Holocarboxylase Synthetase-dependent Biotinylation of Histone H4

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysine (K) residues in histones H3 and H4. Histone biotinylation marks play important roles in the repression of genes and retrotransposons. Preliminary studies suggested that K16 in histone H4 is a target for biotinylation by HCS. Here we demonstrated that H4K16bio is overrepresented in repeat regions {pericentromeric alpha satellite repeats; long terminal repeats (LTR)} compared with euchromatin promoters. H4K16bio was also enriched in the repressed interleukin-2 gene promoter. The enrichment at LTR22 and promoter 1 of the sodium-dependent multivitamin transporter (SMVT) depended on biotin supply; and was significantly lower in fibroblasts from an HCS-deficient patient compared with an HCS wild-type control. We conclude that H4K16bio is a real phenomenon and plays a role in the transcriptional repression of repeats and genes. HCS catalyzes the covalent binding of biotin to carboxylases, in addition to its role as a histone biotinyl ligase. HCS null individuals are not viable whereas HCS deficiency is linked to developmental delays and phenotypes such as short life span and low stress resistance. Here, we developed a 96-well plate assay for high-throughput analysis of HCS based on the detection of biotinylated p67 using IRDye-streptavidin and infrared spectroscopy. We demonstrated that the catalytic activity of rHCS depends on temperature and time, and proposed optimal substrate and enzyme concentrations to ensure ideal measurement of rHCS activity and its kinetics. Additionally, we demonstrated that this assay is sensitive enough to detect biotinylation of p67 by endogenous HCS from Jurkat lymphoid cells.

Evaluation of IDDM8 susceptibility locus in a Russian simplex family data set

Type 1 diabetes (TID) susceptibility locus, IDDM8, has been accurately mapped to 200 kilobases at the terminal end of chromosome 6q27. This is within the region which harbours a cluster of three genes encoding proteasome subunit beta 1 (PMSB1), TATA-box binding protein (TBP) and a homologue of mouse programming cell death activator 2 (PDCD2). In this study, we evaluated whether these genes contribute to TID susceptibility using the transmission disequilibrium test of the data set from 114 affected Russian simplex families. The A allele of the G/A1180 single nucleotide polymorphism (SNP) at the PDCD2 gene, which was significant in its preferential transfer from parents to diabetic children (75 transmissions vs. 47 non-transmissionS, x(2) = 12.85, P corrected = 0.0038), was found to be associated with T1D. G/A1180 dimorphism and two other SNPs, C/T771 TBP and G/T(-271) PDCD2, were shown to share three common haplotypes, two of which (A-T-G and A-T-T) have been associated with higher development risk of TID. The third haplotype (G-T-G) was related to having a lower risk of disease. These findings suggest that the PDCD2 gene is a likely susceptibility gene for TID within IDDM8. However, it was not possible to exclude the TBP gene from being another putative susceptibility gene in this region. (c) 2005 Elsevier Ltd. All rights reserved.

Elevated interleukin 6 is induced by prostaglandin E2 in a murine model of inflammation: possible role of cyclooxygenase-2.

Injection of mineral oils such as pristane into the peritoneal cavities of BALB/c mice results in a chronic peritonitis associated with high tissue levels of interleukin 6 (IL-6). Here we show that increased prostaglandin E2 (PGE2) synthesis causes induction of IL-6 and that expression of an inducible cyclooxygenase, Cox-2, may mediate this process. Levels of both PGE2 and IL-6 are elevated in inflammatory exudates from pristane-treated mice compared with lavage samples from untreated mice. The Cox-2 gene is induced in the peritoneal macrophage fraction isolated from the mice. A cause and effect relationship between increased macrophage PGE2 and IL-6 production is shown in vitro. When peritoneal macrophages are activated with an inflammatory stimulus (polymerized albumin), the Cox-2 gene is induced and secretion of PGE2 and IL-6 increases, with elevated PGE2 appearing before IL-6. Cotreatment with 1 microM indomethacin inhibits PGE2 production by the cells and reduces the induction of IL-6 mRNA but has no effect on Cox-2 mRNA, consistent with the fact that the drug inhibits catalytic activity of the cyclooxygenase but does not affect expression of the gene. Addition of exogenous PGE2 to macrophages induces IL-6 protein and mRNA synthesis, indicating that the eicosanoid stimulates IL-6 production at the level of gene expression. PGE2-stimulated IL-6 production is unaffected by addition of indomethacin. Taken together with the earlier finding that indomethacin diminishes the elevation of IL-6 in pristane-treated mice, the results show that PGE2 can induce IL-6 production in vivo and implicate expression of the Cox-2 gene in the regulation of this cytokine.

Efeito da dieta hipocalórica no perfil metabólico e composição corporal de mulheres com e sem Síndrome Metabólica e genótipo Pro12Pro do gene PPARγγ2

A obesidade é uma doença crônica não transmissível, caracterizada pelo excesso de gordura corporal. Então, a gordura acumulada na região abdominal promove resistência à insulina e conseqüentemente alterações metabólicas as quais em conjunto configuram o quadro de síndrome metabólica (SM). O genótipo Pro12Pro parece estar relacionado à menor sensibilidade à insulina, desencadeando o processo fisiopatológico da SM. Então, o objetivo deste trabalho foi avaliar o efeito de uma dieta hipocalórica sobre o perfil metabólico e composição corporal de mulheres com e sem SM com genótipo Pro12Pro no gene PPARγ2. O presente estudo trata-se de um ensaio clínico, onde mulheres entre 30 e 45 anos, obesas grau I, sem SM (n=23) e com SM (n=7) foram submetidas à dieta hipocalórica por 90 dias. A identificação do genótipo foi realizada por reação em cadeia da polimerase (PCR). No início e nos dias 30, 60 e 90 foram avaliados peso corporal, massa magra (MM), massa gorda (MG), componentes da SM, uricemia, insulinemia, leptinemia, adiponectinemia, os índices HOMA-IR e QUICKI. O consumo energético foi avaliado nas 12 semanas de tratamento. Foi utilizado o teste t de Student para amostras independentes foi utilizado para comparar os grupos entre si, e o modelo pareado para comparar a evolução dentro de cada grupo em relação ao início do estudo. Todas as mulheres apresentaram genótipo Pro12Pro. O grupo com SM apresentou menor HDL-c (44,43,2 vs. 56,82,4 mg/dL, p=0,013), e maior triglicerídeo (180,926,7 vs. 89,76,6mg/dL, p=0,014) e VLDL-c (36,25,3 vs. 17,91,3mg/dL, p=0,014) no início do estudo. Ambos os grupos apresentaram redução ponderal (-3,30,7% grupo sem SM e - 4,20,9% grupo com SM) e da circunferência da cintura (-2,40,5% grupo sem SM e - 5,91,4% grupo com SM) significativas. O grupo sem SM reduziu da MG progressivamente até os 90 dias (37,00,8 para 36,60,5%, p=0,02), e com isso aumentou MM (62,00,5 para 63,40,5%, p=0,01), o grupo com SM também reduziu MG ao longo do estudo (32,62,3 para 29,62,4%, p<0,01) e aumentou MM significativamente (62,21,0 para 64,31,3%). A pressão arterial sistólica reduziu no primeiro mês de tratamento no grupo sem SM (de 120,41,8 para 112,32,1 mmHg, p<0,01). No que diz respeito aos parâmetros metabólicos, o grupo sem SM mostrou redução da insulinemia (32,54,2 para 25,92,4U/mL, p=0,05) e aumento da adiponectinemia (4,70,6 para 5,10,8 ng/mL, p=0,02) aos 30 dias, do colesterol total (180,25,8 para 173,85,4 mg/dL, p=0,04), e da leptina (27,01,9 para 18,21,4 ng/mL, p<0,01) aos 60 dias, porém, houve redução do QUICKI aos 90 dias (0,390,03 para 0,350,01, p=0,01). No grupo com SM, a leptinemia reduziu aos 60 dias (20,31,9 para 14,71,1 ng/mL, p=0,01) e a adiponectinemia aos 90 dias (5,71,2 para 7,11,4 ng/mL, p<0,01), também houve remissão de 57,1% dos casos de SM. Sugerimos que, a dieta hipocalórica foi eficaz na redução do peso corporal e da MG, principalmente a localizada na região abdominal. Conseqüentemente, houve melhora considerável do perfil metabólico relacionado à obesidade no grupo sem SM, e também dos marcadores de sensibilidade à insulina e cardioprotetores relacionados à SM, além da remissão dos casos de SM.

Isolation and characterization of Argonaute 2: A key gene of the RNA interference pathway in the rare minnow, Gobiocypris rarus

Argonaute 2 gene plays a pivotal role in RNAi in many species. Herein is the first report of the cloning and characterization of Argonaute 2 gene in fish. The full-length cDNA of Gobiocypris rarus Argonaute 2 (GrAgo2) consisted of 3073 nucleotides encoding 869 amino acid residues with a calculated molecular weight of 98.499 kDa and an estimated isoelectric point of 9.18. Analysis of the deduced amino acid sequence showed the presence of two signature domains, PAZ and Piwi. RT-PCR analysis indicated that GrAgo2 mRNA expression could be detected in widespread tissues. After infection with grass carp reovirus, GrAgo2 expression was up-regulated from 12 h post-injection (p < 0.05) and returned to control levels at 48 h post-injection (p > 0.05). These data imply that GrAgo2 is involved in antiviral defense in rare minnow. (C) 2008 Published by Elsevier Ltd.

Gene structures and promoter characteristics of interferon regulatory factor 1 (IRF-1), IRF-2 and IRF-7 from snakehead Channa argus

Three interferon regulatory factor (IRF) genes, CaIRF-1, CaIRF-2 and CaIRF-7, and their promoters of snakehead (Channa argus) were cloned and characterized. The CaIRF-1 gene consists of ten exons, spans 4.3 kb and encodes a putative peptide of 299 aa. The CaIRF-2 gene consists of nine exons, spans 8 kb and encodes a putative peptide of 328 aa. The gene organizations of CaIRF-1 and CaIRF-2 are very similar to that of human IRF-1 and IRF-2 except more compact. Comparison of exon-intron organization of the two genes indicated a common evolutionary structure, notably within the exons encoding the DNA binding domain (DBD) of the two factors. The CaIRF-7 gene spans 4.1 kb and encodes a putative peptide of 437 aa. However, the gene organization of CaIRF-7 consisting of ten exons is different to human IRF-7a gene which has an intron in 5' UTR. Three CaIRFs share homology in N-terminal encompassing the DBD that contains a characteristic repeat of tryptophan residues. The promoters of CaIRF-1 and CaIRF-2 genes contain the conserved sites for NF-kappa B and Sp1. The gamma-IFN activation sites (GAS) were found in the promoters of CaIRF-1 and CaIRF-7. The promoter of CaIRF-7 contains conserved interferon stimulating response element (ISRE) which is characteristic of IFN-induced gene promoter, and suggests that there also exist intracellular amplifier circuit in fish IFN signal pathway. Moreover, the element GAAANN oriented in both directions is repeated in CaIRF promoter regions, which confers to further inducibility by IFN. The constitutive expression of CaIRF genes were found to increase obviously in response to induction by the known IFN-inducer poly I:C. (c) 2008 Published by Elsevier Ltd.

Gene structure and transcription of IRF-2 in the mandarin fish Siniperca chuatsi with the finding of alternative transcripts and microsatellite in the coding region

The gene of interferon regulatory factor-2 (IRF-2) has been cloned from the mandarin fish (Siniperca chuatsi). The IRF-2 gene has 6,418 nucleotides (nt) and contains eight exons and seven introns, encoding two mRNAs. The two IRF-2 mRNAs each contained an open reading frame of 873 nt, which both translate into the same 291 amino acids but differed in their 5' untranslated region: one mRNA was transcribed initially from the exon 1 bypassing exon 2, while the other was transcribed from the exon 2. The microsatellites (CA repeats) could be found in the carboxyl terminal region of mandarin fish IRF-2, which result in the truncated form molecules. The microsatellites' polymorphism was investigated, and eight alleles were found in 16 individuals. The microsatellites were also examined in IRF-2 of several freshwater perciform fishes. The transcription of the IRF-2 in different tissues with or without poly inosine-cytidine stimulation was analyzed by real-time PCR, and the constitutive transcription of both molecules could be detected in all the tissues examined.

Glycogen synthase 2 is a novel target gene of peroxisome proliferator-activated receptors.

Glycogen synthase 2 (Gys-2) is the ratelimiting enzyme in the storage of glycogen in liver and adipose tissue, yet little is known about regulation of Gys-2 transcription. The peroxisome proliferator-activated receptors (PPARs) are transcription factors involved in the regulation of lipid and glucose metabolism and might be hypothesized to govern glycogen synthesis as well. Here, we show that Gys-2 is a direct target gene of PPARalpha, PPARbeta/delta and PPARgamma. Expression of Gys-2 is significantly reduced in adipose tissue of PPARalpha-/-, PPARbeta/delta-/- and PPARgamma+/- mice. Furthermore, synthetic PPARbeta/delta, and gamma agonists markedly up-regulate Gys-2 mRNA and protein expression in mouse 3T3-L1 adipocytes. In liver, PPARalpha deletion leads to decreased glycogen levels in the refed state, which is paralleled by decreased expression of Gys-2 in fasted and refed state. Two putative PPAR response elements (PPREs) were identified in the mouse Gys-2 gene: one in the upstream promoter (DR-1prom) and one in intron 1 (DR-1int). It is shown that DR-1int is the response element for PPARs, while DR-1prom is the response element for Hepatic Nuclear Factor 4 alpha (HNF4alpha). In adipose tissue, which does not express HNF4alpha, DR-1prom is occupied by PPARbeta/delta and PPARgamma, yet binding does not translate into transcriptional activation of Gys-2. Overall, we conclude that mouse Gys-2 is a novel PPAR target gene and that transactivation by PPARs and HNF4alpha is mediated by two distinct response elements.

Effects of cyclosporin, phenytoin, and nifedipine on the synthesis and degradation of gingival collagen in tufted capuchin monkeys (Cebus apella): Histochemical and MMP-1 and -2 and collagen I gene expression analyses

Background: The purpose of this experimental study was to evaluate the collagen fiber distribution histologically after phenytoin, cyclosporin, or nifedipine therapy and to correlate it with collagen I and matrix metalloproteinase (MMP)-1 and -2 gene expression levels.Methods: Gingival samples from the canine area were obtained from 12 male monkeys (Cebus apella). The mesial part of each sample was assessed by reverse transcription-polymerase chain reaction, whereas the distal part was processed histologically for picrosirius red and hematoxylin and eosin stainings, as well as for collagen IV immunostaining. One week after the first biopsy, the animals were assigned to three groups that received daily oral dosages of cyclosporin, phenytoin, or nifedipine for 120 days. Additional gingival samples were obtained on days 52 and 120 of treatment from two animals from each group on the opposite sides from the first biopsies.Results: Picrosirius red staining showed a predominance of mature collagen fibers in the control group. Conversely, there was an enlargement of areas occupied by immature collagen fibers in all groups at days 52 and 120, which was not uniform over each section. There was a general trend to lower levels of MMP-1 gene expression on day 52 and increased levels on day 120. Phenytoin led to increased levels of MMP-2 and collagen I gene expression on day 120, whereas the opposite was observed in the nifedipine group.Conclusion: Cyclosporin, phenytoin, and nifedipine led to phased and drug-related gene expression patterns, resulting in impaired collagen metabolism, despite the lack of prominent clinical signs.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Background: Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods: In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results: DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion: The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases. © 2009 Santos et al; licensee BioMed Central Ltd.

Capacidade combinatória de linhagens S4 de milho super-doce (Zea mays L.), portadoras do gene shrunken-2

Pós-graduação em Agronomia (Agricultura) - FCA

Safety and therapeutic efficacy of adoptive p53-specific T cell antigen receptor (TCR) gene transfer

Immunotherapy with T cells genetically modified by retroviral transfer of tumor-associated antigen (TAA)-specific T cell receptors (TCR) is a promising approach in targeting cancer. Therefore, using a universal TAA to target different tumor entities by only one therapeutic approach was the main criteria for our TAA-specific TCR. Here, an optimized (opt) αβ-chain p53(264-272)-specific and an opt single chain (sc) p53(264-272)-specific TCR were designed, to reduce mispairing reactions of endogenous and introduced TCR α and TCR β-chains, which might lead to off-target autoimmune reactions, similar to Graft-versus-host disease (GvHD). rnIn this study we evaluated the safety issues, which rise by the risk of p53TCR gene transfer-associated on/off-target toxicities as well as the anti-tumor response in vivo in a syngeneic HLA-A*0201 transgenic mouse model. We could successfully demonstrate that opt sc p53-specific TCR-redirected T cells prevent TCR mispairing-mediated lethal off-target autoimmunity in contrast to the parental opt αβ-chain p53-specific TCR. Since the sc p53-specific TCR proofed to be safe, all further studies were performed using sc p53-specific TCR redirected T cells only. Infusion of p53-specific TCR-redirected T cells in Human p53 knock-in (Hupki) mice after lymphodepletion-preconditioning regimen with either sublethal body irradiation (5Gy) or chemotherapy (fludarabine and cyclophosphamide) in combination with vaccination (anti-CD40, CpG1668 and p53(257-282) peptide) did not result in a depletion of hematopoietic cells. Moreover, adoptive transfer of high numbers of p53-specific TCR-redirected T cells in combination with Interleukin 2 (IL-2) also did not lead to toxic on-target reactions. The absence of host tissue damage was confirmed by histology and flow cytometry analysis. Furthermore, p53-specific TCR-redirected T cells were able to lyse p53+A2.1+ tumor cells in vitro. However, in vivo studies revealed the potent suppressive effect of the tumor microenvironment (TME) mediated by tumor-infiltrating myeloid-derived suppressor cells (MDSC). Accordingly, we could improve an insufficient anti-tumor response in vivo after injection of the sc p53-specific TCR-redirected T cells by additional depletion of immunosuppressive cells of the myeloid lineage.rnTogether, these data suggest that the optimized sc p53(264-272)-specific TCR may represent a safe and efficient approach for TCR-based gene therapy. However, combinations of immunotherapeutic strategies are needed to enhance the efficacy of adoptive cell therapy (ACT)-mediated anti-tumor responses.

Prognostic relevance of Bcl-2 overexpression in surgically treated prostate cancer is not caused by increased copy number or translocation of the gene

Overexpression of anti-apoptotic Bcl-2 plays a role in prostate cancer progression, particularly in transformation to androgen-independent disease. Androgen-independent prostate cancers have been shown to harbor Bcl-2 gene copy number gains frequently suggesting that this genetic alteration might play a role in Bcl-2 overexpression. The relation of Bcl-2 overexpression and copy number gains or translocation of the BCL-2 gene in prostate cancer under hormone-naïve conditions is unknown.

Molecular characterization of the equine collagen, type IX, alpha 2 (COL9A2) gene on horse chromosome 2p16-->p15

The mammalian collagen, type IX, alpha 2 gene (COL9A2) encodes the alpha-2 chain of type IX collagen and is located on horse chromosome 2p16-->p14 harbouring a quantitative trait locus for osteochondrosis. We isolated a bacterial artificial chromosome (BAC) clone containing the equine COL9A2 gene and determined the complete genomic sequence of this gene. Cloning and characterization of equine COL9A2 revealed that the equine gene consists of 32 exons spanning approximately 15 kb. The COL9A2 transcript encodes a single protein of 688 amino acids. Thirty two single nucleotide polymorphisms (SNPs) equally distributed in the gene were detected in a mutation scan of eight unrelated Hanoverian warmblood stallions, including one SNP that affects the amino acid sequence of COL9A2. Comparative analyses between horse, human, mouse and rat indicate that the chromosomal location of equine COL9A2 is in agreement with known chromosomal synteny relationships. The comparison of the gene structure and transcript revealed a high degree of conservation towards the other mammalian COL9A2 genes. We chose three informative SNPs for association and linkage disequilibrium tests in three to five paternal half-sib families of Hanoverian warmblood horses consisting of 44 to 75 genotyped animals. The test statistics did not reach the significance threshold of 5% and so we could not show an association of COL9A2 with equine osteochondrosis.