Structural and Gene Characterization of a New Antifungal Peptide Obtained from Penicillium crustosum FP11 Strain

A novel antifungal peptide, PcAFP (6.48 kDa, pI 8.83 ), was obtained from the culture supernatant of the fungus Penicillium crustosum . The gene encoding the PcAFP peptide was isolated b ased on its homologue in Penicillium chrysogenum , PgAFP. PcAFP is a small, cystine-rich peptide, and th e mature peptide consists of 58 amino acid residues. The i mmature P. crustosum antifungal protein (AFP) showed 95.65% identity to the antifungal prote in of P. chrysogenum , while the mature peptide showed 98.28% identity with PgAFP. Molecular modeling of the tertiary structure of the mature peptide revealed details of the conserved stru cture of the AFPs, such as the ? -barrel motif stabilized by three disulfide bonds and the ? -core motif. Analysis of the extract by 16% tricine SD S- PAGE showed a 6.9 kDa peptide, which was close to the pr edicted molecular mass of the mature peptide of 6.48 kDa. Assays of antimicrobial activity , performed by broth microdilution using the crude extract obtained from the culture medium, showe d activity against Candida albicans . These results demonstrate the conservation of the PcAPF gene and the high level of identity with the PgAFP antifungal protein of P. chrysogenum . Given these structural and biochemical characteristics, PcAFP could be a potential candidate for future investigations that may aid in the development of new antifungal compounds.

Enhance of Cellulase Production and Biomass Degradation by Transformation of the Trichoderma reesei RUT-C30∆ zface1 Strain

Abstract The second-generation bioethanol employs lignocellulosic materials degraded by microbial cellulases in their production. The fungus Trichoderma reesei is one of the main microorganisms producing cellulases, and its genetic modification can lead to the optimization in obtaining hydrolytic enzymes. This work carried out the deletion of the sequence that encodes the zinc finger motif of the transcription factor ACE1 (cellulase expression repressor I) of the fungus T. reesei RUT-C30. The transformation of the RUT-C30 lineage was confirmed by amplification of the 989 bp fragment relative to the selection marker, and by the absence of the zinc finger region amplification in mutants, named T. reesei RUT-C30Δzface1. The production of cellulases by mutants was compared to RUT-C30 and measured with substrates carboxymethylcellulose (CMC), microcrystalline cellulose (Avicel®) and Whatman filter paper (PF). The results demonstrated that RUT-C30Δzface1 has cellulolytic activity increased 3.2-fold in Avicel and 2.1-fold in CMC and PF. The mutants presented 1.4-fold higher sugar released in the hydrolysis of the biomass assays. These results suggest that the partial deletion of ace1 gene is an important strategy in achieving bioethanol production on an industrial scale at a competitive price in the fuel market.

High levels of ß-xylosidase in Thermomyces lanuginosus: potential use for saccharification

ABSTRACT A new strain of Thermomyces lanuginosus was isolated from the Atlantic Forest biome, and its β-xylosidases optimization in response to agro-industrial residues was performed. Using statistical approach as a strategy for optimization, the induction of β-xylosidases activity was evaluated in residual corn straw, and improved so that the optimum condition achieved high β-xylosidases activities 1003 U/mL. According our known, this study is the first to show so high levels of β-xylosidases activities induction. In addition, the application of an experimental design with this microorganism to induce β-xylosidases has not been reported until the present work. The optimal conditions for the crude enzyme extract were pH 5.5 and 60 °C showing better thermostability at 55 °C. The saccharification ability of β-xylosidase in the presence of hemicellulose obtained from corn straw raw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50 °C. Our data strongly indicated that the β-xylosidases activities was not subjected to the effects of potential enzyme inhibitors often produced during fermentation process. These data suggest the application of this enzyme studied for saccharification of hemicellulose, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production.

DOSE RESPONSE EFFECT OF Paracoccidioides brasiliensis IN AN EXPERIMENTAL MODEL OF ARTHRITIS / Efeito da dose resposta de Paracoccidioides brasiliensis no modelo experimental de artrite

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb) and corresponds to prevalent systemic mycosis in Latin America. The aim of the present work was to evaluate the dose response effect of the fungal yeast phase for the standardization of an experimental model of septic arthritis. The experiments were performed with groups of 14 rats that received doses of 103, 104 or 105 P. brasiliensis (Pb18) cells. The fungi were injected in 50 µL of phosphate-buffered saline (PBS) directly into the knee joints of the animals. The following parameters were analyzed in this work: the formation of swelling in knees infused with yeast cells and the radiological and anatomopathological alterations, besides antibody titer by ELISA. After 15 days of infection, signs of inflammation were evident. At 45 days, some features of damage and necrosis were observed in the articular cartilage. The systemic dissemination of the fungus was observed in 11% of the inoculated animals, and it was concluded that the experimental model is able to mimic articular PCM in humans and that the dose of 105 yeast cells can be used as standard in this model.

A paracoccidioidomicose (PCM) é causada pelo fungo dimórfico Paracoccidioides brasiliensis (Pb) e corresponde à micose sistêmica de maior prevalência na América Latina. O objetivo do presente trabalho foi avaliar a dose resposta de leveduras do fungo para padronização do modelo experimental de artrite séptica. Os experimentos foram realizados com grupos de 14 ratos que receberam doses de 103, 104 ou 105 células de P. brasiliensis (Pb18). Os fungos foram injetados em 50 µL de solução salina em tampão fosfatado (PBS) diretamente na articulação do joelho dos animais. Os seguintes parâmetros foram analisados neste trabalho: a formação de edema nos joelhos infundidos com as células das leveduras e alterações radiológicas, anatopalógicas além de titulação de anticorpos por Elisa. Após 15 dias de infecção, os sinais de inflamação foram evidentes. Aos 45 dias, algumas características de dano e necrose foram observadas na cartilagem articular. A disseminação sistêmica do fungo foi observada em 11% dos animais inoculados, concluiu-se que o modelo experimental é capaz de mimetizar a PCM articular em humanos e que a dose de 105 leveduras representa a dose padrão para o desenvolvimento do modelo.

New aspects on atrazine biodegradation

The world practice of using agrochemicals for long periods, in an indiscriminated and abusive way, has been a concern of the authorities involved in public health and sustainability of the natural resources, as a consequence of environmental contamination. Agrochemicals refer to a broad range of insecticides, fungicides and herbicides, and among them stands out atrazine, a herbicide intensively used in sugarcane, corn and sorghum cultures, among others. Researches have demonstrated that atrazine has toxic effects in algae, aquatic plants, aquatic insects, fishes and mammals. Due to the toxicity and persistence of atrazine in the environment, the search of microbial strains capable of degrading it is fundamental to the development of bioremediation processes, as corrective tools to solve the current problems of the irrational use of agrochemicals. This review relates the main microbial aspects and research on atrazine degradation by isolated microbial species and microbial consortia, as well as approaches on the development of techniques for microbial removal of atrazine in natural environments.

A prática mundial do uso de agroquímicos por períodos extensos, de maneira indiscriminada e abusiva, tem mobilizado as autoridades envolvidas em saúde pública e sustentabilidade de fontes naturais, como uma conseqüência da contaminação ambiental. Agroquímicos referem-se a uma ampla variedade de inseticidas, fungicidas e herbicidas, entre estes a atrazina, um herbicida intensivamente usado em culturas de cana-de-açúcar, milho, sorgo, entre outros. Pesquisadores têm demonstrado que a atrazina tem efeitos tóxicos em algas, plantas aquáticas, insetos aquáticos, peixes e mamíferos. Devido à toxicidade e à persistência da atrazina no ambiente, a busca de linhagens microbianas capazes de degradá-la é fundamental para o desenvolvimento de processos de biorremediação, com uma ferramenta corretiva para solucionar problemas decorridos do uso irracional de agroquímicos. Esta revisão relata os principais aspectos microbianos e pesquisas da degradação da atrazina por espécies microbianas isoladas e consórcio microbiano, bem como avanços no desenvolvimento de técnicas para remoção microbiana da atrazina no ambiente natural.

Vacina de DNA contra doenças infecciosas / DNA vaccines against infectious diseases

A imunização ativa é um importante aliado da medicina moderna para o controle de doenças infecciosas. Muitas doenças infecciosas já foram controladas ou mesmo erradicadas através do uso de vacinas. No entanto muitas ainda permanecem resistentesàs tentativas de controle. O desenvolvimento recente da Imunologia e da Biologia Molecular permitiu o desenvolvimento da vacina de DNA, a qual apresenta seqüências do material genético do agente infeccioso que codificam antígenos imunodominantes. Ao contrário das vacinas tradicionais, as vacinas de DNA têm a capacidade de gerar resposta celular e humoral. Discutiremos nesse artigo as vacinas de DNA que estão sendo desenvolvidas para algumas doenças infecciosas causadas por vírus, bactéria e parasitas e também algumas considerações sobre segurança para uso destas vacinas nos seres humanos.

Active immunization is an important ally of modern medicine for the control of infectious diseases. Many infectious diseases have already been either controlled or eradicated through the use of vaccines. However, there are many that remain resistant to attempts at controlling them. The recent progress in Molecular Immunology and Biology have led to the development of the DNA vaccination. It presents sequences of genetic material of the infected agent which codifies immuno-dominant antigens. Differently from traditional vaccines, DNA vaccines are able to generate humoral and cellular responses. In this article, I discuss DNA vaccines which are being developed against certain infectious diseases caused by virus, bacteria and parasites. I also consider safety aspects with regard to the use of these vaccines in human beings.

Validação do método de detecção de Chlamydia trachomatis por Reação em Cadeia da Polimerase em tempo real / Validation of a Chlamydia trachomatis detection method by Real Time Polymerase chain reaction

Infecções causadas por Chlamydia trachomatis são recentemente reconhecidas como as mais prevalentes e estão entre as mais prejudiciais doenças sexualmente transmissíveis (DST) no mundo. A ausência de sintomas dificulta o diagnóstico clínico das infecções clamidiais. Um dos desafios na prevenção das infecções causadas por C. trachomatis é a disponibilidade de diagnósticos laboratoriais com alta sensibilidade, especificidade e reprodutibilidade. No presente estudo, foi comparado o desempenho do testes de Reação em Cadeia da Polimerase (PCR) COBAS Amplicor® Chlamydia trachomatis (Roche) e PCR em tempo real (SYBR® – Green), usados para detecção de Chlamydia trachomatis em espécimes de urina e secreções urogenitais. O COBAS Amplicor® (Roche) é umteste de PCR convencional e foi usado como ferramenta para controle e teste de padrão ouro para diagnóstico no presente trabalho. Um total de 136 amostras de secreção endocervical (61), secreção uretral (14) e urina (61), foram obtidas de pacientes submetidos a testes de detecção de Chlamydia trachomatis no Laboratório Alvaro S/A. Os métodos de PCR em tempo real e COBAS Amplicor® (Roche)foram igualmente sensíveis para o diagnóstico de C. trachomatis em amostras de secreção uretral. Entretanto, em análises de urinae secreção endocervical o teste COBAS Amplicor® (Roche) mostrou 57% e 33% de positividade, respectivamente, contra 47% e 24% de positividade obtidos para as mesmas amostras usando a PCR em tempo real. A maior sensibilidade (100%) obtida usando-seo ensaio de PCR em tempo real, foi em análises das amostras de secreção endocervical. A PCR em tempo real, comparada com o teste padrão ouro, mostrou uma alta especificidade (100%) e um valor preditivo positivo de 100% para as diferentes amostras clínicas estudadas. Em conclusão, os dados apresentados neste estudo indicam que a PCR em tempo real mostrou um alto desempenho para detecção de C. trachomatis em diferentes espécimes clínicos...

Infections caused by Chlamydia trachomatis are now recognized as the most prevalent and are among the most damaging of all sexually transmitted diseases (STD) worldwide. Lack of symptoms makes clinical diagnosis of chlamydial infection difficult. One of the challenge in the prevention of C. trachomatis infections is the availability of laboratory diagnostics with high sensitivity, specificity and reliable. In the present study, the performance of conventional polymerase chain reaction (PCR) and Real-time PCR (SYBRR –Green) tests used for detection of Chlamydia trachomatis in urine and urogenital specimens were compared. The COBAS AmplicorR (Roche) is a conventional PCR and was used as a tool of control and gold standard test for diagnosis in this work. A total of 136 samples of endocervical secretion (61), uretral secretion (14) and urine (61), were obtained from patients submitted of Chlamydia trachomatis detection tests of Laboratório Alvaro S/A. Real-time PCR (SYBRR –Green) and COBAS AmplicorR Chlamydia trachomatis Test (Roche) methods were equally sensitivity for diagnostic of C. trachomatis in uretral secretion samples. However, in analyses of the urine and endocervical secretion samples the COBAS AmplicorR test displayed 57% and 33% of positivity, recpectively, against 47% and 24% of positivity obtained for the same samples using Real-time PCR. The highest sensibility (100%) using Real-timePCR assay was obtained in analyses of endocervical secretion samples. Real-time PCR, comparing with our gold standard showed a high spedificity (100%) and a positive predictive value of 100% for different clinical samples studied. In conclusion, the data presented in this study indicate that PCR - Real time have shown a high performance for detection of C. trachomatis in different clinical specimens. Moreover, this method can be used as a powerful tool in diagnostic and epidemiological investigation of infection associated with C. trachomatis.

Estrutura, expressão e análise funcional do gene codificado Calmodulina no fungo aquático Blastocladiella emersonii / Structure, expression and functional analysis of the gene encoding Calmodulin in the aquatic fungus Blastocladiella emersonii

O único gene para Calmodulina (CaM) e o cDNA correspondente do quitridiomiceto Blastocladiella emersonii foram isolados e caracterizados. O gene CaM é interrompido por três introns e transcrito como uma única espécie de mRNA de 0,7 kb, codificando uma proteína 91 por cento idêntica à CaM humana. A Calmodulina de B. emersonii foi expressa em Escherichia coli como uma proteína de fusão com Glutationa-S-transferase (GST), purificada por cromatografia de afinidade e clivada da porção GST usando uma protease sítio específico. na presença de `Ca²+ï, a CaM de B. emersonii exibiu uma alteração na mobilidade eletroforética semelhante à CaM bovina e foi capaz de ativar a autofosforilação da proteína quinase dependente de `Ca²+ï-CaM (CaMKII) de cérebro de rato...