine the potential application of these isolates in biological control, growth promotion and enzyme production for biotechnology.

Pseudorabies virus can be classified into five genotypes using partial sequences of UL44

Suid herpesvirus 1 (SuHV-1) is the causative agent of pseudorabies (PR), a disease of great importance due to the huge losses it causes in the swine industry. The aim of this study was to determine a method for genotyping SuHV-1 based on partial sequences of the gene coding for glycoprotein C (gC) and to elucidate the possible reasons for the variability of this region. A total of 109 gCsequences collected from GenBank were divided into five major groups after reconstruction of a phylogenetic tree by Bayesian inference. The analysis showed that a portion of gC (approximately 671 bp) is under selective pressure at various points that coincide with regions of protein disorder. It was also possible to divide SuHV-1 into five genotypes that evolved under different selective pressures. These genotypes are not specific to countries or continents, perhaps due to multiple introduction events related to the importation of swine.

PIK3CA exon 20 mutations are associated with poor prognosis in breast cancer patients

OBJECTIVES: The phosphatidylinositol 3-kinase/AKT axis is an important cell-signaling pathway that mediates cell proliferation and survival, two biological processes that regulate malignant cell growth. The phosphatidylinositol 3-kinase CA gene encodes the p110α subunit of the phosphatidylinositol 3-kinase protein. There are phosphatidylinositol 3-kinase CA mutations in several types of human tumors, and they are frequently observed in breast cancer. However, these mutations have not been investigated in Brazilian breast cancer patients. METHODS: PCR-SSCP and direct DNA sequencing were performed to identify phosphatidylinositol 3-kinaseCA exon 9 and exon 20 mutations in 86 patients with sporadic breast cancer. The relationships between PIK3CA mutations and patient clinicopathological characteristics and survival were analyzed. The presence of the TP53 mutation was also examined. RESULTS: Twenty-three (27%) of the 86 primary breast tumors contained PIK3CA mutations. In exons 9 and 20, we identified the hotspot mutations E542K, E545K, and H1047R, and we identified two new missense mutations (I1022V and L1028S) and one nonsense (R992X) mutation. Phosphatidylinositol 3-kinase CA exon 20 mutations were associated with poor overall survival and TP53 gene mutations. CONCLUSIONS: Phosphatidylinositol 3-kinase CA mutations are common in tumors in Brazilian breast cancer patients, and phosphatidylinositol 3-kinase CA and TP53 mutations are not mutually exclusive. Phosphatidylinositol 3-kinase CA exon 20 mutations are associated with poor survival, and they may be useful biomarkers for identifying breast cancer patients with aggressive tumors and for predicting the response to treatment with PI3K pathway inhibitors.

HCV: similitudes y diferencias con el HIV / HCV: similarities and differences with HIV

El virus de la hepatitis C (HCV) ha sido caracterizado en profundidad a nivel molecular en la última década. La partícula viral envuelta alberga una nucleocápside, estructura constituida principalmente por una proteína básica que está en estrecha interacción con el genoma viral representado por una molécula de ARN de cadena simple con polaridad positiva. La organización genómica del HCV es similar a la de Pestivirus y Flavivirus. Diferentes receptores celulares se han postulado en su participación para el ingreso del virus a la célula blanco. Su estrategia de multiplicación deja avizorar los blancos de acción de nuevas drogas para controlar la replicación. Si bien comparte con el HIV - desde su naturaleza de ARN virus - entre otras características virológicas la magnífica plasticidad genómica, otras por el contrario revisten claras diferencias. Ambos virus constituyen un enorme desafío en Salud

The hepatitis C virus (HCV) has been deeply characterized at molecular level during the last decade. The enveloped viral particle protects the nucleocapsid that is essentially constituted by a basic protein that interacts with the viral genome, a single strand RNA with positive polarity. The genomic organization of the HCV is similar to the Pestivirus and Flavivirus. Different cellular receptors have been postulated to play a role to the virus entry in the cellular target. The replication strategy exhibit the different plausible target of antiviral action with new drugs in order to control the replication. The HCV shares with the HIV the vast genomic plasticity because both are RNA viruses but other characteristics are different between them. Both viruses are an enormous trial for human health

Cloning and characterization of a gene encoding novel zinc finger protein transcription factor induced under water deficit stress from rice (Oryza sativa) cv. N-22.

A gene OsZnI encoding Cys3/His1-type zinc finger protein was isolated from the water stress-induced cDNA library of rice (Oryza sativa) cv. N-22, an early maturing, deep-rooted, drought-tolerant genotype adapted to upland conditions. The in-silico analysis revealed an insert of 800 bp with an ORF of 663 nucleotides, encoding 221 amino acids. OsZnI had three distinct features — nuclear localization signal (NLS) present in Arg152-Arg168, Zn finger domain between 185-193 amino acids and 12 amino acids conserved domain in 71-82 amino acids homologous to LEA motif, and belonged to C-type family of Zn finger protein. OsZnI showed induced expression under water deficit stress.

Signals in the promoter regions of several cancerous genes.

The eukaryotic core promoter regions are complex and fuzzy, usually lacking any conserved regions. However, they contain signals in the form of short stretches of nucleic acid sequences, for transcription start sites (TSS) that are recognized by the transcription factors (TFs). The core promoter region thus plays an important role in biological pathways (gene network and activation). It has been reported that these signals are composed of nucleotide hexamers in the promoter sequence (smaller sequences are likely to have too little information to be useful and longer sequences are too complex to be recognized by proteins) reasonably close to the TSS. The signals (nucleotide hexamers) have been identified by a similarity search on the eukaryotic promoter database (EPD, Homo sapiens). The signals have been classified, depending on their base composition. They have been have clustered using an algorithm, such that there are two and three nucleotide differences between the classes and a single nucleotide difference within a class. We have reclassified the hexamers taking the highest frequent hexamers present in the EPD (Homo sapiens) as the class representatives. Also we have tried to find whether the same composition is reflected on the miRNAs but found that they probably have other functions unrelated to promoter recognition. In this report melanoma carcinoma pathway has been chosen as the reference pathway and the promoters of the driver genes has been searched for the presence of the major classes. A few of these were found and are reported here. Several non-cancerous genes have also been studied as reference and comparison.

Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20 percent O2) and hypoxia (less than 5 percent O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50 percent lower under hypoxia, while the HRE plasmid was about 50 percent higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

Fixations of the HIV-1 env gene refute neutralism: New evidence for pan-selective evolution

We examined 103 nucleotide sequences of the HIV-1 env gene, sampled from 35 countries and tested: I) the random (neutral) distribution of the number of nucleotide changes; II) the proportion of bases at molecular equilibrium; III) the neutral expected homogeneity of the distribution of new fxated bases; IV) the hypothesis of the neighbor infuence on the mutation rates in a site. The expected random number of fxations per site was estimated by Bose-Einstein statistics, and the expected frequencies of bases by matrices of mutation-fxation rates. The homogeneity of new fxations was analyzed using χ2 and trinomial tests for homogeneity. Fixations of the central base in trinucleotides were used to test the neighbor infuence on base substitutions. Neither the number of fxations nor the frequencies of bases ftted the expected neutral distribution. There was a highly signifcant heterogeneity in the distribution of new fxations, and several sites showed more transversions than transitions, showing that each nucleotide site has its own pattern of change. These three independent results make the neutral theory, the nearly neutral and the neighbor infuence hypotheses untenable and indicate that evolution of env is rather highly selective.

Occurrence of Aggregatibacter actinomycetemcomitans in Brazilian indians from Umutina Reservation, Mato Grosso, Brazil

Aggregatibacter actinomycetemcomitans is associated with periodontal disease, especially localized aggressive periodontitis, produces a potent leukotoxin and its distribution is influenced by ethnic characteristics of the population. Objective: Using culture and polymerase chain reaction (PCR) techniques, this study evaluated the occurrence of this microorganism and the distribution of leukotoxic strains isolated from Indians belonging to the Umutima Reservation, Mato Grosso, Brazil. MATERIAL AND METHODS: Forty-eight native Brazilians with gingivitis and 38 with chronic periodontitis, belonging to Umutina, Paresi, Bororo, Bakairi, Kayabi, Irantxe, Nambikwara and Terena ethnicities, were studied. Subgingival, supragingival and saliva samples of each patient were collected and transferred to VMGA III medium and to ultra pure Milli Q water. Bacteria were grown on TSBV agar and incubated in anaerobiosis (90 percent N2 + 10 percent CO2) at 37ºC for 72 h. The presence of the ltx promoter was determined by PCR, and a 530 bp deletion in the promoter was evaluated by using specific primers. RESULTS: A. actinomycetemcomitans was isolated from 8.33 percent of saliva, supragingival and subgingival samples from patients with gingivitis and from 18.42 percent of saliva and supragingival biofilm, and 26.32 percent subgingival biofilm from patients with chronic periodontitis. By PCR, the bacterial DNA was detected in 8.33 percent of saliva, supragingival and subgingival biofilms from patients with gingivitis and from 23.68 percent of saliva, 28.95 percent supragingival biofilm and 34.21 percent subgingival biofilm from patients with periodontitis. All strains were grouped as non-JP2 clones based on the absence of deletion in the leukotoxin promoter. Differences among the microbial and clinical parameters in patients were analyzed by using the Mann-Whitney, Chi-square or Fisher's exact tests. CONCLUSIONS: The present results suggest that A. actinomycetemcomitans ...

Isolation and characterization of a novel strain of Stenotrophomonas maltophilia possessing various dioxygenases for monocyclic hydrocarbon degradation / Isolamento e caracterização de uma nova cepa de Stenotrophomonas maltophilia com várias dioxigenases para degradação de hidrocarbonetos monocíclicos

A Gram-negative bacterium, designated as strain KB2, was isolated from activated sludge and was found to utilize different aromatic substrates as sole carbon and energy source. On the basis of morphological and physiochemical characteristics and 16S rRNA gene sequence analysis, the isolated strain KB2 was identified as Stenotrophomonas maltophilia. Strain KB2 is from among different Stenotrophomonas maltophilia strains the first one described as exhibiting the activities of three types of dioxygenases depending on the structure of the inducer. The cells grown on benzoate and catechol showed mainly catechol 1,2- dioxygenase activity. The activity of 2,3-dioxygenase was detected after phenol induction. Protocatechuate 3,4-dioxygenase was found in crude cell extracts of this strain after incubation with 4-hydroxybenzoic acid, protocatechuic acid and vanillic acid. Because of broad spectrum of dioxygenases' types that Stenotrophomonas maltophilia KB2 can exhibit, this strain appears to be very powerful and useful tool in the biotreatment of wastewaters and in soil decontamination.

Uma bactéria Gram-negativa, denominada KB2, foi isolada de lodo ativado, verificando-se ser capaz de utilizar substratos aromáticos com única fonte de carbono e energia. Com base nas características morfológicas e físico-químicas, e na análise da sequencia do gene 16SrRNA, esta bactéria foi identificada como Stenotrophomonas maltophilia. Entre as diversas cepas de S. maltophilia já descritas, essa cepa é a primeira com atividade de três tipos de dioxigenases, dependendo da estrutura do indutor. As células cultivadas em benzoato e catecol apresentaram atividade de catecol 1,2-dioxigenase principalmente. A atividade de 2,3-dioxigenase foi detectada após indução com fenol. Após incubação com ácidos 4-hidrobenzoico, ácido protocatecuico evanílico, encontrou-se protocatecuato 3,4-dioxigenase no extrato celular. Devido ao amplo espectro de atividade das diferentes dioxigenases de S. maltophilia KB2, esta cepa parece ser uma ferramenta poderosa e útil para o biotratamento de efluentes e descontaminação do solo.

Populations analysis of the Brazilian Sharpnose Shark Rhizoprionodon lalandii (Chondrichthyes: Carcharhinidae) on the São Paulo coast, Southern Brazil: inferences from mt DNA sequences

Sharks of the genus Rhizoprionodon can be considered some of the most important predators along the trophic coastal marine ecosystems and represent an important economic resource for the small-scale fisheries, especially on the Brazilian coastline. In order to analyze the population structure of the shark Rhizoprionodon lalandii of São Paulo, Southeastern coast of Brazil, levels of genetic diversity were identified by nucleotide sequence analyses of the mitochondrial DNA control region. The results obtained from this study present moderate values of haplotype diversity and low nucleotide diversity. Although the AMOVA tests (ΦST = 0.08394, P < 0.01) had shown slightly differences among the studied samples, evidence for the occurrence of population structuring was not found, which may be a general feature of sharks living in coastal areas.

Tubarões do gênero Rhizoprionodon são considerados predadores de grande importância ao longo da cadeia trófica nos ecossistemas costeiros e marinhos, também representando um importante recurso econômico para a pesca, especialmente no litoral brasileiro. A fim de analisar a estrutura populacional do tubarão Rhizoprionodon lalandii no litoral de São Paulo, sudeste do Brasil, foram identificados os níveis de diversidade genética a partir da análise de sequências nucleotídicas da região controladora do DNA mitocondrial. Os dados obtidos neste estudo apresentam valores moderados de diversidade haplotípica e baixos índices de diversidade nucleotídica. Embora os testes de AMOVA (ΦST = 0,08394, P < 0,01) tenham revelado uma pequena diferença entre as amostras estudadas, evidências sobre a ocorrência de estruturação populacional não foram encontradas o que pode representar uma característica geral para tubarões vivendo em áreas costeiras.

Molecular cloning and expression of a putative crustacean hyperglycemic hormone of Litopenaeus vannamei in Pichia pastoris

Crustacean hyperglycemic hormone (CHH) is the most abundant and best studied member of the CHH/MIH/GIH neuropeptide hormone family. CHH plays a major role in controlling glucose levels in the hemolymph, and it also has significance in regulating molting, reproduction, and osmoregulation. In contrast, molt-inhibiting hormone (MIH) is responsible for maintaining animals in an intermolt stage. In this study, Liv-MIH-1 cDNA, which encodes a mature neuropeptide from the eyestalk of white shrimp, Litopenaeus vannamei, was expressed in methylotrophic yeast (Pichia pastoris KM71) under the control of an alcohol oxidase promoter. Recombinant Liv-MIH-1 was secreted into the culture medium using the á-factor prepro-sequence without Glu-Ala repeats. The expected protein, which had an apparent molecular mass of 12.1 kDa, was detected by Tricine-SDS-PAGE analysis and confirmed by Western blot. Pure recombinant Liv-MIH-1 was obtained by affinity chromatography, and N-terminal sequence analysis confirmed expression of the protein. Biological assays for CHH and MIH activity were also performed. Purified recombinant Liv-MIH-1 showed the ability to elevate the glucose level of hemolymph of L. vannamei, but molting was unaffected. Since these results are in agreement with the high structural and phylogenetic similarity that has been observed between Liv-MIH-1 and other CHH neuropeptides we propose to rename the protein Liv-CHH-SG1.