Comparação entre dois métodos de detecção de DNA de papilomavírus humano em carcinoma epidermoide de lábio

Introdução: Recentemente o papilomavírus humano (HPV) tem sido associado à carcinogênese oral. A metodologia empregada na detecção do vírus é uma das maiores causas observadas da grande variabilidade nas taxas de detecção do HPV. Objetivo: Este estudo comparou a sensibilidade de detecção do DNA do HPV em casos de carcinoma epidermoide de lábio utilizando a amplificação do DNA viral por reação em cadeia da polimerase (PCR) ou nPCR. Material e método: Foram utilizadas 33 amostras provenientes de casos de carcinoma epidermoide de lábio. Para as extrações do DNA utilizou-se o sistema QIAamp DNA Mini Kit. Como controle interno utilizou-se o gene da b-globina. Das 33 amostras iniciais, 30 foram positivas para o gene b-globina, sendo utilizadas para detectar o DNA viral. Comparou-se a amplificação do DNA viral pelos métodos da PCR com os oligonucleotídeos MY09/MY11 e nPCR, empregando-se os pares de oligonucleotídeos iniciadores MY09/MY11 e, na segunda etapa, o par GP5+/GP6+. O controle positivo para a presença do DNA do HPV utilizado foi a linhagem de células HeLa e, como controle negativo, a mistura de amplificação sem DNA. A análise dos produtos de PCR e nPCR para HPV foi realizada por eletroforese em gel de poliacrilamida a 8%. Resultados: Utilizando-se o método da PCR, a amplificação do DNA do HPV foi constatada em dois casos. Com a nPCR foi verificada presença de DNA viral em 13 das 30 amostras. Conclusão: Com a utilização da nPCR, a detecção do HPV nos casos estudados aumentou mais de seis vezes.

5S rDNA characterization in twelve Sciaenidae fish species (Teleostei, Perciformes): depicting gene diversity and molecular markers

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

Extensive antigenic and genetic variation among foot-and-mouth disease type A viruses isolated from the 1994 and 1995 foci in São Paulo, Brazil

Nine foot-and-mouth disease virus (FMDV) type A isolates recovered from the field FMD foci in São Paulo State, Brazil, during 1994 and 1995 (a period preceding the last reported focus of FMD in 1996 in this state) were compared among themselves and with the reference vaccine strain A(24)Cruzeiro. The techniques used were sandwich ELISA, virus neutralization (VN), polyacrylamide gel electrophoresis (PAGE) of the structural polypeptides and direct sequencing of the VP1-coding region (1D gene). Results of VN were recorded as serological relationships R and those from ELISA were expressed as percentage of the homologous reaction r. ELISA and VN gave comparable results (correlation coefficient, 0.936) allowing assignment of these field viruses to four groups which were distinct from the A(24)Cruzeiro strain. PAGE and ID nucleotide sequencing were also able to distinguish between these viruses. The high level:of genetic and antigenic variation found when comparing the A(24)Cruzeiro vaccine strain and type A strains recovered, from the last identified foci of FMD came from a formerly endemic area where vaccination with polyvalent vaccines (O(1)Campos, A(24)Cruzciro and C(3)Indaial) had been extensively applied. The similarity between the results of the serological and genetic analyses suggest that the antigenic differences found are mainly located in the 1D protein. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Characterisation of protease activity in extracellular products secreted by Giardia duodenalis trophozoites treated with propolis

Results from our laboratory revealed propolis activity on Giardia trophozoites proliferation. Since therapeutic agents can inhibit the activity of proteases related to relevant biologic and physiologic processes of parasites, this study was undertaken to characterise the proteolytic activity of excretory/secretory products (ESP) of trophozoites treated with propolis. ESP was obtained from culture supernatants of trophozoites exposed to 250 and 500 mu g mL(-1) of propolis. ESP were tested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the protein profiles and the protease activity was assayed in gelatin-containing gels. Synthetic inhibitors were used to characterise the protease classes. Treated and non-treated ESP showed a similar protein and hydrolysis pattern. A simple pattern of protein composed by five evident bands of approximately 167, 132, 79, 61 and 51 kDa was found, and the zymograms comprised hydrolysis zones distributed from > 170 to 23 kDa. No inhibition was seen on protease activity of propolis-treated trophozoites, whose hydrolysis pattern was similar to control. One may conclude that both ESP degraded gelatin and the activity was predominantly due to cysteine proteases. Although propolis had no effect on the proteolytic activity, further studies could identify the active constituents responsible for propolis antigiardial activity and their mechanisms of action.

Down-regulation of MyoD gene expression in rat diaphragm muscle with heart failure

Diaphragm myopathy has been described in patients with heart failure (HF), with alterations in myosin heavy chains (MHC) expression. The pathways that regulate MHC expression during HF have not been described, and myogenic regulatory factors (MRFs) may be involved. The purpose of this investigation was to determine MRF mRNA expression levels in the diaphragm. Diaphragm muscle from both HF and control Wistar rats was studied when overt HF had developed, 22 days after monocrotaline administration. MyoD, myogenin and MRF4 gene expression were determined by RT-PCR and MHC isoforms by polyacrylamide gel electrophoresis. Heart failure animals presented decreased MHC IIa/IIx protein isoform and MyoD gene expression, without altering MHC I, IIb, myogenin and MRF4. Our results show that in HF, MyoD is selectively down-regulated, which might be associated with alterations in MHC IIa/IIx content. These changes are likely to contribute to the diaphragm myopathy caused by HF.

Giardia duodenalis: protein substrates degradation by trophozoite proteases

The present investigation was undertaken to identify and characterize trophozoite proteases of five axenic strains of Giardia duodenalis isolated in Brazil and the reference strain Portland 1 isolated in the United States. Trophozoite cell lysates of each strain were analysed for the pattern of proteins and for proteolytic activity. Samples were tested in SDS-polyacrylamide gel electrophoresis for the protein profiles, and the detection of proteases in cell lysates was performed using substrate gel electrophoresis [gelatin, collagen, bovine serum albumin (BSA) and haemoglobin] and azocasein assays. Indeed, synthetic inhibitors were included in the assays to characterize the protease classes. Differences on the hydrolysis patterns of protein substrates were observed in relation to the substrate composition as much as the Giardia trophozoite strain. The substrate-containing gels revealed hydrolysis bands with molecular masses ranging from > 97 to 20-15 kDa, and most zones were common to the five strains. However, some pronounced differences could be detected in the BTU-11 pattern. Azocasein was also degraded; however, depending on the lysate assayed, the degree of substrate degradation was variable. It was observed that inhibitory effects are substrate-dependent since the activity was predominantly due to cysteine proteases against gelatin, collagen, BSA and azocasein substrates and due to serine against haemoglobin. The presence of aspartic protease and aminopeptidase activity in the lysates was also indicated.

Heparin-binding proteins of seminal plasma in Nellore bulls

O objetivo deste estudo foi identificar proteínas ligadoras à heparina no plasma seminal de touros Nelore (Bos taurus indicus). Para tanto, foram selecionados quatro touros entre 30 e 36 meses de idade e peso aproximado de 500-550kg. Após centrifugação, amostras do plasma seminal foram misturadas e as proteínas ligadoras à heparina foram isoladas por meio da cromatografia por afinidade. As frações após a eluição foram agrupadas para caracterização das bandas protéicas (SDSPAGE, 12,5%). Foram identificadas oito bandas protéicas variando entre 15 e 63kDa. Duas proteínas com 22 e 25kDa foram similares às descritas em touros Bos taurus taurus. Outras proteínas identificadas com 39, 53, 58 e 63kDa ainda não foram descritas e possivelmente sejam específicas para Bos taurus indicus.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

O presente estudo consiste em uma caracterização preliminar da atividade proteolítica de frações de proteínas purificadas a partir de lisados de trofozoítos de cepa isolada e axenizada no Brasil. Frações obtidas por cromatografia líquida (FPLC) foram analisadas quanto ao perfil eletroforético em géis de poliacrilamida (SDS-PAGE) e a atividade proteolítica foi avaliada em géis contendo gelatina como substrato. A caracterização das enzimas foi realizada a partir da análise do efeito de inibidores sintéticos de cisteína-proteases (E-64, IAA), serina-proteases (PMSF), serina e cisteína-proteases (TPCK, TLCK, elastatinal), metalo-proteases (EDTA) e aspartil proteases (pepstatina) sobre a degradação do substrato. Entre 30 frações eluídas, bandas de proteínas foram observadas em oito delas, entretanto, atividade proteolítica foi detectada apenas nas frações 23, 24, 25 e 26. O perfil eletroforético das proteínas revelou poucas bandas distribuídas na faixa de 45 a 18 kDa. Os zimogramas revelaram zonas de proteólise na faixa de aproximadamente 62 a 35 kDa, entretanto destacaram-se as bandas de hidrólise de 62, 55, 53, 50, 46 e 40 kDa. Nos ensaios de inibição, a proteólise foi marcantemente inibida por E-64, TPCK, TLCK e elastatinal. Redução discreta da proteólise foi observada com IAA e PMSF, enquanto que EDTA e pepstatina não promoveram alteração dos perfis de hidrólise. Estas observações são relevantes, especialmente se considerarmos que para elucidar o envolvimento das proteases na relação parasita-hospedeiro, a purificação dessas moléculas é um requisito importante.

Identification and characterization of excretory/secretory products of Giardia duodenalis trophozoites

One of the major questions concerning Giardia is the understanding of pathophysiological processes associated with small intestine abnormalities. There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in the host intestinal epithelium. The present investigation was undertaken to examine the protease activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). E/S products from trophozoites of each strain in conditioned medium were tested with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for the protein profiles, and the protease activity was analyzed using substrate-impregnated SDS-PAGE (gelatin and collagen) and hemoglobin assay. The proteases characterization was based on inhibition assays including synthetic inhibitors. Electrophoresis analysis of E/S products revealed a banding pattern composed by few bands (4 to 6 bands) in the migration region of 123 to 28 kDa. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted substrate degradation and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibitor assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases, although the presence of serine proteases was also indicated. Degradation of substrates including collagen and hemoglobin could lead us to speculate different functions of Giardia excreted/secreted proteases in vivo, but to confirm this possibility and to elucidate its implication on host-parasite interactions, further experiments applying protocols for the purification of proteases are necessary. Even so, our observations are relevant and hold the perspective for the understanding about protease activity in Giardia trophozoites of axenic strain isolated in an endemic area.

Analytical approach to the metallomic of Nile tilapia (Oreochromis niloticus) liver tissue by SRXRF and FAAS after 2D-PAGE separation: Preliminary results

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

Comparative study on extracts from the tissue covering the stingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays

Stingrays are elasmobranchs found along the seacoast and in some rivers of Brazil. Pain is the most conspicuous symptom observed in patients wounded by the bilaterally retroserrate stingers located in the tail, which are covered by glandular and integument tissues. In addition, cutaneous necrosis is commonly observed in injuries caused by freshwater stingrays. The aim of this work was to characterize and compare certain properties of tissue extracts obtained from the glandular tissues covering the stinger apparatus of Potamotrygon falkneri and Dasyatis guttata stingrays. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), tissue extracts have similar bands above 80kDa, but most differences were observed below this molecular mass. Lethal, dermonecrotic and myotoxic activities were detected only in P. falkneri tissue extract. Edematogenic activity was similar and dose dependent in both tissue extracts. Nociceptive activity was verified in both tissue extracts, but P. falkneri presented a two-fold higher activity than D. guttata tissue extract. No direct hemolysis, phospholipase A(2) and coagulant activities were observed in both tissue extracts. Antigenic cross-reactivity was noticed by ELISA and Western blotting, using antisera raised in rabbits. Species-specific sera reacted with several components of both tissue extracts, noticeably above 22 kDa. Both tissue extracts presented gelatinolytic, caseinolytic and fibrinogenolytic activities, which were not caused by the action of metalloproteinases. Hyaluronidase activity was detected only in P. falkneri tissue extract. Our experimental observations suggest that P. falkneri tissue extract is more toxic than D. guttata tissue extract. These results may explain why injuries caused by freshwater stingrays are more severe in human accidents. (c) 2007 Elsevier Ltd. All rights reserved.

Vitellogenin content in fat body and ovary homogenates of workers and queens of Melipona quadrifasciata anthidioides during vitellogenesis

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

Purification of papain from fresh latex of Carica papaya

No presente trabalho apresenta-se um método de cristalização da papaína oriunda do látex fresco de mamão, o qual apresenta uma alta produtividade em relação aos métodos previamente descritos. A metodologia aqui descrita não envolve o uso de reagentes sulfidrílicos, a papaína foi obtida de forma praticamente pura, apresentando uma simples banda quando submetida a eletroforese, e com propriedades idênticas àquelas obtidas por outros métodos. A atividade específica foi determinada utilizando Z-gly-pNP e BAEE como substrato. A papaína obtida por essa metodologia, sem uso de substâncias tais como cisteína e ditiotreitol, apresenta-se na forma de um complexo com inibidores naturais, os quais podem ser removidos por diálise.

Purification and Characterization of Two Extracellular Xylanases from Penicillium sclerotiorum: A Novel Acidophilic Xylanase

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Purification and properties of an acid beta-xylosidase from Penicillium sclerotiorum

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)