Background: The rumen wall plays a major role in efficient transfer of digested nutrients in the rumen to peripheral tissues through the portal venous system. Some of these substrates are metabolised in the epithelium...Background: The rumen wall plays a major role in efficient transfer of digested nutrients in the rumen to peripheral tissues through the portal venous system. Some of these substrates are metabolised in the epithelium during this process. To identify the specific proteins involved in these processes, we used proteomic technologies. Protein extracts were prepared from ventral rumen tissue of six sheep fed a fibrous diet at 1.5× maintenance energy requirements.Using a newly developed method, we were able to enzymatically isolate the epithelial cells from underlying tissue layers, thus allowing cytosol and membrane fractions to be independently analysed using liquid chromatography tandem mass spectrometry(LC MS/MS).Results: Using our procedure we identified 570 epithelial proteins in the Ovis aries sequence database. Subcellular locations were largely cytosolic(n = 221) and extracellular(n = 85). However, a quarter of the proteins identified were assigned to the plasma membrane or organelle membranes, some of which transport nutrients and metabolites. Of these 91 were transmembrane proteins(TMHMM), 27 had an N-terminal signal peptide(signalP) and TMHMM motif,13 had a glycosylphosphatidylinositol(GPI) anchor and signalP sequence, 67 had beta(β) strands or 17 β strands and a transit peptide sequence, indicating the identified proteins were integral or peripheral membrane proteins. Subunits of the 5 protein complexes involved in mitochondrial cellular energy production were well represented. Structural proteins(15%), proteins involved in the metabolism of lipids and proteins(26%) and those with steroid or cytokine action were a feature of the proteome.Conclusion: Our research has developed a procedure to isolate rumen epithelium proteins from the underlying tissue layers so that they may be profiled using proteomic technologies. The approach improves the number of proteins that can be profiled that are specific to the epithelium of the rumen wall. It provides new insights into the proteins of structural and nutritional importance in the rumen epithelium, that carry out nutrient transport and metabolism, cell growth and signalling.展开更多
Isolated ruminal epithelia from caudal blind sacs of dairy goats were incubated with butyrate and insulin-like growth factor-1（IGF-1） at different concentrations. Proportions of ruminal epithelium in different phase...Isolated ruminal epithelia from caudal blind sacs of dairy goats were incubated with butyrate and insulin-like growth factor-1（IGF-1） at different concentrations. Proportions of ruminal epithelium in different phases of the cell division cycle were determined by flow cytometric analysis. The proportion of epithelial cells in S phase and G2-M phase（PS＆G2-M） increased significantly（P〈0.01） whereas the proportion of epithelial cells in G0-G1 phase（PG0-G1） decreased after incubation with IGF-1. PS＆G2-M decreased whereas PG0-G1 increased markedly（P〈0.01） after incubation with sodium butyrate. PS＆G2-M incubated with IGF-1 and butyrate sodium together increased more than that incubated with IGF-1 alone; PG0-G1, however, decreased significantly（P〈0.01）. Our results indicate that IGF-1 enhances whereas sodium butyrate inhibits the proliferation of rumen epithelial cells. Furthermore, butyrate and IGF-1, together, have a synergic effect on the proliferation of rumen epithelium.展开更多
Lactation in dairy cattle is coupled with increased nutrient requirements for milk synthesis. Therefore, dairy cattle metabolism has to adapt to meet lactation-associated challenges and requires major functional adjus...Lactation in dairy cattle is coupled with increased nutrient requirements for milk synthesis. Therefore, dairy cattle metabolism has to adapt to meet lactation-associated challenges and requires major functional adjustments of the rumen and whole digestive system. This report describes the use of next-generation sequencing technology for assembly and profiling of the transcriptome of cattle rumen epithelial tissues from cattle in both dry and lactation periods. Transcriptomics profiling and comparison revealed extensive changes in gene expression related to metabolism in rumen epithelial tissue due to the adaptation to lactation. Ruminal epithelial adaptation to the challenges of metabolism and high nutrient requirements during lactation is presumably the primary triggers for these alterations in gene expression. Principal component analysis (PCA) indicated that the gene expression profiles of the rumen epithelia from dry and lactating cattle fall into two very distinct clusters. Gene Ontology (GO) enrichment analysis revealed that the most GO terms were related to various metabolic processes in lactating cattle. The most significantly (false discovery rate (FDR) p-value < 0.05) enriched GO term in biological processes was “carbohydrate derivative metabolic process”, followed by “nucleoside metabolic process”. Up-stream regulators, such as PPARA (Peroxisome proliferator-activated receptor alpha) gene, and up-regulated genes of molecular transporters are the focal points of this report.展开更多
本试验旨在采用体外法研究p H与脂多糖(LPS)或组胺(HIS)的交互作用对奶山羊瘤胃上皮细胞紧密连接蛋白mRNA表达量的影响。选用8只体况良好,体重、泌乳量相近的萨能奶山羊作为瘤胃上皮供体。采用3×3双因素试验设计,山羊屠宰后采集瘤...本试验旨在采用体外法研究p H与脂多糖(LPS)或组胺(HIS)的交互作用对奶山羊瘤胃上皮细胞紧密连接蛋白mRNA表达量的影响。选用8只体况良好,体重、泌乳量相近的萨能奶山羊作为瘤胃上皮供体。采用3×3双因素试验设计,山羊屠宰后采集瘤胃上皮,插入到尤斯灌流仪器(Ussing chamber)半室中央,在浆膜侧半室加入5 m L缓冲液,黏膜侧加入配制好的不同处理的培养液5 m L,每个处理3个重复。试验1:因素1为p H,分别为7.4、5.5、5.2;因素2为LPS浓度,分别为0、30、60 k EU/m L。试验2:因素1为p H,分别为7.4、5.5、5.2;因素2为HIS浓度,分别为0、0.5、10.0 ng/m L。采集培养80 min后的瘤胃上皮,测定其紧密连接蛋白Claudin-1、Claudin-4、Claudin-7、Occludin、zonula occludens-1(ZO-1)mRNA表达量。结果显示:1)p H与LPS的交互作用对Claudin-1、Claudin-7、ZO-1 mRNA表达量影响显著(P<0.05)。与p H 7.4×0 k EU/m L LPS组相比,降低p H或添加LPS均显著降低了Claudin-1、Claudin-7 mRNA表达量(P<0.05),ZO-1 mRNA表达量有整体降低的趋势,在p H 5.2×60 k EU/m L LPS组最高。2)p H与HIS的交互作用对Claudin-1、Claudin-7、ZO-1 mRNA表达量影响显著(P<0.05)。p H 5.5×0.5 ng/m L HIS组Claudin-1 mRNA表达量最低,但与p H 5.2×0.5 ng/m L HIS组差异不显著(P>0.05)。p H 7.4×10.0 ng/m L HIS组Claudin-7 mRNA表达量显著低于p H 7.4×0 ng/m L HIS组(P<0.05),但与p H 5.5×10.0 ng/m L HIS组差异不显著(P>0.05)。与p H 7.4×0 ng/m L HIS组相比,降低p H或添加LPS有提高ZO-1 mRNA表达量的趋势,且p H 5.2×10.0 ng/m L HIS组显著提高(P<0.05)。结果提示,亚急性瘤胃酸中毒(SARA)发生后,p H与LPS或HIS交互作用于瘤胃上皮,降低瘤胃上皮紧密连接蛋白mRNA表达量,进而增大瘤胃上皮黏膜通透性。展开更多
基金funded by Meat and Livestock Australia(MLA)and Commonwealth Government Department of Agriculture Forestry and Fisheries(DAFF grant no.1193857–31)
文摘Background: The rumen wall plays a major role in efficient transfer of digested nutrients in the rumen to peripheral tissues through the portal venous system. Some of these substrates are metabolised in the epithelium during this process. To identify the specific proteins involved in these processes, we used proteomic technologies. Protein extracts were prepared from ventral rumen tissue of six sheep fed a fibrous diet at 1.5× maintenance energy requirements.Using a newly developed method, we were able to enzymatically isolate the epithelial cells from underlying tissue layers, thus allowing cytosol and membrane fractions to be independently analysed using liquid chromatography tandem mass spectrometry(LC MS/MS).Results: Using our procedure we identified 570 epithelial proteins in the Ovis aries sequence database. Subcellular locations were largely cytosolic(n = 221) and extracellular(n = 85). However, a quarter of the proteins identified were assigned to the plasma membrane or organelle membranes, some of which transport nutrients and metabolites. Of these 91 were transmembrane proteins(TMHMM), 27 had an N-terminal signal peptide(signalP) and TMHMM motif,13 had a glycosylphosphatidylinositol(GPI) anchor and signalP sequence, 67 had beta(β) strands or 17 β strands and a transit peptide sequence, indicating the identified proteins were integral or peripheral membrane proteins. Subunits of the 5 protein complexes involved in mitochondrial cellular energy production were well represented. Structural proteins(15%), proteins involved in the metabolism of lipids and proteins(26%) and those with steroid or cytokine action were a feature of the proteome.Conclusion: Our research has developed a procedure to isolate rumen epithelium proteins from the underlying tissue layers so that they may be profiled using proteomic technologies. The approach improves the number of proteins that can be profiled that are specific to the epithelium of the rumen wall. It provides new insights into the proteins of structural and nutritional importance in the rumen epithelium, that carry out nutrient transport and metabolism, cell growth and signalling.
基金supported by the National Natural Science Foundation of China(30960252)the China Agriculture Research System(CARS-37)
文摘Isolated ruminal epithelia from caudal blind sacs of dairy goats were incubated with butyrate and insulin-like growth factor-1（IGF-1） at different concentrations. Proportions of ruminal epithelium in different phases of the cell division cycle were determined by flow cytometric analysis. The proportion of epithelial cells in S phase and G2-M phase（PS＆G2-M） increased significantly（P〈0.01） whereas the proportion of epithelial cells in G0-G1 phase（PG0-G1） decreased after incubation with IGF-1. PS＆G2-M decreased whereas PG0-G1 increased markedly（P〈0.01） after incubation with sodium butyrate. PS＆G2-M incubated with IGF-1 and butyrate sodium together increased more than that incubated with IGF-1 alone; PG0-G1, however, decreased significantly（P〈0.01）. Our results indicate that IGF-1 enhances whereas sodium butyrate inhibits the proliferation of rumen epithelial cells. Furthermore, butyrate and IGF-1, together, have a synergic effect on the proliferation of rumen epithelium.
文摘Lactation in dairy cattle is coupled with increased nutrient requirements for milk synthesis. Therefore, dairy cattle metabolism has to adapt to meet lactation-associated challenges and requires major functional adjustments of the rumen and whole digestive system. This report describes the use of next-generation sequencing technology for assembly and profiling of the transcriptome of cattle rumen epithelial tissues from cattle in both dry and lactation periods. Transcriptomics profiling and comparison revealed extensive changes in gene expression related to metabolism in rumen epithelial tissue due to the adaptation to lactation. Ruminal epithelial adaptation to the challenges of metabolism and high nutrient requirements during lactation is presumably the primary triggers for these alterations in gene expression. Principal component analysis (PCA) indicated that the gene expression profiles of the rumen epithelia from dry and lactating cattle fall into two very distinct clusters. Gene Ontology (GO) enrichment analysis revealed that the most GO terms were related to various metabolic processes in lactating cattle. The most significantly (false discovery rate (FDR) p-value < 0.05) enriched GO term in biological processes was “carbohydrate derivative metabolic process”, followed by “nucleoside metabolic process”. Up-stream regulators, such as PPARA (Peroxisome proliferator-activated receptor alpha) gene, and up-regulated genes of molecular transporters are the focal points of this report.
文摘本试验旨在采用体外法研究p H与脂多糖(LPS)或组胺(HIS)的交互作用对奶山羊瘤胃上皮细胞紧密连接蛋白mRNA表达量的影响。选用8只体况良好,体重、泌乳量相近的萨能奶山羊作为瘤胃上皮供体。采用3×3双因素试验设计,山羊屠宰后采集瘤胃上皮,插入到尤斯灌流仪器(Ussing chamber)半室中央,在浆膜侧半室加入5 m L缓冲液,黏膜侧加入配制好的不同处理的培养液5 m L,每个处理3个重复。试验1:因素1为p H,分别为7.4、5.5、5.2;因素2为LPS浓度,分别为0、30、60 k EU/m L。试验2:因素1为p H,分别为7.4、5.5、5.2;因素2为HIS浓度,分别为0、0.5、10.0 ng/m L。采集培养80 min后的瘤胃上皮,测定其紧密连接蛋白Claudin-1、Claudin-4、Claudin-7、Occludin、zonula occludens-1(ZO-1)mRNA表达量。结果显示:1)p H与LPS的交互作用对Claudin-1、Claudin-7、ZO-1 mRNA表达量影响显著(P<0.05)。与p H 7.4×0 k EU/m L LPS组相比,降低p H或添加LPS均显著降低了Claudin-1、Claudin-7 mRNA表达量(P<0.05),ZO-1 mRNA表达量有整体降低的趋势,在p H 5.2×60 k EU/m L LPS组最高。2)p H与HIS的交互作用对Claudin-1、Claudin-7、ZO-1 mRNA表达量影响显著(P<0.05)。p H 5.5×0.5 ng/m L HIS组Claudin-1 mRNA表达量最低,但与p H 5.2×0.5 ng/m L HIS组差异不显著(P>0.05)。p H 7.4×10.0 ng/m L HIS组Claudin-7 mRNA表达量显著低于p H 7.4×0 ng/m L HIS组(P<0.05),但与p H 5.5×10.0 ng/m L HIS组差异不显著(P>0.05)。与p H 7.4×0 ng/m L HIS组相比,降低p H或添加LPS有提高ZO-1 mRNA表达量的趋势,且p H 5.2×10.0 ng/m L HIS组显著提高(P<0.05)。结果提示,亚急性瘤胃酸中毒(SARA)发生后,p H与LPS或HIS交互作用于瘤胃上皮,降低瘤胃上皮紧密连接蛋白mRNA表达量,进而增大瘤胃上皮黏膜通透性。
