Purificação de lacases PPO-I de Botryosphaeria rhodina

Laccases are glycoprotein polyphenol oxidases which are involved in fungal pathogenicity and they are also useful for biotechnological applications. The ligninolytic ascomycete, Botryosphaeria rhodina, has been studied as producer of exopolysaccharide and PPO-I and PPO-II laccases induced by veratryl alcohol. However, as the induced laccases have not been isolated, the aim of this study was to purify the enzyme and to identify the carbohydrates constituents of the glycosidic moiety. The fungus was cultivated on broth Vogel, 1% glucose and 30.4mM veratryl alcohol during 4.5 days at 28°C/180 rpm. The extracellular fluid showed high carbohydrate concentration and the stability of PPO-I laccase under conditions of refrigeration and freezing at 4°C-18°C over 40 days. The purification was developed by ultrafiltration using a NMWL 100 and 30 kDa membrane, gelfiltration on Sephadex G-100, and ion-exchange chromatography on DEAE-cellulose. The purified laccase was identified as a glycoprotein, weight molecular 113 kDa, consisting of 40% protein and 60% carbohydrate identified by HPAEC-PAD as fucose, galactose, mannose, glucose and glucosamine.

Network reconfiguration and loss allocation in a deregulated environment of distribution systems

Low flexibility and reliability in the operation of radial distribution networks make those systems be constructed with extra equipment as sectionalising switches in order to reconfigure the network, so the operation quality of the network can be improved. Thus, sectionalising switches are used for fault isolation and for configuration management (reconfiguration). Moreover, distribution systems are being impacted by the increasing insertion of distributed generators. Hence, distributed generation became one of the relevant parameters in the evaluation of systems reconfiguration. Distributed generation may affect distribution networks operation in various ways, causing noticeable impacts depending on its location. Thus, the loss allocation problem becomes more important considering the possibility of open access to the distribution networks. In this work, a graphic simulator for distribution networks with reconfiguration and loss allocation functions, is presented. Reconfiguration problem is solved through a heuristic methodology, using a robust power flow algorithm based on the current summation backward-forward technique, considering distributed generation. Four different loss allocation methods (Zbus, Direct Loss Coefficient, Substitution and Marginal Loss Coefficient) are implemented and compared. Results for a 32-bus medium voltage distribution network, are presented and discussed.

Multiplex PCR use for Staphylococcus aureus identification and oxacillin and mupirocin resistance evaluation

Oxacillin-resistant Staphylococcus aureus represents a serious problem in hospitals worldwide, increasing infected patients' mortality and morbidity and raising treatment costs and internment time. In this study, the results of using the Multiplex PCR technique to amplify fragments of the genes femA (specific-species), mecA (oxacillin resistance) and ileS-2 (mupirocin resistance) were compared with those of tests conventionally used to identify S. aureus isolates and ascertain their resistance to drugs. Fifty S. aureus strains were isolated from patients receiving treatment at UNOESTE University Hospital in Presidente Prudente, SP, Brazil. The 686 bp fragment corresponding to the gene femA was amplified and detected in all the isolates. On the other hand, the 310 bp fragment corresponding to the mecA gene was amplified in 29 (58%) of the isolates. All of the isolates showed sensitivity to mupirocin in the agar diffusion test, which was corroborated by the lack of any amplicon of the 456 bp fragment corresponding to the ileS-2 gene, in the PCR bands. The conventional tests to identify S. aureus and detect resistance to oxacillin and mupirocin showed 100% agreement with the PCR Multiplex results. The use of techniques for rapid and accurate identification of bacteria and assessment of their resistance may be valuable in the control of infection by resistant strains, allowing the rapid isolation and treatment of an infected patient. However, the results demonstrate that traditional phenotypic tests are also reliable, though they take more time.

Pathological and molecular analysis of Turkey coronavirus replication in captive wild Turkey embryos (broad-breasted bronze breeder)

The degree of genetic and pathologic variation exhibited by a turkey Coronavirus (TCoV) strain was investigated after nine serial passages in 25-day-old turkey embryos obtained from wild broad-breasted bronze breeders. In spite of spleen, liver, kidneys, cloacal bursa and thymus have been collected and analysed, the main histopathological changes were only documented in the intestine sections. Microscopic lesions were characterized as mild enteritis, low degree of enterocyte vacuolization and detachment of the intestinal villous after five consecutive passages and were considered absent in the last passages. Genealogic analysis based on S1 and S2 DNA sequences suggested that Brazilian isolate might be considered as originated from TCoV strains circulating in the United States, as 100% identity with TCoV-Gl strain. Although S1 S2 sequences from each passage revealed no significant point mutations, and no correlation could be speculate between S2 nucleotide changes and pathologic features in infected embryos. This is the first demonstration of wild turkey embryos as a model for TCoV isolation and propagation.

Impacts of enemy-mediated effects and the additivity of interactions in an insect trophic system

In this study, we used data from both experiments and mathematical simulations to analyze the consequences of the interacting effects of intraguild predation (IGP), cannibalism and parasitism occurring in isolation and simultaneously in trophic interactions involving two blowfly species under shared parasitism. We conducted experiments to determine the short-term response of two blowfly species to these interactions with respect to their persistence. A mathematical model was employed to extend the results obtained from these experiments to the long-term consequences of these interactions for the persistence of the blowfly species. Our experimental results revealed that IGP attenuated the strength of the effects of cannibalism and parasitism between blowfly host species, increasing the probability of persistence of both populations. The simulations obtained from the mathematical model indicated that IGP is a key interaction for the long-term dynamics of this system. The presence of different species interacting in a tri-trophic system relaxed the severity of the effects of a particular interaction between two species, changing species abundances and promoting persistence through time. This pattern was related to indirect interactions with a third species, the parasitoid species included in this study. © 2012 The Society of Population Ecology and Springer Japan.

No evidence for association of the CD40, CD40L and BLYS polymorphisms, B-cell co-stimulatory molecules, with Brazilian endemic Plasmodium vivax malaria

Background: Plasmodium vivax is the most prevalent malaria species in Brazil. The parasite-host coevolutionary process can be viewed as an 'arms race', in which adaptive genetic changes in one are eventually matched by alterations in the other. Methods: Following the candidate gene approach we analyzed the CD40, CD40L and BLYS genes that participate in B-cell co-stimulation, for associations with P. vivax malaria. The study sample included 97 patients and 103 controls. We extracted DNA using the extraction and purification commercial kit and identified the following SNPs: 21C.T in the CD40 gene, 2726T.C in the CD40L gene and the 2871C.T in the BLyS gene using PCR-RFLP. We analyzed the genotype and allele frequencies by direct counting. We also compared the observed with the expected genotype frequencies using the Hardy-Weinberg equilibrium. Results: The allele and genotype frequencies for these SNPs did not differ statistically between patient and control groups. Gene-gene interactions were not observed between the CD40 and BLYS and between the CD40L and BLYS genes. Overall, the genes were in Hardy-Weinberg equilibrium. Significant differences were not observed among the frequencies of antibody responses against P. vivax sporozoite and erythrocytic antigens and the CD40 and BLYS genotypes. Conclusions: The results of this study show that, although the investigated CD40, CD40L and BLYS alleles differ functionally, this variation does not alter the functionality of the molecules in a way that would interfere in susceptibility to the disease. The variants of these genes may influence the clinical course rather than simply increase or decrease susceptibility. © Royal Society of Tropical Medicine and Hygiene 2013. All rights reserved.

Characterization of flavonoids and phenolic acids in Myrcia bella cambess. Using FIA-ESI-IT-MSn and HPLC-PAD-ESI-IT-MS combined with NMR

The leaves of Myrcia DC. ex Guill species are used in traditional medicine and are also exploited commercially as herbal drugs for the treatment of diabetes mellitus. The present work aimed to assess the qualitative and quantitative profiles of M. bella hydroalcoholic extract, due to these uses, since the existing legislation in Brazil determines that a standard method must be developed in order to be used for quality control of raw plant materials. The current study identified eleven known flavonoid-O-glycosides and six acylated flavonoid derivatives of myricetin and quercetin, together with two kaempferol glycosides and phenolic acids such as caffeic acid, ethil galate, gallic acid and quinic acid. In total, 24 constituents were characterized, by means of extensive preparative chromatographic analyses, along with MS and NMR techniques. An HPLC-PAD-ESI-ITMS and FIA-ESI-IT-MSn method were developed for rapid identification of acylated flavonoids, flavonoid-O- glycosides derivatives of myricetin and quercetin and phenolic acids in the hydroalcoholic M. bella leaves extract. The FIA-ESI-IT-MS techinique is a powerful tool for direct and rapid identification of the constituents after isolation and NMR characterization. Thus, it could be used as an initial method for identification of authentic samples concerning quality control of Myrcia spp extracts. © 2013 by the authors.

Estudo químico de Jatropha curcas e de Jatropha gossypifolia nativas e cultivadas: avaliação de ciclopeptídeos em função de habitat e hábito : prospecção e atividade biológica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A comparison of microbiological and molecular detection of vaginal Lactobacillus spp. between mares and women

A possible explanation for endometritis in mares is ascendant contamination from the vagina. The presence of Lactobacillus spp. is considered to be important in women for a healthy vaginal environment; however, there are few studies in mares related to the presence of Lactobacillus in the vaginal flora of healthy mares. The present work aims to determine the occurrence of Lactobacillus spp. in the vaginal micro-environment of mares. A total of 35 crossbred multiparous mares, aged between 4 and 12 years, with no history of reproductive problems and with healthy reproductive tracts, were used. Two vaginal swabs were obtained from the mares during estrus for Lactobacillus isolation and PCR evaluation. Ten human female volunteers, aged between 24 and 35 years, sexually active, with no history of gynecological diseases and treatments in the past two years were used. Lactobacillus spp. were isolated from 5.7% of the mares' vaginal samples and from 90% of the women's vaginal samples. Lactobacillus DNA was detected by PCR in 22.9% of the mares' vaginal samples and in all of the vaginal samples from the healthy women. The primers used here were demonstrated to have in silico specificity for the detection of L. equi (AB425924.1), L. pantheris (DQ471798.1) and L. mucosae (DQ471799.1), but they did not anneal on Enterococcus faecalis (EU887827.1) or E. faecium (EU887814.1). In conclusion, this study showed a low occurrence of Lactobacillus spp. in mares, suggesting that this bacterium may not play a fundamental role in the equilibrium of the vaginal micro- environment of normal mares.

Commercial Bovine Proteoglycan Is Highly Arthritogenic and Can Be Used as an Alternative Antigen Source for PGIA Model

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

HPLC-DAD based method for the quantification of flavonoids in the hydroethanolic extract of Tonina fluviatilis Aubl. (Eriocaulaceae) and their radical scavenging activity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determinants of bird species richness, endemism, and island network roles in Wallacea and the West Indies: is geography sufficient or does current and historical climate matter?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inter- and intra-specific diversity of Cuban Pinguicula (Lentibulariaceae) based on morphometric analyses and its relation with geographical distribution

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Species limits, phylogeographic and hybridization patterns in Neotropical leaf frogs (Phyllomedusinae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Produção de anticorpos monoclonais murinos dirigidos contra células-tronco mesenquimais de coelho

Mesenchymal stem cells (MSCs) are a heterogeneous population of cells that proliferate in vitro as plastic-adherent cells, have fibroblast-like morphology and can differentiate into bone, cartilage and fat cells. Therapeutic potential of MSCs have been studied in experimental models, such as rabbit, in Laboratory of Cell Engineering of Botucatu. However, no specific markers have been reported for expanded rabbit MSCs, which hampers the isolation of pure MSC populations by immunophenotypic characterization. Thus, the objective of this study was to produce monoclonal antibodies (mAbs) to rabbit MSCs. MSCs derived from rabbit bone marrow (BM) were isolated, cultured, expanded ex vivo, and immunized into three BALB/c mices, and spleen cells subsequently harvested were used to generate hibridoma cell lines secreting antibodies against MSCs. Hybridoma cells were screened by flow cytometry and antibody-producing cells were subjected to subsequent rounds of retests. MSC1-160 obtained the best positivity for IgG expression and was cloned by limiting dilutions and micromanipulation. Ascitic fluid from ten best clones was purified by affinity chromatography in Protein A-sepharose CL-4B column and purification control was performed by electrophoresis in agarose gels. The purified IgG were tested against rabbit MSCs, obtaining high positivity by flow Cytometry. In conclusion, we developed 10 mAbs, MSC1-160 A20, A30, A41, A47, A55, A60, A63, A69, A81, and A82, that recognize rabbit MSC cell surface antigens showing potential for immunophenotypic characterization of rabbit MSC cell lines