猪STCH基因的Bi-PASA遗传标记及多态性研究

微粒体应激 70蛋白三磷酸腺苷酶 (STCH)基因属于应激 70蛋白基因伴侣家族 ,在机体免疫反应和疾病抵抗力等方面起重要作用。根据人和小鼠STCH基因的保守序列设计引物 ,PCR扩增到猪STCH基因第5外显子 4 4 5bp片段。序列测定显示 ,猪STCH基因与人和小鼠STCH基因分别具有 87 13%和 80 4 5 %的同源性。通过测定和比较中国梅山猪、欧洲约克夏猪及PIC商品猪的STCH基因序列 ,发现在猪STCH基因编码区第 5外显子 10 5 0位点上存在一个单碱基突变位点。利用双向特定等位基因PCR扩增法 (Bi PASA)建立了检测猪STCH基因变异的遗传标记 ,并用该标记分析了STCH基因在中国家猪 (梅山猪、荣昌猪和金华猪 )、欧洲家猪 (约克夏猪、大白猪 )、商品猪 (PIC合成系 )以及欧洲野猪的基因频率和多态性。本研究建立的Bi PASA遗传标记和基因变异信息 ,将为进一步分析猪STCH基因变异与经济性状的相关分析提供基础资料。     

GADD45beta/GADD45gamma and MEKK4 comprise a genetic pathway mediating STAT4-independent IFNgamma production in T cells

The stress-inducible molecules GADD45beta and GADD45gamma have been implicated in regulating IFNgamma production in CD4 T cells. However, how GADD45 proteins function has been controversial. MEKK4 is a MAP kinase kinase kinase that interacts with GADD45 in vitro. Here we generated MEKK4-deficient mice to define the function and regulation of this pathway. CD4 T cells from MEKK4-/- mice have reduced p38 activity and defective IFNgamma synthesis. Expression of GADD45beta or GADD45gamma promotes IFNgamma production in MEKK4+/+ T cells, but not in MEKK4-/- cells or in cells treated with a p38 inhibitor. Thus, MEKK4 mediates the action of GADD45beta and GADD45gamma on p38 activation and IFNgamma production. During Th1 differentiation, the GADD45beta/GADD45gamma/MEKK4 pathway appears to integrate upstream signals transduced by both T cell receptor and IL12/STAT4, leading to augmented IFNgamma production in a process independent of STAT4.     

脂联素基因SNP45 T/G多态性与2型糖尿病相关性研究

目的:探讨脂联素基因(APM1)SNP45 T/G多态性与湖北汉族人群2型糖尿病的相关性.方法:采用聚合酶链反应-限制性片断长度多态性(PCR-RFLP)方法分析了479例样本的APM1基因SNP45T/G多态性,并测定身高、体重、腰围、臀围、血压和空腹血糖等生理指标.结果:两种实验设计中对照组与病例组基因型和等位基因频率差异均无统计学意义.结论:脂联素基因SNP45T/G多态性在湖北汉族人群2型糖尿病的发生发展中可能不起主要作用.     

脂联素基因SNP45T／G多态性与2型糖尿病相关性研究

目的：探讨脂联素基因（APM1）SNP45T／G多态性与湖北汉族人群2型糖尿病的相关性。方法：采用聚合酶链反应．限制性片断长度多态性（PCR—RFLP）方法分析了479例样本的APM1基因SNP45T／G多态性,并测定身高、体重、腰围、臀围、血压和空腹血糖等生理指标。结果：两种实验设计中对照组与病例组基因型和等位基因频率差异均无统计学意义。结论：脂联素基因SNP45T／G多态性在湖北汉族人群2型糖尿病的发生发展中可能不起主要作用。     

Molecular Characterization and SNP Development for the Porcine Il6 and Il10 Genes

Different cytokines are secreted in response to specific microbial molecules referred to as pathogen associated molecular patterns (PAMPs). Interleukin 6 (IL6) and interleukin 10 (IL10), both secreted by macrophages and lymphocytes, play a central role in the immunological response. In this work we obtained the genomic structure and complete DNA sequence of the porcine IL6 and IL10 genes and identified polymorphisms in the genomic sequences of these genes on a panel of ten different pig breeds. Comparative intra- and interbreed sequence analysis revealed a total of eight polymorphisms in the porcine IL6 gene and 21 in the porcine IL10 gene, which include single nucleotide polymorphisms (SNPs) and insertion deletion polymorphisms (indels). Additionally, the chromosomal localization of the IL10 gene was determined by FISH and RH mapping.     

Paired box 5 is a frequently methylated lung cancer tumour suppressor gene interfering β‐catenin signalling and GADD45G expression

Recent studies suggest that paired box 5 (PAX5) is down‐regulated in multiple tumours through its promoter methylation. However, the role of PAX5 in non‐small cell lung cancer (NSCLC) pathogenesis remains unclear. The aim of this study is to examine PAX5 expression, its methylation status, biological functions and related molecular mechanism in NSCLC. We found that PAX5 was widely expressed in normal adult tissues but silenced or down‐regulated in 88% (7/8) of NSCLC cell lines. PAX5 expression level was significantly lower in NSCLC than that in adjacent non‐cancerous tissues (P = 0.0201). PAX5 down‐regulation was closely associated with its promoter hypermethylation status and PAX5 expression could be restored by demethylation treatment. Frequent PAX5 promoter methylation in primary tumours (70%) was correlated with lung tumour histological types (P = 0.006). Ectopic expression of PAX5 in silenced lung cancer cell lines (A549 and H1975) inhibited their colony formation and cell viability, arrested cell cycle at G2 phase and suppressed cell migration/invasion as well as tumorigenicity in nude mice. Restoration of PAX5 expression resulted in the down‐regulation of β‐catenin and up‐regulation of tissue inhibitors of metalloproteinase 2, GADD45G in lung tumour cells. In summary, PAX5 was found to be an epigenetically inactivated tumour suppressor that inhibits NSCLC cell proliferation and metastasis, through down‐regulating the β‐catenin pathway and up‐regulating GADD45G expression.     

The Effect of GADD45a on Furazolidone‐Induced S‐Phase Cell‐Cycle Arrest in Human Hepatoma G2 Cells

In this study, overexpression of GADD45a induced by furazolidone in HepG2 cells could arouse S‐phase cell cycle arrest, suppress cell proliferation, and increase the activities of cyclin D1, cyclin D3, and cyclin‐dependent kinase 6 (CDK6). To the opposite, GADD45a knockdown cells by RNAi could reduce furazolidone‐induced S‐phase cell cycle arrest, increase the cell viability, decrease the activities of cyclin D1, cyclin D3, and CDK6; however, cyclin‐dependent kinase 4 (CDK4) showed no change. Moreover, data from our current studies show that cyclin D1, cyclin D3, and CDK6 are target genes functioning at the downstream of the GADD45a pathway induced by furazolidone. These results demonstrate that the GADD45a pathway is partially responsible for the furazolidone‐induced S‐phase cell cycle arrest. GADD45a influences furazolidone‐induced S‐phase cell cycle arrest in human hepatoma G2 cells via cyclin D1, cyclin D3, and CDK6, but not CDK4.     

Implication of mitogen-activated protein kinase in the induction of G1 cell cycle arrest and gadd45 expression by the carotenoid fucoxanthin in human cancer cells

Background

The precise mechanism of the anti-tumor action of fucoxanthin has yet to be elucidated. We previously reported that gadd45a and gadd45b might play a role in the G1 arrest induced by fucoxanthin. In the present study, we show that several MAPKs modulate the induction of gadd45 and G1 arrest.

Methods

HepG2 and DU145 cells were used. The cell cycle was analyzed using flow cytometry. Expression of gadd45 was assayed by Northern blot and/or quantitative RT-PCR analyses. Activation of MAPK was assayed by Western blot analysis.

Results

Inhibition of p38 MAPK enhanced the induction of gadd45a expression and G1 arrest by fucoxanthin in HepG2 cells. Inhibition of ERK enhanced gadd45b expression but had no effect on the induction of G1 arrest by fucoxanthin in HepG2 cells. Inhibition of SAPK/JNK suppressed the induction of gadd45a expression and G1 arrest by fucoxanthin in DU145 cells.These data suggest that gadd45a is closely related with the G1 arrest induced by fucoxanthin, and that the pattern of MAPK involvement in the induction of gadd45a and G1 arrest by fucoxanthin differs depending on the cell type.

General significance

The implication of GADD45 and MAPK involvement in the anti-tumor action of carotenoids is first described.     

GADD45A inhibits autophagy by regulating the interaction between BECN1 and PIK3C3

GADD45A is a TP53-regulated and DNA damage-inducible tumor suppressor protein, which regulates cell cycle arrest, apoptosis, and DNA repair, and inhibits tumor growth and angiogenesis. However, the function of GADD45A in autophagy remains unknown. In this report, we demonstrate that GADD45A plays an important role in regulating the process of autophagy. GADD45A is able to decrease LC3-II expression and numbers of autophagosomes in mouse tissues and different cancer cell lines. Using bafilomycin A₁ treatment, we have observed that GADD45A regulates autophagosome initiation. Likely, GADD45A inhibition of autophagy is through its influence on the interaction between BECN1 and PIK3C3. Immunoprecipitation and GST affinity isolation assays exhibit that GADD45A directly interacts with BECN1, and in turn dissociates the BECN1-PIK3C3 complex. Furthermore, we have mapped the 71 to 81 amino acids of the GADD45A protein that are necessary for the GADD45A interaction with BECN1. Knockdown of BECN1 can abolish autophagy alterations induced by GADD45A. Taken together, these findings provide the novel evidence that GADD45A inhibits autophagy via impairing the BECN1-PIK3C3 complex formation.     

猪精氨酸-丝氨酸蛋白激酸3(SRPK3)基因的克隆、表达及多态性分析

通过对猪SRPK3基因初步的研究,为猪分子遗传育种提供基础分子生物学信息,为猪的遗传育种提供分子标记。以大白猪为实验材料,采用RT-PCR方法克隆了精氨酸-丝氨酸蛋白激酶3 (serine/arginine-rich specific kinase 3,SRPK3) 的全长基因CDS区;采用生物信息学方法分析了SRPK3基因核酸序列并对其所编码的的蛋白序列进行了预测与分析编码蛋白序列的结构特点;采用PCR-SSCP方法对大白猪,野猪,民猪及野家杂交猪的SRPK3基因的多态性进行了检验;采用实时荧光定量PCR (Real-time) 方法检测了SRPK3在1日龄和30日龄大白猪及杜洛克的心脏、肌肉、脾脏、肝脏、肾脏、肺脏、胃、小肠、大肠、脑的表达情况;采用皮下注射的方式构建猪骨骼肌损伤模型用于研究在骨骼肌修复过程中SRPK3基因表达特性。经拼接所得到的1 708bp核苷酸片段,涵盖了SRPK3基因的全长CDS (1 701bp),该基因编码含567个氨基酸片段;蛋白存在两个S_TKc结构域,猪SRPK3蛋白序列与人和牛的相似性较高。PCR-SSCP检测发现第6外显子上A⁶²⁹→G⁶²⁹,T⁶⁵³→T⁶⁵³的突变,氨基酸变化为Pro→His,Ile→Thr;第9外显子处的G¹⁰⁵⁹→ A¹⁰⁵⁹,氨基酸无突变。利用荧光定量PCR研究发现,表达结果显示该基因表达具有组织和种间特异性。SRPK3基因的表达在整个骨骼肌细胞损伤修复过程中逐渐升高。SRPK3基因主要在肌肉和心肌内表达,骨骼肌损伤修复过程中伴随骨骼肌细胞分化SRPK3的表达持续升高,推测其可能与骨骼肌细胞发育相关。     

The role of D-GADD45 in oxidative,thermal and genotoxic stress resistance

There is a relationship between various cellular stress factors and aging. In earlier studies, we demonstrated that overexpression of the D-GADD45 gene increases the life span of Drosophila melanogaster. In this study, we investigate the relationship between D-GADD45 activity and resistance to oxidative, genotoxic and thermal stresses as well as starvation. In most cases, flies with constitutive and conditional D-GADD45 overexpression in the nervous system were more stress-resistant than ones without overexpression. At the same time, most of the studied stress factors increased D-GADD45 expression in the wild-type strain. The lifespan-extending effect of D-GADD45 overexpression was also retained after exposure to chronic and acute gamma-irradiation, with doses of 40 сGy and 30 Gy, respectively. However, knocking out D-GADD45 resulted in a significant reduction in lifespan, lack of radiation hormesis and radioadaptive response. A dramatic decrease in the spontaneous level of D-GADD45 expression was observed in the nervous system as age progressed, which may be one of the causes of the age-related deterioration of organismal stress resistance. Thus, D-GADD45 expression is activated by most of the studied stress factors, and D-GADD45 overexpression resulted in an increase of stress resistance.     

The role of D-GADD45 in oxidative,thermal and genotoxic stress resistance

There is a relationship between various cellular stress factors and aging. In earlier studies, we demonstrated that overexpression of the D-GADD45 gene increases the life span of Drosophila melanogaster. In this study, we investigate the relationship between D-GADD45 activity and resistance to oxidative, genotoxic and thermal stresses as well as starvation. In most cases, flies with constitutive and conditional D-GADD45 overexpression in the nervous system were more stress-resistant than ones without overexpression. At the same time, most of the studied stress factors increased D-GADD45 expression in the wild-type strain. The lifespan-extending effect of D-GADD45 overexpression was also retained after exposure to chronic and acute gamma-irradiation, with doses of 40 сGy and 30 Gy, respectively. However, knocking out D-GADD45 resulted in a significant reduction in lifespan, lack of radiation hormesis and radioadaptive response. A dramatic decrease in the spontaneous level of D-GADD45 expression was observed in the nervous system as age progressed, which may be one of the causes of the age-related deterioration of organismal stress resistance. Thus, D-GADD45 expression is activated by most of the studied stress factors, and D-GADD45 overexpression resulted in an increase of stress resistance.     

Molecular Cloning of Porcine SUN5 Gene and Association between a SNP with Litter Size Trait

SUN domain-containing protein 5 (SUN5) is an important reproduction related gene. In this study, we cloned the full-length coding sequence of porcine SUN5 gene through RT-PCR. Sequence analysis of this gene revealed that the pig SUN5 gene encodes a protein of 383?amino acids that has high homology with the SUN5 protein of eight species: wild Bactrian camel (95%), alpaca (95%), Yangtze River dolphin (94%), sperm whale (94%), sheep (93%), black flying fox (93%), goat (92%), and horse (91%). This gene is structured into 13 exons and 12 introns as revealed by computer-assisted analysis. The prediction of transmembrane helices showed that pig SUN5 protein might be a transmembrane protein. PCR-Taq I-RFLP was established to detect the GU475008:c.138 G>A substitution of porcine SUN5 gene coding sequence and eight pig breeds displayed obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n?=?200) and Landrace (n?=?200) pig populations, and the results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows (P?相似文献   

Tissue factor pathway inhibitor induces expression of JUNB and GADD45B mRNAs

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that regulates tissue factor-triggered blood coagulation. It has previously been reported that TFPI inhibits the proliferation of human umbilical vein endothelial cells (HUVECs), suggesting that TFPI may act as more than just a mediator of coagulation through changes in gene expression. By using DNA-array techniques and Northern blot analysis, we here revealed that TFPI transiently induced the mRNA expression of JUNB and GADD45B. The inducible effects were not observed in TFPIdeltaC (lacking the C-terminal basic region) or antithrombin (heparin-binding anticoagulant protease inhibitor). Moreover, the TFPI-induced expression of GADD45B was blocked by receptor-associated protein, which masks the ligand-binding domain of very low density lipoprotein receptor (VLDL-R). In conclusion, this is the first report to show an effect of TFPI on mRNA expression, and suggests that TFPI modulates cellular functions by inducing JUNB and GADD45B expression through binding to VLDL-R.     

Molecular Cloning, Mapping, and Polymorphism of the Porcine SCG2 gene

The secretogranin II (SCG2) gene is associated with the synthesis and secretion of follicle-stimulating hormone and luteinizing hormone. In the present study, we have determined the complete cDNA sequence of pig SCG2, which was submitted to GenBank with accession no. AY870646. Its complete open reading frame of 1,851 nucleotides encodes 616 amino acids. The predicted protein shares 80–87% identity with mouse, human, and bovine SCG2 proteins, and all four species share almost complete identity in the secretoneurin and EM66 domains. Pig SCG2 is a protein of 589 amino acids and 68,132 Da, preceded by a signal peptide of 27 residues. It contains nine pairs of dibasic residues, which are used as potential cleavage sites for generation of physiologically active peptides. Analysis of the SCG2 gene across the INRA-Minnesota porcine radiation hybrid panel indicates close linkage with microsatellite marker SW2608, located on Sus scrofa chromosome 15 (SSC15) q25, which harbors several QTL for ovulation rate and meat quality. Comparative sequencing and EST analysis revealed nine SNPs in porcine SCG2 cDNA, including seven SNPs in the coding region and two SNPs in the 3′ UTR. Four nonsynonymous SNPs (G622A, G1671T, C1718T, and A1790C) resulted in amino acid substitutions of Ala→Thr, Glu→Asp, Pro→Leu, and Asn→Thr, respectively.     

The effects of cucurbitacin E on GADD45β‐trigger G2/M arrest and JNK‐independent pathway in brain cancer cells

Cucurbitacin E (CuE), an active compound of the cucurbitacin family, possesses a variety of pharmacological functions and chemotherapy potential. Cucurbitacin E exhibits inhibitory effects in several types of cancer; however, its anticancer effects on brain cancer remain obscure and require further interpretation. In this study, efforts were initiated to inspect whether CuE can contribute to anti‐proliferation in human brain malignant glioma GBM 8401 cells and glioblastoma‐astrocytoma U‐87‐MG cells. An MTT assay measured CuE's inhibitory effect on the growth of glioblastomas (GBMs). A flow cytometry approach was used for the assessment of DNA content and cell cycle analysis. DNA damage 45β (GADD45β) gene expression and CDC2/cyclin‐B1 disassociation were investigated by quantitative real‐time PCR and Western blot analysis. Based on our results, CuE showed growth‐inhibiting effects on GBM 8401 and U‐87‐MG cells. Moreover, GADD45β caused the accumulation of CuE‐treated G2/M‐phase cells. The disassociation of the CDC2/cyclin‐B1 complex demonstrated the known effects of CuE against GBM 8401 and U‐87‐MG cancer cells. Additionally, CuE may also exert antitumour activities in established brain cancer cells. In conclusion, CuE inhibited cell proliferation and induced mitosis delay in cancer cells, suggesting its potential applicability as an antitumour agent.