Reduced protein kinase C activity and endogenous protein phosphorylation in ethionine-induced fatty liver in cows

Protein kinase C (PKC) activity was evaluated and the phosphorylation of its endogenous substrates was explored in fatty liver induced by administration of ethionine (an analogue of methionine) to cows in order to assess the relevance of PKC-dependent phosphorylation in the development of fatty liver. PKC activity was decreased in both the cytosolic and the total particulate fractions from fatty livers, compared to the corresponding fractions from control liver. The mode of activation by the PKC cofactors (1-oleoyl-2-acetyl-sn-glycerol, 12-O-tetradecanoylphorbol-13-acetate, phosphatidylserine and Ca²⁺) was similar in both control and fatty livers, suggesting a quantitative but not a qualitative change in PKC in fatty liver. At least three substrate proteins (34 kDa, 26 kDa and 19 kDa) were found in the cytosolic fraction and their phosphorylation was reduced in fatty liver. These results suggest that impairment of the signal transduction pathway mediated by PKC is involved in the pathogenesis of fatty liver in cows.Abbreviations ATP adenosine triphosphate - EGTA ethylene glycol bis(-aminoethylether)-N,N,N,N-tetraacetic acid - NEFA non-esterified fatty acid - OAG 1-oleoyl-2-acetyl-sn-glycerol - PKC protein kinase C - PS phosphatidylserine - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TG triglyceride - TPA 12-O-tetradecanoylphorbol-13-acetate     

Decreased respiratory burst activity in neonatal bovine neutrophils stimulated by protein kinase C agonists.

Newborn calves have a high susceptibility to bacterial infections, which may be related to the impaired neutrophil defense functions in newborns. The oxygen-dependent production of the free radical superoxide anion (O2-) represents an important part of the leukocyte respiratory burst central to neutrophil-directed defenses against bacterial infection. Because protein kinase C (PKC) activation is considered to be an important step in the signal transduction pathway for the O2- generating system, we compared O2- production by newborn and adult bovine neutrophils stimulated with 3 different PKC agonists. When the phorbol ester phorbol 12-myristate 13-acetate (PMA) was used, PKC-dependent O2- generation from newborn neutrophils was significantly reduced (P less than 0.01) for all concentrations of PMA tested (10, 100, and 500 ng/ml). In addition, newborn neutrophils had a significantly (P less than 0.01) reduced lag time for O2- generation. Similar significantly (P less than 0.01) reduced O2- generation from newborn neutrophils was observed with an additional phorbol ester (phorbol 12,13-dibutyrate); lag times were not calculated for phorbol 12,13-dibutyrate. When O2- generation was stimulated with a synthetic diacylglycerol analogue (1,2-dioctanoyl-sn-glycerol), less O2- was generated from both adult and newborn neutrophils than was obtained with the phorbol esters, and newborn neutrophils produced significantly (P less than 0.01) less O2- only at 50 microM 1,2-dioctanoyl-sn-glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of phytohemagglutinin-stimulated bovine mononuclear cell proliferation, interleukin-2 production and protein kinase C activity by a protein kinase C inhibitor, H-7

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a potent and selective inhibitor of protein kinase C (PKC), inhibited PHA-stimulated bovine peripheral blood mononuclear cell (PBMC) proliferation, interleukin-2 (IL-2) production, and cytosolic PKC activity without affecting the cell viabilities. Presence of exogenous cytokines, such as purified human IL-2 or recombinant bovine IL-2 (rbovIL-2), reversed the H-7 inhibitory effects on PHA-stimulated PBMC proliferation. We conclude that the PKC enzyme plays an important role as a second messenger in bovine PBMC proliferation in the early stages of cell activation.     

Nutritional and management strategies for the prevention of fatty liver in dairy cattle

Fatty liver occurs in dairy cattle during periods of elevated blood non-esterified fatty acids (NEFAs). Elevated blood NEFAs are associated with hormonal changes at parturition and negative energy balance. Approaches for preventing fatty liver include inhibition of fatty acid mobilization from adipose tissues and altering hepatic metabolism to enhance fatty acid oxidation or export as a constituent of very low-density lipoproteins (VLDL). Nutritional and management strategies to implement these approaches have been examined. Increasing energy density of diet, either by increasing non-fiber carbohydrate or fat, has failed to prevent fatty liver. Two nutritional supplements, ruminally-protected choline and propylene glycol, have proven effective at preventing fatty liver. Choline probably enhances hepatic VLDL secretion. Propylene glycol most likely reduces fatty acid mobilization from adipose tissue. Shortening or eliminating the dry period is a management strategy that reduces the magnitude of negative energy balance after calving and triglyceride accumulation in the liver.     

黄芪多糖对小鼠脾淋巴细胞蛋白激酶C活性的影响

采用反相离子对高效液相色谱法测定脾淋巴细胞蛋白激酶C（PKC）活力，观察黄芪多糖（APS）体外对小鼠脾淋巴细胞蛋白激酶C活性的影响。结果表明：ASP（100μg/ml）能引起小鼠脾淋巴细胞PCK活性明显升高，提示APS的免疫增强作用与升高脾淋巴细胞蛋白激酶活性有关。     

鸡肝脏型脂肪酸结合蛋白基因启动子活性分析

PCR扩增鸡L-FABP基因5′侧翼区约2kb的DNA片段,进行克隆并测序,构建了鸡L-FABP基因报告基因系列缺失载体,瞬时转染进入人肝癌细胞系,利用双荧光素酶报告基因系统测定了荧光素酶活性。在线分析软件发现鸡L-FABP基因启动子区存在HNF-1、SREBP-1、AP-1、C/EBP、Oct-1、TATA、CCAAT、GATA-1等调控元件,没有发现CpG岛。报告基因结果表明鸡L-FABP基因启动子-2 076bp/-20bp区域具有最强的启动子活性,-522bp/-20bp区域启动子活性最弱;C/EBPα可以显著的抑制鸡L-FABP基因的表达,这些结果为深入研究鸡L-FABP的表达调控机制奠定了基础。     

An immunohistochemical study of protein kinase C in the bovine retina

The expression of protein kinase C (PKC) was studied in the bovine retina by immunohistochemical analysis. Western blot analysis showed that PKC isoforms, including alpha, betaI, delta and theta, were detected in the bovine retina. By immunohistochemistry, both PKC alpha and betaI were expressed in all retinal layers, with an intense localization of both PKC alpha and betaI detected in bipolar cells in the inner nuclear cell layer and in some glial cells in ganglion cell layers. The immunoreactivity of both PKC delta and theta was quite weak in the retinal layers, compared with that of PKC alpha and betaI. These findings suggest that both conventional and novel PKCs are differentially expressed in the bovine retina.     

牛源化脓隐秘杆菌PLO蛋白的原核截短表达及其溶血活性检测

为研究牛源的化脓隐秘杆菌溶血素(PLO)生物学功能,本研究应用PCR方法扩增牛源化脓隐秘杆菌(A.pyogenes)PLO全基因序列,通过生物信息学方法分析其与猪源A.pyogenes的PLO蛋白差异,并构建了溶血功能区重组表达质粒pQE30-PLO585,在E.coli XL1Blue中用IPTG诱导表达。结果表明,扩增到PLO蛋白基因ORF为1 605 bp,编码535个氨基酸,与猪源PLO的核苷酸序列同源性为97.4%,氨基酸同源性为97.2%,在生物学活性功能区没有发生改变。表达的PLO蛋白溶血功能区重组蛋白能够被阳性血清识别,而且具有溶解绵羊红细胞的活性,产生β溶血现象。本研究获得了牛源A.pyogenes截短重组PLO蛋白,并证明PLO具有β溶血功能与较好的抗原性。     

Plasma protein alterations in cattle suffering from acute peritonitis.

Modification of the normal plasma protein picture has been studied in plasma samples from cows suffering from spontaneously arisen (17 cases) or experimentally induced acute peritonitis (5 cases) using polyacrylamide electrophoresis. Considerable differences were found in the postalbumin region during peritonitis. One protein component increased in size, while another disappeared. Two weak components in normal samples were replaced by four discrete protein bands. These modifications were not detected in any of 10 plasma samples from cows suffering from other diseases than peritonitis or in any of 35 samples from clinically healthy animals. The modifications were visible 12-16 h after injection of the provoking agent and were remarkably alike from one case to the next.     

Adiposity, fatty acid composition, and delta-9 desaturase activity during growth in beef cattle

Oleic acid (18:1n‐9) is the most abundant fatty acid in bovine adipose tissue. Because most of the lipid in bovine muscle is contributed by intramuscular adipocytes, oleic acid also is the predominant fatty acid in beef. In many species, the concentration of oleic acid in adipose tissue is dictated by the average concentration of oleic acid in the diet, but in ruminant species such as beef cattle, oleic acid is hydrogenated largely to stearic acid by ruminal microorganisms. In these species, the concentration of oleic acid in adipose tissue is dependent upon the activity of Δ⁹ desaturase, encoded by the stearoyl coenzyme A desaturase (SCD) gene. Expression of the SCD gene is essential for bovine preadipocyte differentiation, and desaturase gene expression and catalytic activity increase dramatically as adipose tissue mass increases after weaning. Feeding a hay‐based diet to American Wagyu steers to a typical Japanese bodyweight endpoint (650 kg) markedly stimulated desaturase enzyme activity as well as the accumulation of both oleic acid and intramuscular lipid, but the increase in oleic acid and intramuscular lipid was much less in hay‐fed Angus steers. Increasing the concentration of oleic acid improves the palatability and healthiness of beef, and Korean Hanwoo and Japanese Black (and American Wagyu) seem especially well adapted to accumulate oleic acid in their adipose tissue.     

Ultrasonography as a diagnostic and prognostic approach in cattle and buffaloes with fatty infiltration of the liver

The aim of the present study was to determine whether ultrasonographic evaluation of the hepatic parenchyma could be used as a diagnostic and prognostic approach in cows and buffaloes with hepatic lipidosis. For this purpose, cows (n=16) and buffaloes (n=10) with fatty infiltration of the liver were examined by ultrasonography. Treated cows and buffaloes were monitored for hepatic changes ultrasonographically, biochemically and histologically. Clinical findings were non-specific and included anorexia, recumbency, muzzle necrosis, and icteric mucosal membranes. Laboratory data revealed neutrophilia, hyper gamma-globulinemia, elevated activities of aspartate aminotransferase, gamma-glutamyl transpeptidase, creatine kinase and lactate dehydrogenase, and high concentrations of insulin, total bilirubin, non-esterified fatty acids and beta-hydroxyl butyric acid. Laboratory results 7, and 21 days after treatment showed progressive improvement in the chemistry profile. On admission, ultrasonographic examination of the hepatic parenchyma in cows and buffaloes revealed either increased or decreased hepatic echogenicity; histologic examination revealed marked fatty infiltration of the hepatocytes. One week after treatment, the hepatic parenchyma was visualized easily, liver boundaries were clearly imaged, and histologic examination of hepatic specimen showed a moderate degree of fatty infiltration. Three weeks after treatment, the hepatic parenchyma was almost similar to normal, the hepatic and portal blood vessels could be easily imaged, and the histologic picture had greatly improved where the liver resembled the normal organ. Six cows and seven buffaloes made a full recovery while the remaining ten cows and three buffaloes were slaughtered and thoroughly examined postmortem. Ultrasonography showed a good correlation with histologic and laboratory findings.     

Porcine leptin inhibits protein breakdown and stimulates fatty acid oxidation in C2C12 myotubes

This study evaluated the potential mechanism(s) by which leptin treatment inhibits loss of muscle mass with fasting. Cultures of C2C12 myoblasts were differentiated into myotubes with 5% (vol/vol) horse serum in Dulbecco's modified Eagle's medium/F12. These myotubes were used to assess 3H-tyrosine incorporation and release following incubation with recombinant porcine leptin (0 to 500 ng/mL). Protein synthesis in myotubes, as measured by 3H-tyrosine incorporation, was not affected by leptin treatment (P > 0.05). Protein breakdown in C2C12 myotubes, as measured by 3H-tyrosine release, was inhibited by leptin treatment. A leptin concentration of 0.5 ng/mL was sufficient to inhibit 3H-tyrosine release by 3.5% (P < 0.05); 50 ng/mL produced a maximal inhibition of 10.2% (P < 0.05). Dexamethasone (1 microM) was used to maximally stimulate protein breakdown. Leptin (50 ng/mL leptin) decreased dexamethasone-induced 3H-tyrosine release by 32% (P < 0.05). The inhibition of 3H-tyrosine release in C2C12 myotubes suggests that leptin produces a protein-sparing effect in vitro by inhibiting protein breakdown. Fatty acid metabolism also was investigated because fatty acids are a major energy source for muscle during periods of reduced intake, as occurs with leptin treatment. Acute (4 h) and chronic (24 h) exposures to porcine leptin (0 to 500 ng/mL) were used to evaluate 14C-palmitate oxidation. Acute leptin treatment had no effect (P > 0.05) on palmitate metabolism. Chronic leptin exposure resulted in up to a 26% increase in palmitate oxidation (P < 0.05). The stimulation of fatty acid oxidation with chronic leptin treatment suggests that leptin spares other energy sources in muscle from oxidation during periods of a leptin-induced decrease in feed intake.     

Adipocyte fatty acid-binding protein and mitochondrial enzyme activities in muscles as relevant indicators of marbling in cattle

Marbling is an important criterion for beef quality grading in many countries. The purpose of the current study was to utilize the natural genetic variation to identify major metabolic indicators of marbling in cattle differing in genotypes. Rectus abdominis (RA, oxidative), semitendinosus (glycolytic), and longissimus thoracis (LT, oxido-glycolytic) muscles were taken from steers of different genotypes that expressed high [Angus, n = 16; and crossbred (Angus x Japanese Black), n = 10] or low (Limousin, n = 12) levels of marbling in their meat. Muscles from Angus and crossbred steers were characterized, as expected, by a greater triacylglycerol (TAG) content (P < 0.001) and also by greater protein contents of fatty acid-binding protein specific for heart and muscles (H-FABP; P < 0.001 for RA and P < 0.05 for LT muscle) or for adipocytes (A-FABP; P < 0.001 for RA and LT muscles). Moreover, oxidative enzyme activities (beta-hydroxyacyl-CoA dehydrogenase, citrate synthase, isocitrate dehydrogenase, cytochrome-c oxidase) were greater (P < 0.01 to 0.001) in the 3 muscles studied, whereas glycolytic enzyme activities (phosphofructokinase and lactate dehydrogenase) were lower (P < 0.001) in RA muscle in Angus and crossbred steers compared with Limousin steers. Significant correlations were observed between TAG content and H- and A-FABP protein contents, and oxidative (r > or = +0.55, P < 0.001) or glycolytic enzyme activities (r > or = -0.47, P < 0.001), when the 3 genotypes and muscles studied were considered as a whole. In addition, A-FABP protein content and some oxidative enzyme activities were significantly correlated with TAG content independently of the genotype and muscle effects. In conclusion, A-FABP protein content, as well as oxidative enzyme activities, may be used as indicators of the ability of steers from extreme genotypes to deposit intramuscular fat.     

Inhibition by melittin of phosphorylation by protein kinase C of annexin I from cow mammary gland

Protein kinase C (PKC) is a phosphotransferase activated by diacylglycerols, phospholipids and Ca(2+), that regulates a wide variety of biological functions by phosphorylating multiple protein substrates such as annexin I. Annexin I is a phospholipid/Ca (2+)-binding protein distributed in various tissues, including the mammary gland, and is thought to mediate the anti-inflammatory actions of glucocorticoids by inhibiting phospholipase A(2). Melittin, a phospholipase A(2) activator in bee venom, is known to inhibit PKC activity when lysine-rich histone is used as the substrate. The purpose of the present study was to examine whether phosphorylation by PKC of annexin I from cow mammary gland was inhibited by melittin. Melittin inhibited annexin I phosphorylation by PKC in a dose-dependent manner, and its IC(50) value (concentration causing 50% inhibition) was 0.8 microM. The phosphorylation of annexin I was also inhibited by the amphiphilic polypeptides mastoparan and polymyxin B, and their inhibitory effects were comparable to that of melittin. The surface-inactive polypeptide bacitracin was less effective. The inhibition by melittin was effectively reversed by the excess addition of phosphatidylserine, but not distinctly by 1-oleoyl-2-acetyl-sn-glycerol or Ca(2+), suggesting that melittin inhibited the phosphorylation of annexin I by interacting with phosphatidylserine. The inhibition by melittin of PKC phosphorylation of annexin I seems to be pathophysiologically important, because a melittin-like phospholipase A(2)-stimulatory protein is present in bovine endothelial cells.     

Functional integrity of precision-cut liver slices from deer and cattle

Precision-cut bovine and cervine liver slices were incubated in RPMI 1640 media for up to 72 h, and cellular integrity was assessed utilizing the leakage of lactate dehydrogenase (LDH) and the formation of the formazan metabolite of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). Leakage of LDH (80%) from the bovine and cervine slices was noted only following 10 h of culture, and similarly, the generation of MTT-formazan declined. Metabolic viability was determined using 7-ethoxycoumarin as the model substrate, which was metabolized by slices from both animal species in a time-dependent manner for at least 6 h to generate 7-hydroxycoumarin, which was secreted into the media primarily as glucuronide and sulphate conjugates. With both cervine and bovine slices metabolic activity decreased markedly after a 4-h preincubation as assessed following a further 2-h incubation in the presence of 7-ethoxycoumarin. Subsequently, ethoxycoumarin metabolism by bovine slices did not decrease further until 24 h and was still detectable at 72 h. In the case of cervine slices, activity declined gradually after 8 h, with no activity being detectable at 72 h. It may be concluded that cervine and bovine slices may be maintained metabolically active for 8-10 h, which should prove sufficient for xenobiotic metabolism studies to be performed.     

Modulation by sphingosine of phosphorylation of substrate proteins by protein kinase C in nuclei from cow mammary gland

Protein kinase C (PKC) is an enzyme activated by diacylglycerols such as 1-oleoyl-2-acetyl-sn-glycerol (OAG), phospholipids (in particular phosphatidylserine; PS) and Ca2+, which regulate a wide variety of intracellular functions by phosphorylating multiple substrate proteins and enzymes. The effect of sphingosine, the backbone moiety of sphingolipids, on PKC activity and phosphorylation of endogenous proteins catalyzed by PKC was investigated in nuclei of cow mammary gland. Sphingosine inhibited nuclear PKC activity when lysine-rich histone was used as the substrate. The sphingosine inhibition of the PKC activity was reversed by the excess addition of PS, but not by OAG or Ca2+. Several nuclear proteins, including 56-kDa, 43-kDa, 38-kDa and 36-kDa proteins, were shown to be substrates for PKC. Of the substrate proteins, the 38-kDa and 36-kDa proteins were identified as annexin I, the Ca2+/phospholipid-binding protein; the 56-kDa and 43-kDa proteins have not yet been identified. Sphingosine inhibited phosphorylation of the 56-kDa protein and the 36-kDa annexin I, whereas it enhanced that of the 43-kDa protein. The 38-kDa annexin I species was unaffected by sphingosine. As with the PKC activity, inhibition by sphingosine of phosphorylation of the 56-kDa protein and 36-kDa annexin I was reversed by the excess addition of PS, but not by OAG or Ca2+. In addition, by the excess addition of PS and not by OAG or Ca2+, the sphingosine-enhanced phosphorylation of the 43-kDa protein was reversed and returned to near the level in the absence of sphingosine. It is suggested that sphingosine is involved in the regulation of PKC-dependent phosphorylation in the nucleus by modulating the association of PKC or its substrates, particularly annexin I, with membrane phospholipids in cow mammary gland.