二肽在仔猪小肠刷状缘膜囊中跨膜转运的动力学特点研究

为研究二肽在仔猪小肠刷状缘膜囊（Brush border membrane vesicles,BBMV）中跨膜转运的动力学特点,采用同位素示踪及体外孵育技术,观察了几种不同条件下Gly-Pro的跨膜转运量。结果表明：（1）在有Na^＋和无Na^＋的条件下,Gly-Pro的转运均受到Leu-Gly、Ala-His、Gly-Leu和His-Leu等二肽的极显著抑制（P〈0.01）,但不受甘氨酸、L-丙氨酸、L-脯氨酸、L-亮氨酸、L-精氨酸、L-谷氨酰胺和L-蛋氨酸的影响;（2）仔猪小肠BBMV对Gly-Pro的转运呈现非线性关系,但是遵从Michaelis-Menten动力学方程,其Kt（亲合常数）值为0.64 mmol/L,而Vmax为7.42 nmol/（min.mg）膜蛋白;（3）仔猪小肠BBMV对Gly-Pro的摄取是由于Gly-Pro直接进入BBMV内部的结果,而不是仅仅结合在BBMV上所引起的。结果提示,Gly-Pro在仔猪小肠BBMV中的跨膜转运具有载体介导的转运特点。     

膜电位对仔猪小肠刷状缘膜囊中二肽跨膜转运的影响

为研究膜电位与仔猪小肠刷状缘膜囊(brush border membrane vesicles,BBMV)中二肽跨膜转运之间的关系,采用膜电位诱导、体外孵育及同位素示踪技术,分别观察由缬氨霉素和羰基氰化物三氟甲氧基苯腙(carbonylcyanide p-(Tri-fluoromethoxy)phenylhydrazone,FCCP)诱导仔猪小肠BBMV产生的K 扩散电位(膜囊内负)和H 扩散电位(膜囊内正)对Gly-Pro二肽跨膜转运的影响。结果表明:在有Na 和无Na 的状况下,K 扩散电位均促进Gly-Pro二肽由膜囊外进入膜囊内部;H 扩散电位抑制Gly-Pro二肽由膜囊外进入膜囊内部。结果提示:在仔猪小肠BBMV转运体系中,Gly-Pro二肽的跨膜转运具有与非Na 的阳离子协同转运的特点。     

内流质子梯度对Gly-Pro在仔猪小肠跨膜转运中的驱动作用

为研究内流质子梯度对仔猪小肠中Gly-Pro跨膜转运的影响,采用放射性同位素示踪及体外孵育技术,观察在不同H＋浓度条件下,Gly-Pro在仔猪小肠刷状缘膜囊（Brush border membrane vesicles,BBMV）的跨膜转运量。结果表明：膜囊内液pH为7.5时,Gly-Pro的转运显著地受到膜囊外液pH值的影响,pH为4.5～5.5时,转运最快;其中pH为5.0时的Gly-Pro跨膜转运速度在1-20 min一直高于pH为7.5时的转运速度;内流质子梯度的存在可以显著促进Gly-Pro的转运,无内流质子梯度时,Gly-Pro的转运不受膜囊外液pH变化的影响。研究结果提示,跨膜内流质子梯度是仔猪小肠中Gly-Pro的跨膜转运的一种驱动力。     

二肽在断奶仔猪小肠刷状缘膜囊中水解状况的研究

通过孵育试验研究了甘-L-亮(Gly-Leu)和L-组-L-亮(His-Leu)2种二肽在断奶仔猪小肠刷状缘膜囊(Brush Border Memlorane Vesides，BBMV)中的水解状况。结果表明，孵育液pH值(分别为3．5、4．0、4．5、5．0、5．5、6．0、6．5、7．0、7．5和8．O)、孵育温度(25℃和37℃)、孵育时间(O．5、1、2、5、10、20、30和40min)以及BBMV(二肽酶)的添加量均不影响此2种二肽在断奶仔猪小肠BBMV孵育体系中的水解。结果提示，Gly-Leu和His-Leu2种二肽在断奶仔猪小肠BBMV体系中均不水解。     

断奶仔猪小肠刷状缘膜囊中小肽水解的研究

通过孵育试验研究了L 亮 甘 (Leu Gly)和 β 丙 组 (Ala His)等两种二肽在断奶仔猪小肠刷状缘膜囊 (BrushBorderMembraneVesicles,BBMV)中的水解状况。结果表明 ,孵育液pH(分别为 3.5 ,4 .0 ,4 .5 ,5 .0 ,5 .5 ,6 .0 ,6 .5 ,7.0 ,7.5和 8.0 )、孵育温度 (2 5℃和 37℃ )、孵育时间 (0 .5 ,1,2 ,5 ,10 ,2 0 ,30和 4 0min)以及BBMV(可能含二肽酶 )的添加量对两种二肽在断奶仔猪小肠BBMV孵育体系中浓度没有影响。结果表明 ,Leu Gly和Ala His在断奶仔猪小肠BBMV体系中均不水解     

仔猪小肠上皮细胞刷状缘膜囊制备方法的研究

采用CaCl2 和MgCl2 两种沉淀法制备了断奶仔猪小肠上皮细胞刷状缘膜囊 (BBMV) ,并通过测定BBMV制备过程中的细胞膜或细胞器标志酶酶活、核糖核酸和脱氧核糖核酸含量等指标对所制备的BBMV的纯度进行了评价。结果表明采用MgCl2 沉淀方法制备的断奶仔猪小肠BBMV纯度优于CaCl2 沉淀法。     

生长猪小肠钠依赖型磷摄入特性的研究

选取6头体重接近的3月龄生长期大白猪,屠宰后刮下肠黏膜,提取3段小肠上皮细胞刷状缘膜囊泡(Brush-border membrane vesicles,BBMV)。在室温条件下,研究各段小肠BBMV磷的摄入量,pH和钠离子依赖性,并测定BBMV磷吸收动力学参数。结果表明,生长猪BBMV对磷的主动吸收和被动扩散在酸性pH下均受到抑制。钠离子水平影响BBMV磷的主动吸收。pH7.4条件下,测得钠依赖型磷转运系统Hill系数(napp)为2.07,即转运1个磷酸根,至少有2个钠离子与其协同转运。不同肠段BBMV磷的主动吸收并没有显著差异。pH7.4条件下,生长猪钠依赖型磷转运系统对磷的亲和力常数(Km)为(0.43±0.09)mmol/L。最大转运速度(Vmax)为(2.25±0.19)nmol/(mg蛋白.15 s)。     

早期断奶对仔猪小肠中谷氨酸/谷氨酰胺转运载体表达的影响

本试验旨在研究早期断奶对仔猪小肠中谷氨酸/谷氨酰胺转运载体表达的影响。试验从40头母猪的仔猪中各选出体重相近、10日龄的杜长大三元杂交仔猪1头,共40头仔猪,随机不配对分为2组,每组20头仔猪,对照组为哺乳仔猪,随母猪喂养;试验组为断奶仔猪,隔离断奶饲养。试验期10 d。饲养结束,每组随机选取12头仔猪,屠宰后取空肠和回肠组织,测定谷氨酸/谷氨酰胺转运载体蛋白和mRNA表达情况,并检测小肠组织形态和小肠氧化应激水平。结果表明:与哺乳仔猪相比,早期断奶显著提高了仔猪空肠和回肠谷氨酸/胱氨酸交换载体(xCT)和中性氨基酸转运载体2(ASCT2)蛋白和mRNA表达量(P0.05);皮尔森相关分析结果显示,断奶仔猪与哺乳仔猪的xCT和ASCT2蛋白表达量在空肠和回肠组织匀浆、细胞内质、细胞顶膜与空肠和回肠中对应mRNA表达量之间均呈正向线性关系,且差异显著(P0.05)。与哺乳仔猪相比,断奶仔猪空肠和回肠绒毛高度降低,隐窝深度增加,差异均显著(P0.05)。与哺乳仔猪相比,断奶仔猪空肠和回肠组织的总抗氧化能力均显著降低(P0.05),且空肠氧化型谷胱甘肽(GSSG)含量显著提高(P0.05),回肠谷胱甘肽(GSH)含量显著降低(P0.05)。结果提示,早期断奶使仔猪小肠处于氧化应激状态,显著提高断奶仔猪小肠组织中xCT和ASCT2蛋白及mRNA表达量,以促进谷氨酸/谷氨酰胺的摄取,降低仔猪小肠的氧化应激水平。     

高效液相色谱法评定二肽水解的研究

用高效液相色谱法测定了仔猪小肠刷状缘膜囊(Brush Border Membrane Vesicles,BBMV)孵育体系中甘-L-亮(Gly-Leu)和L-组-L-亮(His-Leu)等两种二肽的含量.分别以水(pH值为5.55)和乙腈-三氟乙酸(三氟乙酸浓度为0.1 %)为流动相,经Phenomennex ODS分析柱(250 mm×4.60 mm,5 μm,C18)分离,检测波长为190～400 nm.结果表明,两种二肽在0～300 μg mL-1的之间,其峰面积(X)对质量浓度(Y,μg mL-1)回归得方程分别Y＝0.000 1 X+0.871 9,R2＝0.999 9和Y＝0.000 06 X+6.631 28,R2＝0.998 2;回收率依次为99.91 %和100.05 %,保留时间依次为2.07和1.01 min;最大吸收波长依次为197和199 nm.该方法快速、简便和准确,结果稳定,重现性好,可以用来评定该两种二肽在动物小肠BBMV体系中的水解状况.     

牛磺酸跨膜转运的研究进展

牛磺酸(taurine,TAU)是动物组织细胞内含量丰富的游离β-氨基酸,具有广泛的生物学效应,而牛磺酸跨膜转运是其发挥生物学效应的基础。本文综述了牛磺酸转运体(taurine transporter,TAUT)的特征、分布、影响因素等方面,阐明了牛磺酸转运体在牛磺酸跨膜转运中的重要作用。     

In vitro transport of L-alanine by equine cecal mucosa

When sheets of mucosa from the cecum of clinically normal horses were incubated in vitro with radiolabeled L-alanine, they could accumulate this amino acid against an apparent concentration gradient after 60 to 150 minutes of incubation. The active transport system for L-alanine was on the serosal surface of the mucosal sheet only. L-Alanine accumulation at 60 minutes was partly inhibited by 20 mM glycine (P less than 0.01), 0.5 mM ouabain (P less than 0.05), and Na deprivation (P less than 0.02). Anoxia for 60 minutes increased L-alanine accumulation, but had adverse effects on cell structure and intracellular cation distributions. Transmucosal fluxes induced a small, but significant (P less than 0.05), net secretion of L-alanine, and the mean (+/- SEM) transmucosal potential difference was 7.3 +/- 0.7 mV over the period of flux measurement. It was concluded that L-alanine was accumulated by the serosal surface of the cecal mucosa, possibly to provide substrate for tissue metabolism. There was no evidence that the cecal mucosa could actively transport this amino acid from the luminal bathing medium.     

Na(+)-dependent transport of D-xylose by bovine intestinal brush border membrane vesicles (BBMV) is inhibited by various pentoses and hexoses

To detect whether pentoses and hexoses occurring in rumen bacteria or in hemicellulose ingested with feed and partly released in the small intestine have an affinity for the Na(+)-dependent glucose transporter of the bovine intestinal brush border membrane (BBM), we investigated whether these monosaccharides inhibit Na(+)-dependent transport of 14C-labelled D-xylose across the BBM using brush border membrane vesicles (BBMV) isolated from the mid-jejunum of cows. We used D-xylose as the transport substrate, because it has a low affinity for the Na(+)-dependent glucose transporter and thus its uptake into BBMV is more efficiently competitively inhibited by other sugars than that of D-glucose. D-Ribose, D-mannose and L-rhamnose occurring in rumen bacteria significantly inhibited Na(+)-dependent uptake of D-xylose into BBMV, but their inhibitory effect was less than that of D-glucose, D-xylose and phlorizin. This also applied to L-arabinose (and D-arabinose), which is, like D-xylose and D-galactose, a constituent of hemicellulose, and to 2-deoxy-D-glucose. Of all monosaccharides tested, only D-fructose did not affect Na(+)-dependent D-xylose transport. It is concluded that some pentoses and hexoses occurring in rumen bacteria (D-ribose, D-mannose and L-rhamnose) or hemicellulose (L-arabinose and D-xylose) have a low affinity for the Na(+)-dependent glucose transporter of the bovine BBM and may therefore be absorbed from the jejunum when released in the small intestine.     

L-canavanine inhibits L-arginine uptake by broiler chicken intestinal brush border membrane vesicles

1. Intestinal brush border membrane vesicles (BBMV) were prepared from 3-week-old broiler chickens. 2. Electron microscopy of the BBMV fraction showed single membrane vesicles of different sizes with no electron dense material inside. No other organelles were observed. The sucrase and maltase activities were enriched by factors of 16 and 18, respectively, in the BBMV fraction in comparison with the homogenate. On the other hand, the Na+/K+-ATPase sensitivity to ouabain was increased by a factor of 0.8. 3. The BBMV showed a maximum L-[14C]-arginine uptake (944.9 +/- 22.9 pmoles/mg protein) at 45 s and thereafter it declined slowly. In the presence of 0.5 mM L-canavanine, the L-[14C]-arginine uptake by BBMV was reduced by 43.6% at 45 s. 4. It is concluded that L-canavanine inhibits L-arginine Na+-dependent transport across the enterocyte apical membrane in a highly purified intestinal BBMV from broiler chickens.     

Small intestinal morphology and disaccharidase activities in early-weaned piglets fed a diet containing spray-dried porcine plasma

The purpose of this study was to test whether dietary spray-dried porcine plasma (SDPP) in early-weaned piglets prevents small intestinal villus atrophy by trophic or protective activity. Fifty-four weaned, 18-day-old piglets were used to determine the effect of dietary SDPP on small intestinal villus length, crypt depth, enterocyt mitotic activity and brush border enzyme activities during the first week after weaning. The piglets were offered a diet containing either 8% SDPP or 8% casein. At 2 and 7 days after weaning, piglets were anaesthetized to provide samples of the small intestinal wall and killed immediately afterwards. There were no differences in daily gain and daily feed intake between the two dietary treatments. At day 2 after weaning, all piglets showed a marked reduction in villus height when compared with baseline values. In all piglets, small intestinal enterocyte mitotic activity had decreased by day 2 and was increased again on day 7. There were no significant effects of dietary SDPP on small intestinal villus length, crypt depth and enterocyt mitotic activity. This indicates that SDPP has no trophic effect on the small intestinal mucosa and that it does not protect against the damaging effect on the small intestinal villi that is associated with the process of weaning. There was no effect of SDPP on lactase-, sucrase- or maltase-specific activities that are a measure of the digestive function of the small intestine. It can be concluded that SDPP versus casein has no effect on small intestinal morphology and disaccharidase activities in early weaned piglets kept under low infection pressure.     

High intestinal transport activity for nucleosides in cattle: a synopsis

In ruminants large amounts of nucleic acids associated with the microbial cell mass leaving the fore-stomach system via the abomasum enter the small intestine. In dairy cows this amounts to 100-200 g microbial nucleic acids per day. These nucleic acids are very efficiently digested in the small intestine whereby nucleosides were found to be the main degradation products. Therefore, this review centers mainly on the mechanisms of intestinal absorption of nucleosides and their role in nucleic acid digestion. Furthermore, metabolism of nucleosides in intestinal epithelial cells and its bearing for nucleoside absorption and salvage of nucleosides for nucleic acid synthesis in various tissues is considered. According to own studies using isolated intestinal brush-border membrane (BBM) vesicles (BBMV) from dairy cows purine and pyimidine nucleosides are transported actively by two separate Na+ co-transport systems (N1 and N2) across the bovine BBM, whereby transport activity in the small intestine decreases from proximal to distal. Guanosine and inosine appeared to be transported exclusively by N1 while thymidine and cytidine appeared to be transported exclusively by N2. Uridine and adenosine had an affinity to both transporters. In comparison to findings in man and rabbit, transport capacity (Vmax) of N1 and N2 in the BBM of cows was more than 10-fold higher. Similar findings were obtained in BBMV isolated from the small intestine of veal calves with rudimentary forestomach development in regard to nucleoside transport. Therefore, the high intestinal transport activity for nucleosides seems to be a genetically fixed property in the bovine, which is not related to a functional fore-stomach system.     

Body and intestinal growth of broiler chicks on a commercial starter diet. 3. Development and characteristics of tryptophan transport

1. A study was conducted to characterise the development of amino acid transport in broiler chicks, using L-tryptophan as a model. The chicks were maintained on a broiler starter diet between hatch and 21 d of age. 2. There was a significant reduction in the rate of uptake of 0.04 mM L-tryptophan with age in both the jejunum and ileum. Uptake was enhanced in the presence of 50 mM sodium chloride to different degrees depending on age and intestinal site. At both intestinal sites, uptake capacity increased with age while there was a reduction in uptake efficiency with age. 3. At a concentration of 25 mM, both sodium chloride and potasium chloride increased uptake by ileal brush-border membrane vesicles (BBMV) of 7-d-old chicks but uptake was reduced when potassium chloride was included at a concentration of 50 mM. In the presence of valinomycin, uptake by jejunal BBMV was stimulated by 25 mM sodium chloride. In the presence of both sodium chloride and potassium chloride and in the absence of valinomycin, uptake was increased by 42.6% but this was reduced to 23.4% when the ionophore was included in the buffer. 4. The Na+-independent uptake of L-tryptophan into jejunal vesicles of 21-d-old chicks was lower in the presence of D-tryptophan than in the presence of 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH). The inclusion of BCH in the incubation medium at low concentrations significantly enhanced the uptake of 0.04 mM L-tryptophan by jejunal BBMV. 5. At similar concentration (0.04 mM) to L-tryptophan, lysine, methionine and alanine in the presence of Na+ also stimulated L-tryptophan uptake. The uptake of L-tryptophan was reduced at a higher concentration, 25 mM, of these amino acids. 6. The study revealed a decline in rate of amino acid uptake and an increase in total uptake capacity with age. Tryptophan uptake was both Na+-independent and dependent, and occurred more in the ileum than in the jejunum. The uptake of L-tryptophan depended on the concentration of other amino acids and other factors in the diet and digesta.     

Effects of superoxide dismutase on injury induced by anoxia and reoxygenation in equine small intestine in vitro.

Sheets of mucosa from the jejunum of healthy horses were mounted in incubation chambers and bathed with Krebs-Ringer bicarbonate solution. Changes in tissue function and histologic appearance were compared after the following conditions: (1) control conditions for 30 minutes with 95% O2/5% CO2 in the gas phase; (2) same conditions as control, except incubation with superoxide dismutase (300 U/ml) during the last 18 minutes; (3) anoxia for 15 minutes with 95% N2/5% CO2, followed by reoxygenation for 15 minutes; (4) same conditions as 3, except incubation with superoxide dismutase during reoxygenation; and (5) anoxia for 30 minutes. Anoxia reduced the accumulation of radiolabeled L-alanine and caused cell swelling, as indicated by an increase in tissue water and tissue Na contents. Reoxygenation improved the tissue's ability to accumulate L-alanine, but tissue swelling continued after this treatment. Tissue Na content and L-alanine accumulation were restored to control values by reoxygenation with superoxide dismutase in the bathing medium. The grade of structural damage, as indicated by separation of epithelial cells from villi, was equally severe after all, but control, conditions. Superoxide dismutase had no effect on the tissue control conditions. Results of this study suggest that superoxide radicals are involved in the pathogenesis of reperfusion injury in equine jejunal mucosa and that this may be of clinical importance in cases of small intestinal strangulation obstruction.