Differential expression of immune response genes associated with subclinical mastitis in dairy buffaloes

Buffalo milk production has become of significant importance on the world scale, however, there are few studies involving biotechnological tools specifically for buffalo. To verify the effects caused by subclinical mastitis on the components of milk and to study the innate immune system in the udder of dairy buffaloes with subclinical mastitis, we evaluated the levels of expression of the lactoferrin (LTF), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-8 (IL-8), and toll-like receptors 2 (TLR-2) and 4 (TLR-4) genes in buffaloes with and without subclinical mastitis. Milk samples were collected for the determination of milk components: somatic cell score (SCS), fat, protein, lactose, total solids and solids-not-fat (SNF), as well as for RNA extraction of milk cells, complementary DNA synthesis, and expression profile quantification by quantitative real-time PCR. For gene expression, the ΔΔCt was estimated using contrasts of the target genes expression adjusted for the expression of the housekeeping genes between both groups. Linear regression analysis was performed to determine the relationship between the genes studied and the milk components. Subclinical mastitis induced changes in the fat, lactose and SNF in milk of buffaloes, and the messenger RNA abundance was upregulated for TLR-2, TLR-4, TNF-α, IL-1β and IL-8 genes in milk cells of buffaloes with subclinical mastitis, whereas the LTF gene was not differentially expressed. Results of linear regression analysis showed that TLR-2 gene expression most explains the variation in SCS, and the change in a unit of ΔCt of the TNF-α gene would result in a higher increase in SCS. The study of these immune function genes that are active in the mammary gland is important to characterize the action mechanism of the innate immunity that occurs in subclinical mastitis in dairy buffaloes and may aid the development of strategies to preserve the health of the udder.     

Expression of candidate genes for residual feed intake in Angus cattle

Residual feed intake (RFI) has been adopted in Australia for the purpose of genetic improvement in feed efficiency in beef cattle. RFI is the difference between the observed feed intake of an animal and the predicted feed intake based on its size and growth rate over a test period. Gene expression of eight candidate genes (AHSG, GHR, GSTM1, INHBA, PCDH19, S100A10, SERPINI2 and SOD3), previously identified as differentially expressed between divergent lines of high‐ and low‐RFI animals, was measured in an unselected population of 60 steers from the Angus Society Elite Progeny Test Program using quantitative real‐time PCR. Results showed that the levels of gene expression were significantly correlated with RFI. The genes explain around 33.2% of the phenotypic variance in RFI, and prediction equations using the expression data are reasonably accurate estimators of RFI. The association of these genes with economically important traits, such as other feed efficiency‐related traits and fat, growth and carcass traits, was investigated as well. The expression of these candidate genes was significantly correlated with feed conversion ratio and daily feed intake, which are highly associated with RFI, suggesting a functional role for these genes in modulating feed utilisation. The expression of these genes did not show any association with average daily gain, eye muscle area and carcass composition.     

Expression profile of male germ cell-associated genes in mouse embryonic stem cell cultures treated with all-trans retinoic acid and testosterone

Cells that morphologically and functionally resemble male germ cells can be spontaneously derived from ES cells. However, this process is inefficient and unpredictable suggesting that the expression pattern of male germ cell associated genes during spontaneous ES cell differentiation does not mimic the in vivo profiles of the genes. Thus, in the present study, the temporal profile of genes expressed at different stages of male germ cell development was examined in differentiating ES cells. The effect of all-trans retinoic acid (RA) which is a known inducer of primordial germ cell (PGC) proliferation/survival in vitro and testosterone which is required for spermatogenesis in vivo on the expression of these genes was also determined. Each of the 12 genes analyzed exhibited one of four temporal expression patterns in untreated differentiating ES cells: progressively decreased (Dppa3, Sycp3, Msy2), initially low and then increased (Stra8, Sycp1, Dazl, Act, Prm1), initially decreased and then increased (Piwil2, Tex14), or relatively unchanged (Akap3, Odf2). RA-treated cells exhibited increased expression of Stra8, Dazl, Act, and Prm1 and suppressed expression of Dppa3 compared to untreated controls. Furthermore, testosterone increased expression of Stra8 while the combination of RA and testosterone synergistically increased expression of Act. Our findings establish a comprehensive profile of male germ cell gene expression during spontaneous differentiation of murine ES cells and describe the capacity of RA and testosterone to modulate the expression of these genes. Furthermore, these data represent an important first step in designing a plausible directed differentiation protocol for male germ cells.     

抗生素抗性基因连锁传播的侧翼保守元件分析

抗生素在医疗、畜牧和水产养殖业的大量使用造成了环境中耐药细菌和抗性基因的日益增加,也加速了抗性基因在环境细菌间的传播扩散.本研究以环境样本直接提取的总DNA为模板,运用热不对称交错PCR (thermal asymmetric interlaced PCR, Tail-PCR)技术直接扩增抗生素抗性基因上下游序列.通过优化Tail-PCR反应程序,单循环同时扩增出tetW基因的多条侧翼序列,包括6条上游序列和9条下游序列.基于序列的生物信息学分析发现,上游包括一段反向重复序列和已知的一段tetW调节肽序列以及一个已知的插入序列,下游包括一个保守的未知序列和一个开放式阅读框架(the open reading frame,ORF)编码甲基转移酶.结果不仅发现了可能协助tetW基因传播的功能元件,也提供了一个未知侧翼序列高效和便捷的研究方法,即采用Tail-PCR技术,一组样品即能便捷获得多条侧翼序列.     

Optimization and application of real-time PCR method for detecting the expression levels of nitrogen assimilation-related genes in rice

By optimizing the Mg²⁺ concentration, Taq enzyme dosage, SYBR Green I (SGI) concentration, and plate reading temperature in PCR system, we established the method for detecting the expression levels of nitrogen assimilation-related genes in rice by using RT-PCR technique. Based on this qualified method, we investigated the variations of OsAMT1.1 (one of nitrogen uptake genes) and OsGlt1 (one of nitrogen metabolism genes) expression levels in rice seedlings under conditions of varying nitrogen supply. The results show that by optimizing the parameters in the PCR system to fit the characters of target genes best, we can successfully quantify the low-abundant nitrogen transport and metabolism genes in rice quickly and exactly using fluorescence RT-PCR technique. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 4, pp. 625–636. The text was submitted by the authors in English.     

Polymorphisms of the ATP1A1 gene associated with mastitis in dairy cattle

Mastitis affects the concentrations of potassium and sodium in milk. Since sodium-potassium adenosine triphosphatase (Na(+), K(+)-ATPase) is critical for maintaining the homeostasis of these two ions, and is involved in cell apoptosis and pathogenesis, we presumed that polymorphism of the ATP1A1 gene, which encodes the bovine Na(+), K(+)-ATPase α1 subunit could be associated with mastitis. The ATP1A1 gene was analyzed in 320 Holstein cows using PCR low ionic strength single-strand conformation polymorphism (PCR-LIS-SSCP) and DNA sequencing methods. A C/A SNP was identified at nucleotide position -15,739 in exon 17 of the ATP1A1 gene, but it did not induce any change in amino acids. We examined a possible association of polymorphism of the ATP1A1 gene with somatic cell score and 305-day milk yields. Individuals with genotype CC in ATP1A1 had significantly lower somatic cell scores and 305-day milk yields than those with genotype CA. We also examined changes in Na(+), K(+)-ATPase activity of red cell membranes. The Na(+), K(+)-ATPase activity was significantly higher in dairy cows with genotype CC compared to the other two genotypes, and the Na(+), K(+)-ATPase activity of the resistant group was significantly higher than that of the susceptible group in dairy cows. We conclude that this polymorphism has potential as a marker for mastitis resistance in dairy cattle.     

Expression and regulation of genes associated with cell death during murine preimplantation embryo development

The newly fertilized preimplantation embryo depends entirely on maternal mRNAs and proteins deposited and stored in the oocyte prior to its ovulation. If the oocyte is not sufficiently equipped with maternally stored products, or if zygotic gene expression does not commence at the correct time, the embryo will die. One of the major abnormalities observed during early development is cellular fragmentation. We showed previously that cellular fragmentation in human embryos can be attributed to programmed cell death (PCD). Here, we demonstrate that the PCD that occurs during the 1-cell stage of mouse embryogenesis is likely to be regulated by many cell death genes either maternally inherited or transcribed from the embryonic genome. We have demonstrated for the first time the temporal expression patterns of nine cell death regulatory genes, and our preliminary experiments show that the expression of these genes is altered in embryos undergoing fragmentation. The expression of genes involved in cell death (MA-3, p53, Bad, and Bcl-xS) seems to be elevated, whereas the expression of genes involved in cell survival (Bcl-2) is reduced. We propose that PCD may occur by default in embryos that fail to execute essential developmental events during the first cell cycle. Mol. Reprod. Dev. 51:243–253, 1998. © 1998 Wiley-Liss, Inc.     

基因表达转录分析中内参基因的选择

目前基因表达的转录分析多采用单一看家基因作为内参来校正目标基因的表达量.实验中以人肝癌BEL-7402细胞为研究对象,应用实时荧光定量PCR技术,观察了新型三肽化合物酪丝缬肽作用后RPL13A、UBC、EIF4A、B2M、GAPDH和ACTB共6个看家基因mRNA水平的表达情况.经过geNorm程序统计学分析处理,结果表明,这6个看家基因的表达存在差异,确定了RPL13A、UBC2个看家基因用于校正目标基因的表达量.基因表达转录分析中内参基因选择的必要性在实验中得以证明,更重要的是为各种实验因素影响下(尤其是新物质作用下)内参基因的选择介绍和提供了一种行之有效的方法.     

柽柳bZIP基因调控耐盐相关基因的表达

本研究探讨了柽柳（Tamarixhispida）bZIP（basicleucine zipper）基因对抗逆基因表达的调控。我们比较了非盐胁迫和盐胁迫条件下非转基因和转bZIP基因植株的SOD、POD、ATPase、GST、LTP和LEA等基因表达量的变化。结果表明,在非胁迫条件下,bZIP转录因子可能直接调控了poxN1、TOBPXD、TOBLTP和ltp1基因的表达,而其他基因的表达可能不受bZIP转录因子的直接调控。在盐胁迫下,bZIP转录因子能够直接或间接地调控部分抗逆基因的表达,使它们的表达量显著增强。     

细菌感染对家蚕DNA甲基化与组蛋白乙酰化相关基因表达的影响

【目的】探讨DNA甲基化及组蛋白乙酰化是否参与家蚕 Bombyx mori 免疫反应的调控。【方法】对家蚕与其他生物的DNA甲基转移酶 (DNMT)、组蛋白去乙酰化酶（HDAC）与组蛋白乙酰转移酶（HAT）的蛋白序列进行系统进化分析;利用定量PCR检测家蚕5龄第3天幼虫感染病原菌绿脓杆菌 Pseudomonas aeruginosa 和金黄色葡萄球菌 Staphylococcus aureus 后 BmDNMT 1, BmHDACI-1, BmHDACI-2和 BmHAT 1在家蚕脂肪体组织中的表达变化;给家蚕5龄第2天幼虫注射DNMT, HDAC和HAT抑制剂,观察它们对家蚕感染细菌后的存活率的影响。【结果】系统进化分析显示,BmDNMT1在进化上呈现特殊性,独立于其他昆虫DNMT1的进化,而BmHDACs和BmHAT在进化上相对保守。定量PCR检测表明,在两种细菌感染后,BmDNMT1, BmHDACs 和 BmHAT1 在家蚕幼虫脂肪体中的表达水平均有不同程度的上升。然而,DNMT, HDAC和HAT抑制剂对家蚕幼虫感染细菌后的存活率并无明显影响。【结论】本研究发现感染绿脓杆菌和金黄色葡萄球菌后,家蚕幼虫脂肪体中 BmDNMT1, BmHDACs 和 BmHAT1 的表达水平有不同程度的上调,推测DNA甲基化和组蛋白乙酰化/去乙酰化可能参与家蚕免疫反应的调控。     

胡萝卜中DcDofD1转录因子基因的克隆及其对非生物逆境胁迫的响应分析

Dof (DNA-binding with one finger)转录因子是植物特有的一类转录因子,该类转录因子在植物生长发育过程中起重要作用。本实验以胡萝卜‘黑田五寸’和‘君川红’为实验材料,分别从中克隆得到DcDofD1转录因子基因。采用生物信息学方法对DcDofD1转录因子氨基酸组成、理化性质、进化关系进行了分析。并利用实时定量PCR方法对DcDofD1基因在非生物胁迫下的表达量进行了分析。结果表明‘黑田五寸’和‘君川红’中的DcDofD1转录因子具有明显的单锌指结构,保守域的氨基酸序列比较保守,进化分析显示,DcDofD1属于Dof家族的D1亚族。在胡萝卜‘黑田五寸’和‘君川红’中,DcDofD1基因对高温、低温、干旱、盐等非生物胁迫响应。而不同胡萝卜材料中,DcDofD1基因对非生物逆境响应的强度和速度不同。     

Effects of leptin Arg25Cys on peripheral mononuclear cell counts and antibody response to vaccination in beef cattle

A single nucleotide polymorphism (SNP) in the leptin gene that results in Arg25Cys has been associated with beef carcass quality and milk composition in dairy cattle. However, leptin (LEP) also plays a role in immune performance and hence it was important to determine whether selection based on this SNP would negatively affect immune cell numbers or antibody production. LEP c.73C>T was assessed for effects on immune cell counts and antibody titres in 27 beef cattle herds ( n  = 556). A commercial rabies vaccine had been administered to these animals. Prior to being vaccinated, counts of several important mononuclear cells (total and activated B lymphocytes, total and activated T helper and T cytotoxic, WC1 T lymphocytes and monocytes) as well as baseline serum antibody titres were determined for each animal. On day 21, antibody titres were measured and a booster vaccine was administered. Finally on day 42, antibody titres and mononuclear cell types were again counted. Counts of six different cell types were significantly associated with the LEP genotype; however, no consistent patterns were observed between LEP genotype (TT, CT or CC) and peripheral blood mononuclear cell populations. Significant differences in the production of rabies antibodies in response to vaccination were observed relative to LEP genotype. Our results suggest that selection for either the C or T allele would not detrimentally impact on the measured indicators of immune function in beef cattle.     

Molecular cloning and expression analysis of heat-shock protein 70 in orange-spotted grouper Epinephelus coioides following heat shock and Vibrio alginolyticus challenge

In this study, the complementary (c)DNA encoding heat-shock protein 70 (Hsp70) of orange-spotted grouper Epinephelus coioides (OsgHsp70) was cloned. OsgHsp70 was 2206 bp and encoded 652 amino acids with predicted molecular mass of 70·89 kDa and theoretical isoelectric point of 5·48. Three Hsp70 family signatures, bipartite nuclear localization signal sequence (NLS) and cytoplasmic characteristic motif (EEVD) were observed in the OsgHsp70, which shared high similarity in amino-acid sequences with the Hsp70 gene of other vertebrates. The results indicated that the OsgHsp70 is a member of the heat-shock protein 70 family. The Hsp70 messenger (m)RNA expressions were quantified by real-time PCR following heat shock, bacterial infection and immunization with formalin-killed Vibrio alginolyticus, a kind of bacterial pathogen that causes septicaemia. Hsp70 mRNA expression in gill, kidney, spleen, thymus gland, muscle and total-blood samples increased at first and then decreased gradually following heat shock. A similar time-dependent pattern was observed following V. alginolyticus pathogen challenge, in which Hsp70 mRNA expression peaked at 24 h after live bacterial infection and 3 days after dead bacterial vaccination. The results indicated that the Hsp70 gene was inducible and involved in the fish immune response.     

Identification of pivotal genes associated with the prognosis of gastric carcinoma through integrated analysis

Purpose: Detecting and diagnosing gastric cancer (GC) during its early period remains greatly difficult. Our analysis was performed to detect core genes correlated with GC and explore their prognostic values.Methods: Microarray datasets from the Gene Expression Omnibus (GEO) ({"type":"entrez-geo","attrs":{"text":"GSE54129","term_id":"54129"}}GSE54129) and The Cancer Genome Atlas (TCGA)-stomach adenocarcinoma (STAD) datasets were applied for common differentially co-expressed genes using differential gene expression analysis and Weighted Gene Co-expression Network Analysis (WGCNA). Functional enrichment analysis and protein–protein interaction (PPI) network analysis of differentially co-expressed genes were performed. We identified hub genes via the CytoHubba plugin. Prognostic values of hub genes were explored. Afterward, Gene Set Enrichment Analysis (GSEA) was used to analyze survival-related hub genes. Finally, the tumor-infiltrating immune cell (TIC) abundance profiles were estimated.Results: Sixty common differentially co-expressed genes were found. Functional enrichment analysis implied that cell–cell junction organization and cell adhesion molecules were primarily enriched. Hub genes were identified using the degree, edge percolated component (EPC), maximal clique centrality (MCC), and maximum neighborhood component (MNC) algorithms, and serpin family E member 1 (SERPINE1) was highly associated with the prognosis of GC patients. Moreover, GSEA demonstrated that extracellular matrix (ECM) receptor interactions and pathways in cancers were correlated with SERPINE1 expression. CIBERSORT analysis of the proportion of TICs suggested that CD8⁺ T cell and T-cell regulation were negatively associated with SERPINE1 expression, showing that SERPINE1 may inhibit the immune-dominant status of the tumor microenvironment (TME) in GC.Conclusions: Our analysis shows that SERPINE1 is closely correlated with the tumorigenesis and progression of GC. Furthermore, SERPINE1 acts as a candidate therapeutic target and prognostic biomarker of GC.