猪肝过氧化氢酶提取条件的研究

采用水浸提法提取猪肝过氧化氢酶。选择浸提时间、温度、料水比作为单因素进行梯度实验,确定其条件范围,再通过进一步的正交实验得到猪肝过氧化氢酶提取条件的优化组合,即料水质量比1：5,浸提温度25℃,浸提时间8h。     

Hepatic endothelial lipase activity in neonatal rat liver

Hepatic endothelial lipase (HEL) activity is as high in the neonatal (1-day old) rat liver as in adults. Most of the HEL activity is located at the capillaries since 75% of the total activity is released by heparin or collagenase perfusion. The residual activity (non-releasable) is located in hepatocytes and not in hemopoietic cells, which are the major cell type in neonatal liver. Per mg of protein, the HEL activity is 50% higher in neonatal than in adult hepatocytes. We suggest that neonatal hepatocytes have an increased capacity to synthesize and secrete HEL activity, so maintaining a high activity in the whole organ. it might contribute to the hepatic uptake of cholesterol from circulating lipoproteins, in a period in which endogenous cholesterol synthesis is known to be inhibited in the liver.     

Bile salt sulfotransferase in guinea pig liver

Bile salt sulfotransferase from guinea pig liver is purified by the procedures of ammonium sulfate fractionation, Sephadex G-100 column chromatography, agarose-hexane-adenosine 3′,5′-diphosphate affinity chromatography and polyacrylamide gel electrophoresis. The purified enzyme exhibits a pH optimum of 6.8, an isoelectric point of 5.6 and a molecular weight of 7600 estimated by gel filtration technique. The apparent K_m values of the enzyme are 7.7·10^?5 M for taurolithocholate and 1.4·10^?6 M for 3′-phosphoadenosine 5′-phosphosulfate. It requires Mg²⁺ and free sulfohydryl group(s) for activity. The enzyme reacts with hydroxy groups of bile salts at both 3α and 3β positions. No activity is found in the kidney of guinea pig. The purified enzyme does not react with estrone, estradiol, testosterone, dehydroepiandrosterone, cholesterol, phenol, tyramine, and serotonin. The results indicate that bile salt sulfotransferase is distinct from other hepatic sulfotransferases.     

猪sall4b基因的克隆及表达分析

sall4基因是sall基因家族的一个成员,在胚胎发育、器官形成和干细胞多能性的维持以及重建中都起到重要作用,有sall4a和sall4b两种剪切突变体类型。目前猪的sall4基因序列尚未获得。鉴于其在多能性细胞调控中的作用,对猪的sall4基因进行了克隆测序,并对其在各组织及胚胎中的表达进行了初步研究。通过5和3 RACE克隆得到猪sall4基因cDNA全长序列(2 372 bp),序列分析证明此基因编码的蛋白结构更接近于小鼠和人Sall4B亚型,同源性可达70%～80%,而与其他物种的Sall4A相比则缺少一段含锌指结构域的片段,同源性降至30%～55%。Real-time PCR证明猪sall4b基因广泛表达于猪的各种器官,其中除卵巢组织呈高量表达之外,脾、肺、心和睾丸表达量也相对较高;在早期胚胎发育过程中除4-细胞阶段相对表达量较低,其他阶段呈高量表达。免疫荧光跟踪Sall4在猪早期胚胎中的表达情况发现Sall4在着床前胚胎中全程表达并定位于细胞核中,在囊胚阶段基因表达趋向于定位在内细胞团中。表达分析证明sall4b基因与多能性紧密相关,预示着猪sall4b基因将可能作为新的重编程因子用于诱导猪多能干细胞的体系中。     

The oligosaccharyltransferase complex from pig liver: cDNA cloning, expression and functional characterisation

Oligosaccharyltransferase (OST) is an oligomeric protein complex which catalyses the transfer en bloc of Glc₃-Man₉-GlcNAc₂ from Dol-PP to specific asparagine residues in the nascent polypeptide chain. In order to study the function of the pig enzyme subunits, we have cloned OST48, ribophorin I and ribophorin II and characterized these proteins after in vitro translation as well as after expression in COS-1 cells. The individual full-length cDNAs contained open reading frames (ORFs) encoding polypeptides with calculated molecular masses of 48.9[emsp4 ]kDa (OST48), 68.7[emsp4 ]kDa (ribophorin I) and 69.3[emsp4 ]kDa (ribophorin II), respectively. A Kyte and Doolittle hydrophobicity analysis revealed that OST48, ribophorin I and ribophorin II possess a type I membrane topology with the bulk of their polypeptide chains directed towards the ER-lumen. In contrast to OST48, ribophorin I and II contain, respectively, three or two potential N-glycosylation sites of the Asn-Xaa-Thr/Ser type; only one is found to function as the acceptor site in each protein.Transfection of COS-1 cells with vector constructs encoding either OST48, ribophorin I, or a ribophorin I variant tagged with a myc-peptide sequence, resulted in the over-expression of polypeptides whose molecular masses were similar to those calculated from the respective cDNA ORFs. None of these three polypeptides, or ribophorin II, were found to display OST activity when over-expressed alone. By contrast, a modest but reproducible 25% increase of activity was observed when OST48 together with ribophorin I, or OST48 and myc-tagged ribophorin I, were co-expressed, indicating that these two subunits are probably responsible for the catalytic activity in the hetero-oligomeric OST complex. The only modest over-expression of transferase activity suggests that either the dimeric enzyme complex is catalytically unstable, or that the OST48 and ribophorin I polypeptides are unable to fold properly when other subunit components of the hetero-oligomeric OST complex are lacking. OST48 as well as ribophorin I are expressed in COS-1 cells as ER-resident proteins. Whereas OST48 carries a double-lysine motif in the –3/–5 position of its cytosolic C-terminal domain, ribophorin I does not contain recognizable ER-retention information. Replacing the lysine residue in the –3 position by leucine resulted in plasma membrane expression of the OST48-Leu polypeptide, indicating that this sequence motif may be able to influence OST48 localisation. No cell surface staining was observed when OST48-Leu was co-expressed with ribophorin I. This suggests that localisation of OST48 in the ER is mediated by interaction with ribophorin I rather than by the double-lysine motif.     

Molecular cloning, expression and functional characterization of the cytochrome P450 2A6 gene in pig liver

Raising intact male pigs would have a significant economic impact on the pork industry because intact males have improved feed efficiency and a greater lean yield of the carcass compared with barrows. However, the presence of skatole, a major cause of boar taint, in meat from intact male pigs could be highly objectionable to consumers. It has been shown that CYP2A6 is a key enzyme in the hepatic metabolism of skatole and that the activity of CYP2A6 is negatively correlated with skatole accumulation in fat. The aim of this study was to isolate and characterize CYP2A6 from pig liver, as well as identify genetic polymorphisms in the CYP2A6 gene, and examine the association between these polymorphisms and skatole level. We identified a single base deletion in CYP2A6, resulting in a frame shift in the coding region that produces a non-functional enzyme, which was associated with high levels of skatole in fat tissue.     

Purification and properties of pig liver and muscle enolases

Enolases (2-phospho-d-glycerate hydrolase, EC 4.2.1.11) were purified from both pig liver and muscle. Graphs of InC vs.r ² from sedimentation equilibrium experiments are linear, which suggests homogeneous preparations of liver and muscle enolases. From these data the molecular weight of liver enolase is calculated to be approximately 92,000 D and that of muscle enolase to be approximately 85,000 D. SDS-PAGE experiments give a molecular weight value of 46,000 D for liver enolase and a value of 44,000 D for muscle enolase. These molecular weight values for liver and muscle enzymes are within the range for other enolases and show that both of these pig enolases are dimers. Amino acid composition data support the sedimentation equilibrium data and also give a smaller molecule weight (84,968 D) for muscle enolase compared to that of the liver enzyme (89,021 D). The two enzymes differ in their content of lysine [liver enolase (L)=94 residues, muscle enolase (M)=68 residues], histidine (L=13, M=21), serine (L=53, M=36), proline (L=52, M=34), and cysteine (L=4, M=21). Partial specific volumes of 0.737 ml/g for liver enolase and 0.735 ml/g for muscle enolase were calculated from the amino acid composition data. Pig liver and muscle enolases differ radically in their isoelectric points (pI=6.4–6.5 for liver enolase, and pI=8.8–9.0 for muscle enolase), and in their degree of inactivation by 750 mM LiCI (liver enolase is inactivated to a greater degree than the muscle enolase). Despite these physical and chemical differences, the kinetic constantsK _M values for Mg²⁺, 2-phosphoglyceric acid, and phospho(enol)pyruvate appear not to be significantly different for these two forms of enolase. The physical, chemical, and kinetic data for pig liver and muscle enolases are compared to similar data for pig kidney enolase.     

Characterization of the complete porcine MSTN gene and expression levels in pig breeds differing in muscularity

Myostatin (MSTN), a transforming growth factor beta superfamily member, is an essential factor for the growth and development of muscle mass. The protein functions as a negative regulator of muscle growth and is related to the so-called double-muscling phenotype in cattle, where a series of mutations renders the gene inactive. One particular breed of pigs, the Belgian Piétrain, also shows a heavily muscled phenotype. The similarity of muscular phenotypes between the double-muscled cattle and Piétrain pigs indicated that MSTN may be a candidate gene for muscular hypertrophy in pigs. In this study, we sequenced and analysed the complete MSTN gene from 45 pigs of five different breeds, including the heavily muscled Piétrain breed at one extreme and the Meishan and Wild boar breeds at the other extreme. In total, 7626 bp of the porcine MSTN gene were sequenced, including the 5' and 3' UTR. Fifteen polymorphic loci were found, three of which were located in the promoter region, five in intron 1 and seven in intron 2. Most mutations were found when comparing the obtained MSTN sequence with porcine MSTN sequences already published. However, one polymorphism located at position 447 of the porcine MSTN promoter had a very high allele frequency in the Piétrain pig breed and disrupted a putative myocyte enhancer factor 3 binding site. Real-time PCR using Sybr Green showed that this mutation was associated with expression levels of the MSTN gene in m. longissimus dorsi at an age of 4 weeks.     

Enantioselectivity suggests a cytosolic origin for a commercial pig liver esterase preparation

A widely utilized pig liver esterase preparation has been found to be derived essentially exclusively from the cytosolic fraction of pig livers. Esterases in cytosol and microsomes prepared from a fresh pig liver hydrolyzed the S- and R-enantiomers of racemic oxazepam 3-acetate (rac-OXA) with specific activity ratios of approximately 2.3:1 and 1:62, respectively. Product formations were analyzed by chiral stationary phase high-performance liquid chromatography. The commercial pig liver esterase preparation showed greater activity toward S-OXA than did the esterases in the cytosolic fraction prepared from fresh pig liver. The results established that (i) esterases contained in microsomes and cytosol of pig liver have opposite enantioselectivity in the hydrolysis of rac-OXA and (ii) the commercial pig liver esterase preparation has a cytosolic origin. © 1995 Wiley-Liss, Inc.     

Polymorphism of the pig acetyl-coenzyme A carboxylase α gene is associated with fatty acid composition in a Duroc commercial line

Acetyl-coenzyme A carboxylase α (ACACA) catalyses the first committed step in the biosynthesis of long-chain fatty acids (FA) by converting acetyl-CoA into malonyl-CoA. In pigs, the ACACA gene maps to a chromosome 12 QTL with important effects on FA composition. In the present study, we have sequenced the coding region of the pig ACACA gene in 15 pigs, identifying 21 polymorphic sites that were either synonymous or non-coding. Ten of these SNPs segregated in a Duroc commercial population ( n  = 350) for which lipid metabolism and meat and carcass quality trait records were available. Significant associations were found between two linked single nucleotide polymorphisms (c.4899G>A and c.5196T>C) and percentages of carcass lean, intramuscular fat, monounsaturated, saturated (myristic, palmitic and stearic) and polyunsaturated (linoleic) FAs in the longissimus thoracis et lumborum muscle, along with serum HDL-cholesterol concentration. The most important allele substitution effects were observed for the polyunsaturated/saturated FA ratio (13–21% of the phenotypic mean) as well as for the percentages of ω-6 and polyunsaturated FAs, especially linoleic acid (7–16% of the phenotypic mean). These results suggest the existence of a causal mutation, mapping to the chromosomal region containing the pig ACACA gene, with marked effects on FA composition of meat.     

Fetuin A is down-regulated in rats during heterotopic ossification after Achilles tenotomy

We investigated the role of fetuin A during heterotopic ossification (HO) in rats following Achilles tenotomy. We performed a right midpoint Achilles tenotomy on 24 rats. At 5 and 10 h after surgery, we investigated the formation of ectopic bone using X-ray and histological examination. We evaluated the mRNA level of fetuin A using real-time PCR. Presence of fetuin A in the Achilles tendon was assessed by immunohistochemical staining. We also measured the serum concentration of fetuin A using enzyme linked immunosorbent assay (ELISA). The expression of fetuin A was significantly decreased in both the liver and Achilles tendon during HO. ELISA showed a small amount of fetuin A in blood throughout the development of HO. Immunohistochemical staining showed that fetuin A was abundant in the ectopic bone. Fetuin A appears to be involved in the formation of ectopic bone induced by Achilles tenotomy, and a deficiency of fetuin A plays a role in the development of HO.     

Intracellular localization and some properties of the system in guinea pig liver responsible for the aromatization of cyclohexanecarboxylic acid to hippuric acid

Summary The conversion of cyclohexanecarboxylic acid to hippuric acid in subcellular fractions from guinea pig liver was studied using a gas chromatographic-mass spectrometric method employing selected ion monitoring. Comparison of the activities of the cyclohexanecarboxylic acid to hippuric acid converting system (CHC-system) and marker enzymes in the various subcellular fractions showed that the CHC-system is localized exclusively in the mitochondria. No contribution to the total activity of the system was made by microsomal enzymes. The activity of the CHC-system in whole liver homogenate and in isolated mitochondria was similar when the latter were supplemented with ATP, -ketoglutaric acid, glycine, ethylenediaminetetraacetate, PO₄ ^3– and Mg^2–. The formation of hippuric acid in these mitochondrial preparations was linear with respect to time over a period of at least 60 min. Studies designed to optimize the incubation conditions showed that the activity of the CHC-system was reduced by P0₄ ³ concentrations greater than approximately 70 mM. Conversely, both ATP and -ketoglutaric acid stimulated the system. It is possible that two different types of acyl-CoA synthetases, one which is ATP-specific and one which is GTP-specific, may operate in the activation of cyclohexanecarboxylic acid.     

Effect of heat stress and feeding phosphorus levels on pig electron transport chain gene expression

The purpose of this study was to evaluate the effect of temperature and different levels of available phosphorus (aP) on the expression of nine genes encoding electron transport chain proteins in the Longissimus dorsi (LD) muscle of pigs. Two trials were carried out using 48 high-lean growth pigs from two different growth phases: from 15 to 30 kg (phase 1) and from 30 to 60 kg (phase 2). Pigs from growth phase 1 were fed with three different levels of dietary aP (0.107%, 0.321% or 0.535%) and submitted either to a thermoneutral (24°C and RH at 76%) or to a heat stress (34°C and RH at 70%) environment. Pigs from growth phase 2 were fed with three different levels of dietary aP (0.116%, 0.306% or 0.496%) and submitted either to a thermoneutral (22ºC and RH at 77%) or to a heat stress (32ºC and RH at 73%) environment. Heat stress decreased (P<0.001) average daily feed intake at both growth phases. At 24°C, pigs in phase 1 fed the 0.321% aP diet had greater average daily gain and feed conversion (P<0.05) than those fed the 0.107% or 0.535% while, at 34°C pigs fed the 0.535% aP had the best performance (P<0.05). Pigs from phase 2 fed the 0.306% aP had best performance in both thermal environments. Gene expression profile was analyzed by quantitative real-time polymerase chain reaction. Irrespective of growing phase, the expression of six genes was lower (P<0.05) at high temperature than at thermoneutrality. The lower expression of these genes under high temperatures evidences the effects of heat stress by decreasing oxidative metabolism, through adaptive physiological mechanisms in order to reduce heat production. In pigs from phase 1, six genes were differentially expressed across aP levels (P<0.05) in the thermoneutral and one gene in the heat stress. In pigs from phase 2, two genes were differentially expressed across aP levels (P<0.05) in both thermal environments. These data revealed strong evidence that phosphorus and thermal environments are key factors to regulate oxidative phosphorylation with direct implications on animal performance.