A gene expression estimator of intramuscular fat percentage for use in both cattle and sheep

Background

The expression of genes encoding proteins involved in triacyglyceride and fatty acid synthesis and storage in cattle muscle are correlated with intramuscular fat (IMF)%. Are the same genes also correlated with IMF% in sheep muscle, and can the same set of genes be used to estimate IMF% in both species?

Results

The correlation between gene expression (microarray) and IMF% in the longissimus muscle (LM) of twenty sheep was calculated. An integrated analysis of this dataset with an equivalent cattle correlation dataset and a cattle differential expression dataset was undertaken. A total of 30 genes were identified to be strongly correlated with IMF% in both cattle and sheep. The overlap of genes was highly significant, 8 of the 13 genes in the TAG gene set and 8 of the 13 genes in the FA gene set were in the top 100 and 500 genes respectively most correlated with IMF% in sheep, P-value = 0. Of the 30 genes, CIDEA, THRSP, ACSM1, DGAT2 and FABP4 had the highest average rank in both species. Using the data from two small groups of Brahman cattle (control and Hormone growth promotant-treated [known to decrease IMF% in muscle]) and 22 animals in total, the utility of a direct measure and different estimators of IMF% (ultrasound and gene expression) to differentiate between the two groups were examined. Directly measured IMF% and IMF% estimated from ultrasound scanning could not discriminate between the two groups. However, using gene expression to estimate IMF% discriminated between the two groups. Increasing the number of genes used to estimate IMF% from one to five significantly increased the discrimination power; but increasing the number of genes to 15 resulted in little further improvement.

Conclusion

We have demonstrated the utility of a comparative approach to identify robust estimators of IMF% in the LM in cattle and sheep. We have also demonstrated a number of approaches (potentially applicable to much smaller groups of animals than conventional methods) to using gene expression to rank animals for IMF% within a single farm/treatment, or to estimate differences in IMF% between two farms/treatments.     

Pigs fed camelina meal increase hepatic gene expression of cytochrome 8bl,aldehyde dehydrogenase,and thiosulfate transferase

Comelina sotivo is an oil seed crop which can be grown on marginal lands. Camelina seed oil is rich in omega-3 fatty acids （〉35%） and y-tocopherol but is also high in erucic acid and glucosinolates. Camelina meal, is the by-product after the oil has been extracted. Camelina meal was fed to 28 d old weaned pigs at 3.7% and 7.4% unt age 56 d. The camelina meal supplements in the soy based diets, improved feed efficiency but also significantly increased the liver weights. Gene expression analyses of the livers, using intra-species microarrays, identified increased expression of phase 1 and phase 2 drug metabolism enzymes. The porcine versions of the enzymes were confirmed by real time PCR. Cytochrome 8b1 （CYPSB1）, aldehyde dehydrogenase 2 （Aldh2）, and thiosulfate transferase （TST） were all significantly stimulated. Collectively, these genes implicate the camelina glucosinolate metabolite, methyl-sulfinyldecyl isothiocyanate, as the main xeniobiotic, causing increased hepatic metabolism and increased liver weight.     

赖氨酸对奶牛乳腺上皮细胞内乳脂肪合成相关基因和蛋白表达的影响

本试验旨在研究赖氨酸(Lys)对奶牛乳腺上皮细胞(BMECs)内乳脂肪合成相关基因和蛋白表达的影响,探讨Lys影响乳脂肪合成的机理。将第3代BMECs随机分为6组,每组6个重复,每个重复1个培养孔。各组培养基中Lys的浓度分别为0.5(基础培养基,对照)、1.0、2.0、4.0、8.0和16.0mmol/L,37℃、5%CO2培养48h后测定BMECs甘油三酯(TAG)含量、乳脂肪合成相关基因和蛋白的表达量。结果表明:BMECs内TAG含量(P=0.013)以及脂肪酸结合蛋白3(FABP3,P=0.001)、脂蛋白脂酶(LPL,P=0.096)、脂肪酸合成酶(FASN,P=0.003)、乙酰甘油磷酸脂酰转移酶6(AGPAT6,P=0.038)和甘油-3-磷酸酰基转移酶(GPAM,P=0.022)基因表达量对Lys呈显著或趋于显著的浓度依赖效应。FABP3基因表达量以2.0、4.0、8.0、16.0mmol/L组和LPL基因表达量以1.0、2.0、4.0、8.0、16.0 mmol/L组显著高于0.5mmol/L组(P0.05);FASN基因表达量以2.0mmol/L组最高,显著高于16.0mmol/L组(P0.05);硬脂酰辅酶A去饱和酶1(SCD1)基因表达量以2.0、4.0mmol/L组显著高于其他组(P0.05);磷脂酸磷酸酯酶1(LPIN1)、嗜乳脂蛋白亚家族1成员1(BTN1 A1)和黄嘌呤脱氢酶(XDH)基因表达量均以1.0、2.0、4.0、8.0mmol/L组显著高于0.5mmol/L组(P0.05);过氧化物酶体增殖物激活受体γ(PPARγ)基因及蛋白表达量均以2.0、4.0mmol/L组显著高于0.5和8.0、16.0mmol/L组(P0.05);固醇调节元件结合蛋白1(SREBP1)基因表达量以1.0、2.0、4.0mmol/L组显著高于其他组(P0.05),蛋白表达量以1.0 mmol/L组显著高于其他组(P0.05)。但高浓度Lys抑制AGPAT6和GPAM的基因表达,AGPAT6基因表达量以2.0、4.0、8.0、16.0mmol/L组显著低于0.5、1.0mmol/L组(P0.05),GPAM基因表达量以16.0mmol/L组显著低于0.5、1.0、2.0、4.0mmol/L组(P0.05)。可见,Lys对BMECs的乳脂肪合成具有显著的促进效果,但高浓度的Lys抑制了乳脂肪合成相关基因的表达。本试验条件下,培养基中Lys适宜浓度为2.0~4.0mmol/L。     

鸡H-FABP和A-FABP基因表达与肌内脂肪含量相关研究

选用H-FABP和A-FABP基因作为影响鸡肌内脂肪沉积的候选基因,以北京油鸡、矮脚鸡、白莱航鸡和AA肉鸡为研究群体,利用RT-PCR和实时荧光定量PCR技术,分别对56、90、120日龄H-FABP和A-FABP基因mRNA进行定量分析,结合IMF含量及屠体性状测定,分析H-FABP及A-FABP基因表达水平对IMF含量等的影响.结果表明：H-FABP基因mRNA随日龄的增长表达量显著降低,而A-FABP基因mRNA随日龄的增长表达量显著升高,并表现出显著的品种效应（P＜0.01）,性别因素对A-FABP基因表达影响显著.北京油鸡、白莱航鸡和AA鸡群体的H-FABP基因mRNA表达水平与IMF含量及屠体重呈现显著的负相关,而A-FABP基因mRNA表达水平与屠体重显著相关,与IMF含量没有显著的相关性.矮脚鸡A-FABP基因mRNA水平与IMF含量呈现显著的负相关,其H-FABP基因mRNA水平对屠体重影响显著,表现出显著的品种差异.     

3个藏系绵羊品种血清运铁蛋白的多态性比较

本文采用聚丙烯酰胺凝胶垂直平板电泳法,测定了四川省阿坝县草地藏系绵羊红原羊类群、贾洛羊类群及青海欧拉羊血清运铁蛋白的多态性,对其遗传特征进行了比较研究。结果发现：红原羊的运铁蛋白（TF）有AB、BB、BC三种基因型,其基因型频率分别为0.53333、0.33333、0.13333,基因频率分别为0.26667、0.66667、0.06667;贾洛羊的运铁蛋白有AB一种基因型,其基因型频率为1,基因频率分别为0.5、0.5、0;欧拉羊的运铁蛋白有AA、AB、BB三种基因型,其基因型频率分别为0.43243、0.43243、0.13514,基因频率分别为0.64865、0.35135、0。     

（ACP5） in the Ovine Uterus： I. Regulation by Pregnancy and Progesterone

Uteroferrin, also known as type 5 tartrate resistant acid phosphatase （ ACP5 ） or TRAP, is an iron-containing glycoprotein secreted by uterine gland epithelium （GE） in response to progesterone and transported across the placental areoalae into the fetal circulation and aUantoic fluid to deliver iron and to stimulate hematopoeisis in pigs. This study deter- mined if ACP5 was expressed in the ovine uterus in response to pregnancy, progesterone, interferon tau, placental lactogen, and placental growth hormone. ACP5 protein was present in uterine GE of cyclic and early pregnant ewes,particularly between days 18 and 120 of pregnancy. ACP5 mRNA was expressed in uterine GE of cyclic and pregnant ewes in the same temporal and cell-specific manner. ACP5 was present in secretions from uterine glands, i. e. , uterine milk,and allantoic fluid from days 40 to 80 of pregnancy, and in uterine flushings from cyclic and early pregnant ewes. Progesterone induced expression of ACP5 mR- NA and intrauterine infusion of recombinant ovine interferon tau further stimulated ACP5 expression in uterine GE of ewes,but intrauterine injections of ovine placental lactogen and ovine growth hormone had no effect on ACP5 expression in uterine GE. These re- sults indicate that ACP5 is：1 ） expressed only in GE in response to progesterone ;2） secreted into the uterine lumen and transported into the conceptus via placental areolae during pregnancy;and 3） present in secretions from uterine GE and in allantoic fluid. The roles of ACP5 in the ovine uterus may include transport of iron across the placenta and stimulation of hematopoiesis.     

Immune cell populations in the duodenal mucosa of cats with inflammatory bowel disease

The aim of this study was to characterize the phenotype of leukocytes infiltrating the duodenal mucosa of cats with inflammatory bowel disease (IBD) by using immunohistochemistry and computer-aided morphometry to assess whether immunologic markers would aid in characterization of IBD. Frozen and formalin-fixed duodenal biopsies were collected from cats referred for investigation of chronic vomiting, diarrhea, or both (n = 34). Reference ranges were previously established by using duodenal samples from healthy cats (n = 16). No significant difference was found in the number of immunoglobulin G+ (IgG+) or IgA+ in either the villous lamina propria or the crypt lamina propria between cats with IBD and control cats. T cells (CD3+) increased in number from crypt to the tip of the villi in biopsies from both diseased (mean +/- SD for each group was 18.8 +/- 6.6 and 17.7 +/- 4.2 cells/ 10,000 m2 in cryptal areas to 25.2 +/- 9.5 and 29.1 +/- 13.3 cells/10,000m2 in villous areas) and healthy animals (17.9 +/- 3.9 cells/10,000 microm2 in cryptal areas to 24.1 +/- 9.3 cells/10,000 microm2 in villous areas) and no significant difference was found between diseased and control cats. By contrast, major histocompatibility complex (MHC) class II expression by leukocytes with dendritic cell or macrophage morphology in the lamina propria was significantly greater in cats with IBD (13.3 +/- 4.2 cells/10,000 microm2 in cryptal area; P = .016) than in healthy cats (11.9 +/- 3.0 cells/10,000 microm2) and MHC class II expression by enterocytes also was more pronounced in these cats showing an overall intensity of expression of 7.1 +/- 4.0 cells/10,000 microm2 in cats with IBD as opposed to 0.0 +/- 0.0 cells/10,000 microm2 to 0.3 +/- 0.7 cells/10,000 microm2 in healthy cats. These findings suggest that a subtle immunologic dysregulation occurs in spontaneously arising feline IBD.     

Differential expression of CD45 on canine microglial cells

CD45, also called leucocyte common antigen is a transmembrane protein tyrosine phosphatase on the surface of nearly all white blood cells and has a functional role in signal transduction. In the brain, the expression of CD45 can be used to distinguish microglial cells with a characteristic phenotype of CD11b/c+ and CD45(low) from other central nervous system (CNS) macrophages which show an expression of CD11b/c+ and CD45(high). In the course of pathological changes in the CNS, microglia in rodents is known to readily upregulate expression of various surface molecules, such as CD45. Understanding the mechanisms that regulate expression of surface molecules is essential to study the pathogenesis of CNS diseases. In the present study, the expression of CD45 on microglia of 42 dogs was examined ex vivo by means of flow cytometry. The dogs were classified in two groups according to the histopathological diagnosis in the CNS. All dogs without changes in the CNS (group I; n = 22) only showed low percentages of CD45+ microglial cells. In group II consisting of 20 dogs with different intracranial diseases varying results were obtained. Thirteen dogs showed a low percentage of CD45+ microglial cells whereas seven dogs exhibited high percentages of microglial cells expressing CD45. Evaluation of expression intensity in these seven dogs revealed two subpopulations of CD45+ microglial cells: a large subpopulation with CD45(low) and a small subpopulation with CD45(high). The expression intensity of CD45(high) was comparable with that of canine monocytes. It was attempted to correlate these findings to age of the animals, underlying disease, duration of clinical signs, medical treatment, occurrence of seizure activity and the expression of other surface molecules. It appeared that dogs with high percentages of CD45+ suffered from long-lasting CNS disease with seizures. In future studies, the reason and consequences for upregulated CD45 in long-lasting CNS diseases has to be further evaluated.     

Canine lymphoproliferative disease characterized by lymphocytosis: immunophenotypic markers of prognosis

BACKGROUND: Canine lymphoproliferative disease often presents with lymphocytosis and is immunophenotypically diverse. HYPOTHESIS: Immunophenotype predicts prognosis in canine lymphoproliferative disorders involving circulating lymphocytosis. ANIMALS: Dogs that had peripheral blood evaluation performed by flow cytometry by the Clinical Immunology Service at Colorado State University between 2003 and 2005. METHODS: Outcome data regarding treatment and survival were sought on patients with lymphocytosis comprising a single lymphocyte subset. Ninety-six patients that met the inclusion criteria had sufficient follow-up information to be included in the study. RESULTS: Four main phenotypic classifications were found: CD8+ T-cell, CD21+ B-cell, CD4-8-5+ (aberrant T-cell phenotype), and CD34+ (undifferentiated progenitor). Expression of CD34 predicted poor outcome with median survival of 16 days (P < .0001) compared with other phenotypes. Within the CD8+ phenotype, dogs presenting with a lymphocytosis >30,000 lymphocytes/muL had significantly shorter median survival (131 days) than those presenting with <30,000 lymphocytes/muL (1098 days, P < .0008). Within the T-cell leukemias, there was no difference in outcome between dogs with CD4-8-5+ leukemia and dogs with the CD8+ T-cell phenotype nor was the loss of expression of the pan-leukocyte marker CD45 associated with decreased survival time. A CD21+ lymphocytosis composed of large cells was associated with shorter survival time (129 days) than those with smaller circulating cells (median survival not reached, P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE: Immunophenotyping provides an objective method for determining prognosis in lymphoproliferative disorders characterized by lymphocytosis.     

MCU在低氧诱导肉鸡心肌细胞线粒体损伤中的作用

旨在探讨线粒体钙单向转运体（mitochondrial calcium uniporter,MCU）介导线粒体Ca²⁺转运是否参与低氧诱导的肉鸡心肌细胞线粒体损伤。试验通过分离白羽肉鸡鸡胚原代心肌细胞,在低氧条件下（3% O₂,5% CO₂,92% N₂）培养24、48、72 h,同时使用RU360抑制MCU的表达并在低氧条件下处理72 h后,使用流式细胞术检测细胞内Ca²⁺浓度、线粒体Ca²⁺浓度、线粒体活性氧水平和线粒体膜电位,检测MCU及其调节因子的mRNA和蛋白表达。结果显示,通过差速贴壁方法培养的心肌细胞纯度可达90%以上;低氧培养24 h,诱导心肌细胞MCU、MICU1 mRNA表达增加（P<0.05）,胞浆和线粒体内Ca²⁺浓度显著上升（P<0.05）,线粒体膜电位增加（P<0.05）;低氧培养48 h,诱导MCUR1和MICU1 mRNA表达减少（P<0.05）,细胞Ca²⁺浓度上升（P<0.05）;低氧培养72 h,诱导MCU mRNA表达增加（P<0.01）,细胞和线粒体内Ca²⁺增加（P<0.01）,线粒体膜电位下降（P<0.01）,活性氧增加（P<0.01）。低氧处理72 h后,与低氧组相比,RU360预处理组细胞和线粒体内Ca²⁺减少,线粒体膜电位上升,活性氧生成减少（P<0.01）,MCU mRNA表达减少（P<0.01）。结果表明：低氧诱导MCU上调导致线粒体钙超载,促使线粒体功能降低并发生损伤,进而心肌细胞发生损伤;抑制MCU表达可以减轻低氧诱导的心肌细胞线粒体钙超载,保护线粒体。     

Feline large granular lymphocyte (LGL) lymphoma with secondary leukemia: primary intestinal origin with predominance of a CD3/CD8(alpha)(alpha) phenotype

Clinicopathologic and immunophenotypic characteristics of large granular lymphocyte (LGL) neoplasia in 21 cats were examined. All cats were domestic short (19) or long hair (2) with a mean age of 9.3 years at diagnosis. Increased peripheral blood LGL counts were present in 18/21 cats. Neutrophilia (12/21 cats) and increased serum liver enzymes (7/12), total and direct bilirubin (7/13), BUN (5/14), and creatinine (2/14) were observed. Cats usually presented with advanced disease and none survived longer than 84 days (mean 18.8 days) postdiagnosis. Cytologically, LGLs had a mature (6/21), immature (13/21), or mixed (2/21) morphology. Necropsy lesions consisted of neoplastic lymphoid infiltrates in the jejunum, ileum, and duodenum in decreasing order of frequency. In the small intestine, mucosal ulceration (9/13) and epitheliotropism of neoplastic cells (9/13) were common. Neoplastic infiltrates were also present in the mesenteric lymph nodes (13/13), liver (12/13), spleen (8/13), kidneys (5/7), and bone marrow (5/7). A T cell phenotype (CD3epsilon+) characterized LGL neoplasia in 19/21 cases. A CD8alphaalpha+ cytotoxic/suppressor phenotype was present in 12/19 T cell tumors, 2 had a CD4+CD8alphaalpha phenotype, 3 had a CD4-CD8- phenotype, and 2 were CD4+ helper T cells. CD8beta chain expression was not detected in any instance. In two cats, a B or T cell origin could not be established. CD103 was expressed by 11 of 19 (58%) of the lymphomas tested. The immunophenotypic features shared by neoplastic LGLs in the cat and feline intestinal intraepithelial lymphocytes (IELs) support a small intestinal IEL origin for feline LGL lymphoma.     

B7+CTLA4+ T cells engage in T-T cell interactions that mediate apoptosis: a model for lentivirus-induced T cell depletion

Apoptosis in lymph node (LN) T cells of feline immunodeficiency virus (FIV)-infected cats is associated with cells co-expressing B7.1 and B7.2 costimulatory molecules, and their ligand CTLA4. To study the possibility of B7.1/B7.2-CTLA4 mediated T-T interactions and the predicted induction of T cell apoptosis in vitro, costimulatory molecules were up-regulated on CD4+ and CD8+ T cells by mitogen stimulation. B7.1 expression on in vitro stimulated CD4+ and CD8+ cells increased within 24h; B7.2 and CTLA4 expression increased after 48-72 h. Apoptosis, as analyzed by terminal deoxynucleotidyl transferase (transferase nick end labeling, TUNEL)-based staining followed by three color flow cytometric analysis, correlated to the cells expressing B7 and/or CTLA4. Blocking experiments revealed that CD4+ and CD8+ T cell apoptosis could be significantly inhibited with anti-B7 antibodies. As FIV infection results in immune activation with a T cell phenotype similar to that of the in vitro activated T cells, the data support the hypothesis that the chronic expansion of B7+CTLA4+ LN T cells in infected cats allows for T-T cell interactions resulting in T cell depletion and eventually the development of AIDS.     

Prevalence of various phenotypes of Streptococcus suis isolated from swine in the U.S.A. based on the presence of muraminidase-released protein and extracellular factor.

The present study describes the prevalence of muraminidase-released protein (MRP) and extracellular factor (EF) proteins associated with virulence of Streptococcus suis serotype 2 from a collection of USA strains. Sixty-six strains belonging to serotypes 1, 2, 3, 4, 7, and 10, were analyzed with a set of double antibody sandwich ELISAs and Western blots. Nineteen of 34 serotype 2 strains from cases of swine meningitis had the MRP+EF+ phenotype. Five of 7 serotype 2 strains isolated from lungs had an MRP*EF- phenotype. An MRP-EF+ phenotype was found in 4/34 strains isolated from swine meningitis. The MRP*EF- and MRP-EF+ phenotypes have not been reported previously. All strains of serotypes other than 2, including isolates from cases of meningitis, had the MRP-EF- phenotype, suggesting that these strains must have other, as yet undetected, virulence factors.     

dsRNA和Aza-CdR转染猪肾细胞的全基因组差异甲基化区域分布特征的比较

本研究旨在通过比对PolyI：C和Aza-CdR转染猪肾细胞后全基因组差异甲基化峰的分布特征，进而筛选Gene Ontology （GO）特有的差异甲基化基因，分析差异甲基化区域。首先，基于MeDIP-chip技术，采用猪385 K全基因组启动子和CpG岛甲基化芯片，分析3组试验材料（病毒模拟物Poly I：C转染的猪PK15细胞、甲基化酶抑制剂Aza-CdR转染的PK15细胞、无处理的mock细胞），通过Peak DM Value和Peak Score值获得试验组间显著性富集的差异甲基化峰；其次，对差异甲基化基因进行GO注释，筛选差异甲基化区域和差异甲基化基因。最终结合Bisulfite克隆测序和mRNA荧光定量表达试验验证差异甲基化区域DMR。试验初步揭示猪肾细胞全基因组DNA甲基化主要分布于5'调控区域。试验在组间比较后，特别是在P vs.C和A vs.C比较中发现DNA甲基化在基因组上的分布特征与CpG岛密度与距离TSS的位置有关，而在近启动子区域（0―+200 bp） DNA甲基化显著影响基因的表达。Poly I：C对PK15作用使得TSS附近200 bp （-200―+500 bp）低甲基化启动子增多，说明Poly I：C与Aza-CdR的作用相似，均具有潜在的去甲基化作用，特别是位于猪14号染色体上BNIP3L基因的10459946―10460615 bp区段共有669 bp Peak Length CG位点发生去甲基化。研究揭示，PolyI：C和Aza-CdR并不是对猪所有基因具有去甲基化作用，主要针对特有基因的特有启动子，证明这些特有启动子的CpG岛对Poly I：C和Aza-CdR具有特别的敏感性。     

Epigenetically immature oocytes lead to loss of imprinting during embryogenesis

Loss of imprinting (LOI) is occasionally observed in human imprinting disorders. However, the process behind the LOI is not fully understood. To gain a better understanding, we produced embryos and pups from mouse oocytes that lacked a complete methylation imprint using a method that involved transferring the nuclei of growing oocytes into the cytoplasm of enucleated fully grown oocytes following in vitro fertilization (IVF). We then analyzed the imprinting statuses. Our findings show that the incomplete methylation imprint derived from growing oocytes results in epigenetic mosaicism or a loss of methylation imprint (LOM) at maternal alleles in embryos. In some embryos, both hypo- and hypermethylated maternal Kcnq1ot1 alleles were detected, whereas either hypo- or hypermethylated maternal Kcnq1ot1 alleles were detected in others. Such tendencies were also observed at the Igf2r and Mest loci. Gene expression levels of imprinted genes were linked with their methylation statuses in some but not all embryos. Possible explanations of the inconsistency between the data from DNA methylation and gene expression include epigenetic mosaicism in embryos. Pups were successfully produced from growing oocytes at a quite low frequency. They exhibited an obese phenotype and LOI with respect to Igf2r, Snrpn and Mest. Our finding suggests the possibility that LOI/LOM at maternal alleles in human concepti could be derived from epigenetically immature/mutated oocytes.