Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

Avaliaram-se o efeito do IGF-I na maturação in vitro (MIV) (experimento I) e no desenvolvimento embrionário (DE) (experimento II) de oócitos bovinos fecundados in vitro, quanto às taxas de clivagem (TC), de blastocistos (TB) e de eclosão (TE). Para MIV, complexos cumulus-oócitos imaturos foram cultivados em meio TCM-199 suplementado com HEPES, bicarbonato e piruvato de sódio, aditivos, soro fetal bovino (meio B-199) e gonadotrofinas 14U/ml de PMSG e 7U/ml de hCG). Para o desenvolvimento embrionário, os oócitos/zigotos foram cultivados em meio B-199 com células epiteliais do oviduto bovino em suspensão sob óleo de silicone. As condições de cultivo in vitro para ambos os experimentos seguiram os tratamentos: 1- meio B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. Todas as culturas foram realizadas a 38,5° C em atmosfera com 5% de CO2 e os dados foram analisados pelo teste do qui-quadrado. No experimento I, não houve diferença (P>0,05) entre os tratamentos quanto às TC, TB e TE, quando o meio de MIV foi suplementado com IGF-I. No experimento II, a adição de IGF-I ao meio de DE resultou em aumento na TC (P<0,05) mas não influenciou a TB e a TE. A adição de 200 ng/ml de IGF-I ao meio DE melhorou a TC (71,1%) quando comparada com a TC dos grupos de 100 ng/ml de IGF-I (57,6%) ou controle (56,7%), entretanto não houve diferença quando comparada com a dos grupos de 50 ng/ml (69,4%) ou 10 ng/ml (73,1%) de IGF-I. Não houve efeito benéfico na adição de 10 a 200 ng/ml de IGF-I nos meios de MIV e de DE com relação ao desenvolvimento de embriões produzidos a partir de oócitos maturados e fecundados in vitro.

Fecundação in vitro de ovócitos bovinos com sêmen submetido a diferentes diluidores

O objetivo deste trabalho foi avaliar a influência do diluidor do sêmen no desenvolvimento in vitro de ovócitos bovinos após a maturação e fecundação in vitro. Ejaculado de um reprodutor foi fracionado e submetido a três diluidores: Lactose/gema de ovo (LG), Citrato/gema de ovo (CG) e Tris/gema de ovo (TG). Amostras deste material foram envasadas, congeladas e estocadas em N² e, posteriormente, descongeladas; a fração móvel foi separada por gradiente descontínuo de Percoll. A concentração espermática foi ajustada para 10 x 10(6)/mL e a capacitação espermática, induzida com 10 µg/mL de heparina. Após 24 horas de cultura para maturação in vitro, os ovócitos, aspirados de folículos ovarianos, foram inseminados com sêmen diluído em meio TALP e, após 48 horas de cultura, os zigotos foram transferidos para gotas de meio TCM 199, com 5% de soro fetal bovino, 5% de soro de vaca em estro e suspensão de células epiteliais do oviduto bovino, cobertas com óleo de silicone, e mantidos em cultura por nove dias. Todas as culturas foram realizadas a 38,5ºC em atmosfera com 5% de CO2. Os dados foram analisados pelo teste do qui-quadrado e houve diferença com relação à taxa de clivagem (TC), sendo as médias de 66,0; 69,3; e 54,4% para LG, CG e TG, respectivamente. Não houve diferença entre tratamentos com relação às taxas de mórulas/blastocistos ou de eclosão. O diluidor do sêmen não teve efeito sobre o desenvolvimento in vitro de embriões bovinos, embora a TC tenha sido afetada.

The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesin

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein

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

Differential gene expression analysis of Paracoccidioides brasiliensis during keratinocyte infection

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

New antimicrobial therapies used against fungi present in subgingival sites-A brief review

Although the main reservoir of Candida spp. is believed to be the buccal mucosa, these microorganisms can coaggregate with bacteria in subgingival biofilm and adhere to epithelial cells. The treatment of periodontal disease includes scaling and root planning (SR?) associated with proper oral hygiene. However, some patients may have negative responses to different therapeutic procedures, with a continuous loss of insertion, so the use of antimicrobials is needed as an adjuvant to SRP treatment. The use of a broad-spectrum antibiotic, such as tetracycline and metronidazole, as an aid in periodontal treatment has also been a factor for the development of superinfections by resistant bacteria and Candida species, even in patients with HIV. In the dental practice, the most commonly used antifungals are nystatin and fluconazole. However, the introduction of new drugs like the next generation of azoles is essential before the onset of emergent species in periodontal disease. Plants are good options for obtaining a wide variety of drugs. This alternative could benefit a large population that uses plants as a first treatment option. Plants have been used in medicine for a long time and are extensively used in folk medicine, because they represent an economic alternative, are easily accessible and are applicable to various diseases. Herein, we briefly review the literature pertaining the presence of Candida sp. in periodontal pockets, the conventional antifungal resistance and new therapies that include natural antifungal agents are reviewed. (C) 2011 Elsevier Ltd. All rights reserved.

Isolation and partial characterization of a 30 kDa adhesin from Paracoccidioides brasiliensis

The virulence of Paracoccidioides brasiliensis can be attenuated or lost after long periods of repeated subculturing and reestablished after animal inoculation. Only one adhesin (gp43) has been described until now, among the various identified components of P. brasiliensis, and gp43 shows adhesion to laminin. Thus, the present study was designed to isolate and characterize factors putatively related to the capacity of this fungus to adhere to the host by comparing P brasiliensis samples, taken before and after animal inoculation. The two samples differed in their pattern of adhesion and invasion. The sample recently isolated from animals (Pb18b) demonstrated a greater capacity to adhere and to invade the Vero cells than the one subcultured in vitro (Pb18a). Extract from Ph18b also showed higher levels of protein expression than that from Pb18a, when two-dimensional electrophoresis gels were compared. A protein species of 30 kDa, pI 4.9, was more evident in the Pb18b extract and had properties of adhesin. Laminin, but none of the other extracellular matrix (ECM) components, such as fibronectin, collagen I and IV, bound specifically to the P. brasiliensis 30 kDa protein. The roles of 30 kDa and gp43 in cellular interactions were investigated and the adhesion of P. brasiliensis yeast cells was intensively inhibited by pre-treatment of epithelial cells with 30 kDa protein and gp43. Thus, this study presents evidence that adhesion capacity could be related to virulence, and that a 30 kDa adhesin accumulated differentially in samples with different levels of pathogenicity. This protein and its adhesion characteristics are being published for the first time and may be related to the virulence of P brasiliensis. (c) 2005 Elsevier SAS. All rights reserved.

Identification of a gene encoding adaptin-like protein in the Paracoccidioides brasiliensis genome by random amplified polymorphic DNA analysis

Paracoccidioides brasiliensis isolates are not homogeneous in their patterns of pathogenicity in animals and adhesion to epithelial cells. During this investigation, genotypic differences were observed between two samples of P. brasiliensis strain 18 yeast phase (Pbl 8) previously cultured many times, one taken before (Pb18a) and the other after (Pb18b) animal inoculation. Random amplified polymorphic DNA analysis using the primer OPJ4 distinguished Pb18b from Pbl Ba by one 308 bp DNA fragment, which after cloning and sequencing was shown to encode a polypeptide sequence homologous to the protein beta-adaptin. It is suggested, by comparison to other micro-organisms, that this protein might play an important role in the virulence of P. brasiliensis. This result demonstrates the influence of in vitro subculturing on the genotype of this organism.

Testicular structure and description of the seminal pathway in Leporinus macrocephalus (Anostomidae, Teleostei)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise comparativa da imunoexpressão das proteínas hmlh1 e hmsh2 em carcinomas epidermóides de lábio inferior e queilites actínicas com graus variados de displasia

Lip squamous cell carcinoma (SCC) may develop from a premalignant condition, actinic cheilitis (AC) in 95% of the cases. Both premalignant and neoplastic lip diseases are caused mainly by chronic exposure to the ultraviolet component of solar radiation, especially UVB. This exposure causes disruption of the cell cycle and damage to DNA repair systems, like mismatch repair, altering proteins repair as hMLH1 and hMSH2. This research aimed to investigate the immunohistochemical expression of hMLH1 and hMSH2 proteins in lower lip SCCs and ACs, providing additional information about carcinogenesis of the lower lip. The sample consisted 40 cases of ACs and 40 cases of lower lip SCCs. Histological sections of 3 μm were submitted to immunoperoxidase method, for immunohistochemical analysis of lesions were counted in 1000 cells (positive and negative), data were evaluated both in absolute numbers and percentage of immunostained cells, the latter by assigning scores. Associations of the variables and comparative analysis of biomarker expression were performed by Fisher s exact and Pearson s chi-square, "t" student, one-way ANOVA, Mann- Whitney e Kruskal-Wallis tests. The level of significance was 5%. It was found that, in lower lip SCC, the mean of the proteins was higher in female patients (hMLH1= 369,80 + 223,98; hMHS2 = 534,80 + 343,62), less than 50 years old (hMLH1 = 285,50 + 190,65; hMHS2 = 540,00 + 274,79) and classified as low-grade malignancy (hMLH1 = 264,59 + 179,21; hMHS2 = 519,32 + 302,58), in these data only to sex, for hMLH1 protein, was statistically significant (p=0.034). Comparing the different lesions, we observed that for both hMLH1 and hMSH2 protein, the average of positive epithelial cells decreased as the lesion was graded at later stages. The ACs classified without dysplasia or mild dysplasia had the highest average of immunostained cells (hMLH1 = 721.23 + 88.116; hMHS2 = 781.50 + 156.93). The ACs classified as moderate or severe dysplasia had intermediate values (hMLH1 = 532,86 + 197,72; hMHS2 = 611,14 + 172,48) and SSCs of the lower lip had the lowest averages (hMLH1 = 255,03 + 199,47; hMHS2 = 518,38 + 265,68). There was a statistically significant difference between groups (p<0.001). In conclusion, our data support the hypothesis that changes in immunoexpression of these proteins is related to the process of carcinogenesis of the lower lip

Avaliação imuno-histoquímica das galectinas-1, -3, -4 e -7 em carcinoma epidermóide de língua.

Several studies are carried out with aim to establish parameters to determine biologic behavior of oral squamous cell carcinoma, in order this neoplasm presents high rates of morbidity and mortality. The purpose of present research was to performe a clinic, morphologic and immunohistochemical analysis by the expression of galectins 1, 3, 4 and 7 in 65 cases of tongue squamous cell carcinoma, correlating this expression with clinics (outcome of the disease, metastasis and clinical staging) and morphologic parameters (malignancy histologic gradation system). The clinical and morphologic parameters analysed and expression of galectins 1, 3, 4 and 7 were submitted to statistical analysis (Qui2 test), observing that can be utilized as indicators of the biological behavior of the tongue squamous cell carcinoma. The galectin 1 was expressed in 87,7% of cases studied and it exhibit statistically significant correlation with metastasis (p=0,033) and clinical staging (p=0,016), it is located mostly in the citoplasm of the stomal cells. The immunoexpression of galectin 3 in 87,7% of cases was correlated with the presence of metastasis (p=0,033) and malignancy histological gradation system (p=0,031), observed, mostly of cases, in tongue squamous cell carcinoma of malignancy high grading. The galectin 4 showed no statistical significance to any of the parameters evaluated. The expression of galectin 7 in 73,8% of cases showed statistically significant correlation with the malignancy histologic grading (p=0,005), which is marking exclusively found in neoplastic epithelial cells, in the mostly of cases, it is found in cytoplasm and membrane (50%). The expressive immunopositivy of the galectins 1, 3 and 7, observed in this research, leads us to suggest a broad participation of these proteins in oral carcinogenesis, and its possible use as markers of biological behavior and tumor progression in cases of squamous cell carcinoma of the tongue

Sebaceous metaplasia in a canine mammary gland non-infiltrative carcinoma with myoepithelial component

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

Relationship among oxidative DNA damage, gastric mucosal density and the relevance of cagA, vacA and iceA genotypes of Helicobacter pylori

The aim of this study was to evaluate the relationship among oxidative DNA damage, density of Helicobacter pylori and the relevance of cagA, vacA and iceA genotypes of H. pylori. Gastric epithelial cells were isolated from 24 uninfected patients, 42 H. pylori infected patients with gastritis, and 61 patients with gastric cancer. Oxidative DNA damage was analyzed by the Comet assay, the density of H. pylori was measured by real-time polymerase chain reaction (PCR), and allelic variants of cagA, vacA and iceA were identified using the PCR. Infected patients by Helicobacter pylori cagA(+), vacAs1 m1 and iceA1 genotype showed higher levels of oxidative DNA damage than infected patients with H. pylori cagA(-), vacAs2 m2 and iceA2 genotypes and uninfected patients. Density of H. pylori did not influence oxidative DNA damage. Our results indicate that H. pylori genotype is more relevant than density for oxidative DNA damage.

Timoma em cão. Relato de caso

Uma cadela, Dachshund, quatro anos, foi encaminhada ao H.V. da FMVZ-UNESP-Botucatu-SP com histórico de prostração, emagrecimento progressivo, anorexia, cansaço e tosse seca. Ao exame clínico observou-se apatia, secreção nasal serosa bilateral e dispnéia intensa. Radiografia torácica demonstrou efusão pleural, edema pulmonar acentuado, desvio dorsal da traquéia e possível massa mediastinal. A ultra-sonografia, após drenagem torácica, confirmou a presença de massa mediastinal, sendo classificada como neoplasia maligna de origem epitelial pela citologia aspirativa por agulha fina. O animal veio a óbito logo após o diagnóstico de neoplasia. À necropsia constatou-se massa mediastinal encapsulada, consistência macia e superfície de corte com coloração branco-acinzentada e áreas necrótico-hemorrágicas, localizada na região ântero-ventral do tórax. O exame histopatológico demonstrou células epiteliais neoplásicas, células linfóides (timócitos) com morfologia normal, vasos sangüíneos de pequeno e médio calibre, formações císticas com conteúdo eosinofílico e corpúsculos de Hassall. A imunohistoquímica apresentou positividade para citoqueratina AE1/AE3 e UCHL, confirmando o diagnóstico de timoma.