Association of an insulin-like growth factor 1 gene microsatellite with phenotypic variation and estimated breeding values of growth traits in Canchim cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.

Loss of nuclear poly(A)-binding protein 1 causes defects in myogenesis and mRNA biogenesis

The nuclear poly(A)-binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays a critical role in polyadenylation. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing and weakness in the proximal limb muscles. Although significant data from in vitro biochemical assays define the function of PABPN1 in control of poly(A) tail length, little is known about the role of PABPN1 in mammalian cells. To assess the function of PABPN1 in mammalian cells and specifically in cells affected in OPMD, we examined the effects of PABPN1 depletion using siRNA in primary mouse myoblasts from extraocular, pharyngeal and limb muscles. PABPN1 knockdown significantly decreased cell proliferation and myoblast differentiation during myogenesis in vitro. At the molecular level, PABPN1 depletion in myoblasts led to a shortening of mRNA poly(A) tails, demonstrating the cellular function of PABPN1 in polyadenylation control in a mammalian cell. In addition, PABPN1 depletion caused nuclear accumulation of poly(A) RNA, revealing that PABPN1 is required for proper poly(A) RNA export from the nucleus. Together, these experiments demonstrate that PABPN1 plays an essential role in myoblast proliferation and differentiation, suggesting that it is required for muscle regeneration and maintenance in vivo.

Alcoholism and coagulating gland: Androgen and insulin like growth factor-1 receptor features

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

The Leishmania amazonensis TRF ( TTAGGG repeat-binding factor) homologue binds and co-localizes with telomeres

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

Toll-like receptor 2 knockdown modulates interleukin (IL)-6 and IL-8 but not stromal derived factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzymelinked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

Loss of nuclear poly(A)-binding protein 1 (PABPN1) causes defects in myogenesis and mRNA biogenesis

The nuclear poly(A)-binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays a critical role in polyadenylation. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing and weakness in the proximal limb muscles. Although significant data from in vitro biochemical assays define the function of PABPN1 in control of poly(A) tail length, little is known about the role of PABPN1 in mammalian cells. To assess the function of PABPN1 in mammalian cells and specifically in cells affected in OPMD, we examined the effects of PABPN1 depletion using siRNA in primary mouse myoblasts from extraocular, pharyngeal and limb muscles. PABPN1 knockdown significantly decreased cell proliferation and myoblast differentiation during myogenesis in vitro. At the molecular level, PABPN1 depletion in myoblasts led to a shortening of mRNA poly(A) tails, demonstrating the cellular function of PABPN1 in polyadenylation control in a mammalian cell. In addition, PABPN1 depletion caused nuclear accumulation of poly(A) RNA, revealing that PABPN1 is required for proper poly(A) RNA export from the nucleus. Together, these experiments demonstrate that PABPN1 plays an essential role in myoblast proliferation and differentiation, suggesting that it is required for muscle regeneration and maintenance in vivo.

Prognostic value of vascular endothelial growth factor and hypoxia-inducible factor 1 alpha in canine malignant mammary tumors

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

Molecular characterization of interferon regulatory factor 1 in Bubalus bubalis

Interferon regulatory factor 1 (IRF1) is functionally diverse in the regulation of immune response and is considered to be an important candidate gene for studying disease susceptibility in mammals. In this paper, we characterized the whole sequence of the IRF1 gene in river buffalo (Bubalus bubalis) and compared genomic and the amino acid sequences between different species. The buffalo IRF1 gene was 7099 bp long and organized into 10 exons and nine introns. Its molecular structure showed exactly the same number of exons (10) and introns (nine) in bovids, mice, horses, humans, and chickens. However, rats did not have exon 5, but had the largest exon 4, which suggests that exon 5 was incorporated into exon 4. The coding and the amino acid sequences of the gene showed that identity varied from 73 to 99% at the coding sequence level and from 61 to 100% at the amino acid level when compared with other mammals and chickens. Comparative analysis of the gene sequence between two different buffalo breeds, Murrah and Mediterranean, revealed six potential SNPs that are primarily located in the 5' and 3'UTRs.

DNA methylation in the CTCF-binding site I and the expression pattern of the H19 gene: Does positive expression predict poor prognosis in early stage head and neck carcinomas?

Loss of allele-specific expression by the imprinted genes IGF2 and H19 has been correlated with a differentially methylated region (DMR) upstream to the H19 gene. The H19-DMR contains seven potential CCCTC-binding factor (CTCF) binding sites. CTCF is a chromatin insulator and a multifunctional transcription factor whose binding to the H19-DMR is suppressed by DNA methylation. Our study included a group of 41 head and neck squamous cell carcinoma (HNSCC) samples. The imprinting status of the H19 gene was analyzed in 11 out of 35 positive cases for H19 gene expression, and only 1 of them showed loss of imprinting. We detected a significant correlation (P=0.041, Fisher's exact test) between H19 expression and tumor recurrence. Among H19 positive cases, six were T2, in which five developed recurrence and/or metastasis. Inversely, in the group of tumors that showed no H19 gene expression, 5 out of 24 were T2 and only I presented regional recurrence. These data support the hypothesis that H19 expression could be used as a prognostic marker to indicate recurrence in early stage tumors. We also examined the methylation of the CTCF binding site 1 in a subgroup of these samples. The H19 gene silencing and loss of imprinting were not correlated with the methylation pattern of the CTCF binding site 1. However, the significant correlation between H19 expression and tumor recurrence suggest that this transcript could be a marker for the progression of HNSCC. (c) 2005 Wiley-Liss, Inc.