维生素E、K对动物基因表达的影响

本文在介绍维生素E和维生素K营养生理功能的基础上,综述了维生素E和维生素K对相关基因表达的调控及其主要机制。     

Effects of Mannheimia haemolytica leukotoxin on apoptosis and oncosis of bovine neutrophils

OBJECTIVE: To investigate the concentration-dependent effects of Mannheimia haemolytica (formerly Pasteurella haemolytica) leukotoxin (LKT) on apoptosis and oncosis in bovine neutrophils and to examine the role of calcium ions (Ca2+) in LKT-induced apoptosis. SAMPLE POPULATION: Neutrophils isolated from blood samples obtained from healthy calves. PROCEDURE: Neutrophil suspensions were exposed to lytic or sublytic dilutions of LKT and then examined by use of transmission electron microscopy (TEM) or gel electrophoresis. Contribution of extracellular Ca2+ to LKT-induced apoptosis was investigated by incubating neutrophils with LKT or control solutions in buffer containing 1 mM CaCl2 or in Ca2+-free buffer containing 1 mM ethylene glycol-bis (b-aminoethyl ether)-N,N-tetraacetic acid (EGTA) prior to diphenyl amine analysis. RESULTS: Examination by TEM revealed that bovine neutrophils exposed to lytic dilutions of LKT had changes consistent with oncosis, whereas neutrophils exposed to sublytic dilutions of LKT and staurosporin, an inducer of apoptosis, had changes consistent with apoptosis. Effects of sublytic dilutions of LKT on apoptosis were confirmed by gel electrophoresis. Replacement of extracellular Ca2+ with EGTA, a Ca2+ chelator, reduced apoptosis attributable to the calcium ionophore A23187, but it did not have significant effects on apoptosis induced by LKT or staurosporin. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of LKT to cause apoptosis instead of oncosis is concentration-dependent, suggesting that both processes of cell death contribute to an ineffective host-defense response, depending on the LKT concentration in pneumonic lesions. Furthermore, although Ca2+ promotes A23187-induced apoptosis, it is apparently not an essential second messenger for LKT-induced apoptosis.     

高效液相色谱法测定维生素E原料含量

用高效液相色谱法测定饲料添加剂维生素E原料中维生素E含量。采用ODSC18柱(150mm×4.6mm,5μm),以甲醇-水(98+2,V+V)为流动相,流速为1.2ml/min,检测波长为285nm,柱温为(30±2)℃,在此色谱条件下,维生素E在0.104～0.828mg/ml浓度范围内呈良好线性关系(r=0.99994),经方法评估函数的评价表明,方法不存在恒定系统误差和相关性系统误差,方法准确性较好,且方法操作简便易行,系统适用性良好,适用于饲料添加剂维生素E原料中维生素E含量测定。     

Effects of dietary vitamin and fatty acid content in E on muscle vitamin E Aohan fine-wool sheep

Background： Increasing the polyunsaturated fatty acid （PUFA） content and decreasing the saturated fatty acid （SFA） content of mutton can help to improve its nutritional value for consumers. Several laboratories have evaluated the effects of vitamin E on the fatty acid （FA） composition of muscle in sheep. However, little information is available on wool sheep, even though wool sheep breeds are an important source of mutton, especially in northern China where sheep are extensively farmed. The present study was designed to address the effects of vitamin E on muscle FA composition in male Aohan fine-wool sheep. Methods： Forty-two male Aohan fine-wool lambs （5 mo old） with similar initial body weight were randomly divided into seven groups and fed diets supplemented with 0 （control group）, 20, 100, 200, 1,000, 2,000, or 2,400 IU/sheep/d vitamin E for 12 mo. Three lambs from each group were slaughtered to measure vitamin E and FA content in the Iongissimus lumborum （LL） and gluteus medius （GM） muscles. Results： Vitamin E concentrations in the LL and GM increased significantly after 12 mo of vitamin E supplementation （P 〈 0.05）. However, this increase did not occur in a dose-dependent manner because the muscle vitamin E concentration was highest in the 200 IU/sheep/d group. Dietary vitamin E supplementation also caused a significant reduction in SFA content and an increase in monounsaturated FA （MUFA） content in the LL and GM （P 〈 0.05）. All six doses of vitamin E significantly increased cis9 tronsl -conjugated linoleic acid （cgtl -CLA） content in the LL compared with the control group （P 〈 0.05）. Conclusions： Dietary supplementation with vitamin E increased muscle vitamin E content and improved the nutritional value of mutton by decreasing SFA content and increasing MUFA and c9tl 1-CLA contents in Aohan fine-wool sheep. These effects were greatest in sheep fed a diet containing 200 IU/sheep/d vitamin E.     

Influence of conditioned media from bovine cotyledon tissue cultures on function of bovine neutrophils.

Bovine fetal placental (cotyledon) tissue obtained from pregnant cows on days 255, 265, and 275 of gestation, as well as immediately after parturition (n = 5) was incubated in media for 48 hours, and the incubation media were collected. Neutrophils from 4 ovariectomized nonpregnant cows were incubated for 2 hours with conditioned media from placental tissue cultures or medium (control). Immediately after incubation, the neutrophils were subjected to the following leukocyte function assays: chemotaxis against zymosan-activated serum, chemotaxis against undiluted conditioned media (only neutrophils that were incubated in medium only), random migration, ingestion of 125I-iododeoxyuridine Staphylococcus aureus (125I-IdUR-S aureus), iodination of proteins, cytochrome C reduction, and antibody-independent and -dependent cell-mediated cytotoxicity. Conditioned media from cultured cotyledon tissue was chemoattractant for bovine neutrophils, and increased chemotactic response of neutrophils against zymosan-activated serum by 13%. The following neutrophil functions were decreased: random migration by 25%, iodination of proteins by 44%, cytochrome C reduction by 13%, and antibody-dependent cell-mediated cytotoxicity by 5%. Ingestion of 125I-IdUR-S aureus and antibody-independent cell-mediated cytotoxicity were not influenced by coincubation of neutrophils and conditioned media. Time of gestation did not alter the effects of conditioned media on neutrophil function. It was concluded that chemotactic properties of cotyledon tissue extracts, as has been reported earlier, may be attributable to substances released by fetal placental tissue. Those substances might also locally or systemically influence the oxygen-dependent antimicrobial system of neutrophils, thereby causing an increased susceptibility to bacterial infections in the peripartum period.     

Proteomic survey of bovine neutrophils

Mastitis is a major economic concern for the dairy industry. Conditions such as parturition cause a transient immunosuppression that leads to increased incidence of mastitis. One facet of periparturient immunosuppression is a functional impairment of the blood and milk neutrophils in dairy cows. To better understand the biology of the bovine neutrophil we report the first proteomic analysis of the bovine neutrophil. We have identified over 250 proteins using one-dimensional electrophoresis followed by reverse-phase chromatography in line with electrospray tandem mass spectrometry. A large number of metabolic proteins were identified, including most of the enzymes required for generation of NADPH and ATP. In addition, many proteins were identified that participate in cell mobility and phagocytosis. All the bovine members of the cathelicidin family were identified, as well as other proteins with immunological functions. Proteins important for cell signaling, vesicular transport, control of apoptosis and other functions were identified giving an overview of the bovine neutrophil proteome.     

Effect of vitamin E supplementation on various functional properties of macrophages and neutrophils obtained from weaned piglets

Sixteen piglets were used to determine the effect of vitamin E supplementation on several functional properties of macrophages and neutrophils obtained from weaned piglets. Piglets, immediately following weaning, were assigned to one of three experimental groups: control (no vitamin E supplementation), low level of vitamin E supplementation (100 mg DL-alpha-tocopheryl acetate/kg diet) and high level of vitamin E supplementation (300 mg DL-alpha-tocopheryl acetate/kg diet). Supplementation of vitamin E lasted for a period of 36 days, following a 3-day adaptation period after weaning. Blood samples were collected on days 0, 12, 24 and 36 of the experimental period, monocytes/macrophages and neutrophils were isolated and the following parameters were determined in macrophages and neutrophils activated by phorbol myristate acetate: total cell-associated and membrane-bound urokinase plasminogen activator (u-PA) activity and superoxide anion production. Results showed that macrophages and neutrophils isolated from piglets that received supplemental vitamin E had higher (P < 0.05) total and membrane-bound u-PA activities as well as higher (P < 0.05) superoxide anion production compared with the values of the corresponding cells obtained from control piglets on day 12 of the experimental period. Both levels of vitamin E supplementation (low and high) were equally effective. In contrast, vitamin E supplementation had no effect (P > 0.05) on total and membrane-bound u-PA activities and superoxide anion production by porcine macrophages and neutrophils on days 24 and 36 of the experimental period. In conclusion, the low level of vitamin E supplementation is recommended for piglets for the first 2 weeks after weaning.     

Activation-induced mobilization of secretory vesicles in bovine neutrophils.

OBJECTIVE: To characterize mobilization of secretory granules in bovine neutrophils. SAMPLE POPULATION: Neutrophils obtained from four 6- to 18-month-old Holstein cattle. PROCEDURE: Mobilization of secretory granules in bovine neutrophils was determined by measuring changes in cell-surface alkaline phosphatase activity on cells treated with various inflammatory mediators. Subcellular distribution of the alkaline phosphatase activity was determined by analysis of bovine neutrophil homogenates fractionated on density gradients. RESULTS: Alkaline phosphatase-containing secretory granules of bovine neutrophils were readily mobilized by a number of inflammatory agents, including platelet-activating factor, interleukin-8, tumor necrosis factor-alpha, lipopolysaccharide, leukotriene B4, and zymosan-activated plasma. In contrast, N-formyl-methionyl-leucyl-phenylalanine did not have a significant effect. Phorbol myristate acetate induced a biphasic response with up-regulation of cell-surface alkaline phosphatase at low doses and a return to baseline or even a reduction in cell-surface alkaline phosphatase at higher doses (> or = 10 ng/ml). Subcellular fractionation of bovine neutrophil homogenates revealed that alkaline phosphatase activity resided in light-density membrane vesicles (ie, location of secretory granules), which were distinct from specific, azurophil, and large granules. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine neutrophils respond to various inflammatory mediators by mobilizing alkaline phosphatase-containing secretory granules. This suggests that the process is an important early step in the host-defense response of bovine neutrophils.     

维生素E对冷应激雏鸡血清NO含量及GSH-Px活性的影响

用含不同水平维生素E的日粮饲喂慢性冷应激(12℃±1℃)条件下的雏鸡。通过检测雏鸡血清NO含量、谷胱甘肽过氧化物酶(GSH-Px)活性等指标,探索维生素E对其抗氧化功能的影响。结果表明,应用维生素E后冷应激雏鸡血清NO含量极明显下降(P<0.01)、GSH-Px活性极显著升高(P<0.01)。表明维生素E可使冷应激引起的雏鸡血清NO含量、GSH-Px活性明显发生变化,具有缓解应激促进抗氧化功能的作用。     

Expression and role of adhesion molecule CD18 on bovine neutrophils.

Expression of CD18 on bovine neutrophils in response to stimulation by zymosan activated serum (ZAS) and phorbol myristate acetate (PMA) and the effects of monoclonal antibodies (MAB) recognizing CD18 or bovine neutrophil surface antigens (S2G8 and S5F8G10) on adherence, chemotactic responses and phagocytosis of bovine neutrophils were evaluated. CD18 expression of neutrophils was increased after ZAS and PMA treatment by 12.2 and 54.2% respectively, and were significantly (p < 0.05, p < 0.01) different from those of untreated neutrophils. CD18 expression by neutrophils from a Holstein-Friesian heifer affected with leukocyte adhesion deficiency was within negative controls when stimulated by ZAS and PMA. Adherence, chemotactic responses, and phagocytosis were significantly decreased (p < 0.01) in neutrophils continuously treated with anti-CD18 MAB (MHM 23). Adherence was also significantly decreased in anti-CD18 pretreated neutrophils. Significant (p < 0.01) differences of chemotactic responses and phagocytosis of neutrophils were found between neutrophils pretreated and continuously treated with anti-CD18 MAB (MHM 23). Monoclonal antibodies to other surface antigens did not significantly alter neutrophil adherence, chemotaxis or phagocytosis. This study demonstrated that CD18 expression on bovine neutrophils is increased significantly by stimulation with ZAS and PMA and that the adhesion molecule CD18 plays an important role in adhesion-related functions.     

Characterization of arginase expression by equine neutrophils

Neutrophils are the predominant cells recruited in the airways of horses suffering from heaves. These cells have been shown to express arginase in some species. The metabolism of l-arginine is thought to be involved in chronic inflammation, and airway obstruction and remodeling. The aim of this study was to assess the expression, regulation, activity, and functional role of arginase isoforms in equine neutrophils. Arginase I, arginase II, ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) expression were assessed in resting and stimulated (IL-4, LPS/fMLP, PMA; 5 and 18 h) blood neutrophils using quantitative PCR. Arginase expression was also studied by Western blot and enzyme activity assay. The effect of nor-NOHA (1 mM), a specific arginase inhibitor, was assessed on arginase activity in vitro and ex vivo on neutrophil's inflammatory gene expression and viability. Results showed that equine neutrophils constitutively express arginase isoform 2, ODC and OAT. Neutrophil ex vivo stimulation did not induce arginase I or influence arginase II mRNA expression. Ex vivo inhibition of arginase activity by nor-NOHA had no effect on neutrophils inflammatory gene expression induced by LPS/fMLP (5 h) but significantly reversed the cell loss observed after this stimulation.     

Effects of Pasteurella haemolytica A1 leukotoxin on bovine neutrophils: degranulation and generation of oxygen-derived free radicals.

To further define the role of Pasteurella haemolytica A1 leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis, its in vitro effects on bovine neutrophils were investigated. Leukotoxin-containing culture supernatant, from P. haemolytica, stimulated a neutrophil respiratory burst as measured by the generation of oxygen-derived free radicals O2- and H2O2. This effect was immediate because preincubation of neutrophils with the culture supernatant for 5 min or longer substantially suppressed this respiratory burst. This suppression was due to cytolysis of the neutrophils. Prolonged incubation of neutrophils with the same culture supernatant caused further cytolysis and degranulation. Heat-inactivated P. haemolytica culture supernatant that had lost its cytotoxic properties failed to stimulate respiratory burst by neutrophils. Furthermore, the respiratory burst, cytolysis and degranulation were abrogated only by leukotoxin-neutralizing monoclonal and polyclonal antibodies, but not by antibodies against the lipopolysaccharide. These studies show that the leukotoxin component in the culture supernatant was responsible for the generation of oxygen-derived free radicals and proteolytic enzymes from neutrophils which may participate in direct lung injury.     

Effects of vitamins A and E on superoxide production and intracellular signaling of neutrophils in Holstein calves.

The effects of vitamin A and vitamin E treatment on superoxide (O2-) production of neutrophils and their intracellular signaling, including intracellular Ca2+, protein kinase C (PKC), and tyrosine kinase (TK), were examined in Holstein calves. After treatment with vitamin A, heat-aggregated IgG (H-agg.IgG)-induced O2- production in neutrophils ranged from 3.7+/-0.4 to 4.3+/-0.9 nmol (mean +/- SD), and it was significantly (P<0.05) higher than that in the control calves. Opsonized zymosan (OPZ)-induced O2- production was similar with that of control calves. Intracellular signaling of neutrophils in vitamin A-treated calves was enhanced compared with that of control calves when stimulated with H-agg. IgG, but not with OPZ. After treatment with vitamin E, H-agg.IgG- and OPZ-induced O2- production of neutrophils ranged from 4.2+/-0.8 nmol to 5.4+/-0.5 nmol and from 7.8+/-0.6 nmol to 8.1+/-0.4 nmol (mean +/- SD), respectively, and they were significantly (P<0.05) higher than those in control calves. Intracellular signaling was also enhanced compared with that of control calves. These results suggested that the effects of vitamin A and E treatment on O2- production were correlated to changes in intracellular signaling of neutrophils in Holstein calves.     

Secretory virulence factors produced by Staphylococcus aureus isolates obtained from mastitic bovine milk--effect on bovine polymorphonuclear neutrophils

The aim of the research was to test whether exogenic virulence factors secreted by Staphylococcus aureus isolates are involved in mechanisms that allow the bacteria to modulate and evade phagocytosis by bovine polymorphonuclear neutrophils. The research was based on the comparison of the effects of supernatants, prepared from cultures of 30 S. aureus isolates, on the functional properties of bovine neutrophils in vitro. S. aureus isolates were collected from milk samples from cows with clinical mastitis. Supernatants, which were used to treat leukocytes, were prepared from 18 h S. aureus cultures. Exogenic virulence factors secreted by S. aureus isolates significantly influenced the phagocytosis parameters evaluated. Depending on their leukotoxic or superantigenic properties, supernatants could affect the ingestion process, and also showed an influence on the digestion efficiency and phagocytosis carried out by bovine polymorphonuclear neutrophils in vitro.     

加减三黄汤对耐药大肠杆菌acrA mRNA表达水平的影响

为了解加减三黄汤对大肠杆菌耐药性的影响,采用实时荧光定量方法检测了外输泵acrA mRNA表达水平的变化。将100只昆明系小鼠分为阳性组(感染耐药性大肠杆菌2 751株和2 952株),阴性组,攻毒治疗组(2 751株+加减三黄汤,2 952株+加减三黄汤),8d后将鼠随机剖杀,取肝组织抹片培养大肠杆菌,做药敏试验,同时提取质粒,检测大肠杆菌外输泵基因acrA的mRNA表达量。结果显示,经加减三黄汤在体治疗后的大肠杆菌耐药性下降了,每μL中mRNA拷贝数为2 751株最高达1016,2 952株1015;用药后各菌株均有下降;相差极显著(P≤0.01),耐药性越大的拷贝数越多。表明加减三黄汤对耐药大肠杆菌外输泵基因acrA的mRNA表达水平影响与耐药性有一定的相关性。     

Effects of zinc-L-carnosine and vitamin E on aspirin-induced gastroduodenal injury in dogs

Background: Nonsteroidal anti‐inflammatory drugs frequently cause gastrointestinal (GI) injury. Zinc‐l ‐carnosine has antioxidant, anti‐inflammatory, mucosal protective, and healing properties in rodent models and in some human studies of GI injury. Hypothesis: The combination of zinc‐l ‐carnosine and vitamin E attenuates aspirin‐induced gastroduodenal mucosal injury. Animals: Eighteen healthy random‐source Foxhound dogs. Methods: In this randomized, double‐blinded, placebo‐controlled study dogs were treated with placebo (n = 6; 0X group), 30 mg/30 IU (n = 6; 1X group), or 60 mg/60 IU (n = 6; 2X group) zinc‐l ‐carnosine/vitamin E orally every 12 hours for 35 days. Between Day 7 and 35, GI mucosal lesions were induced with aspirin (25 mg/kg PO q8h). Mucosal injury lesions (hemorrhage, erosion, and ulcer) were assessed by gastroduodenoscopy on Days 14, 21, and 35 with a 12‐point scoring scale. Results: At baseline (Day ?1) gastroscopy scores were not significantly different between groups (mean ± SD: 0X, 4.4 ± 0.8; group 1X, 4.4 ± 0.6; group 2X, 4.2 ± 0.3; P= .55). Gastroscopy scores increased significantly in all groups between Day ?1 and Days 14, 21, and 35 (P < .0001). On Day 35, gastroscopy scores were 29.2 ± 5.2 (0X), 27.3 ± 3.7 (1X), and 28.6 ± 3.3 (2X). Mean gastroscopy scores were not significantly different among treatment groups on any of the days (P= .61). Conclusions and Clinical Importance: Administration of the combination of zinc‐l ‐carnosine and vitamin E at 1X or 2X dosing did not attenuate aspirin‐induced gastroduodenal mucosal injury.     

Effects of glycolytic and cytoskeletal inhibitors on phagocytic and nitroblue tetrazolium reductive activities of bovine neutrophils

Phagocytic and oxidative metabolic activities of bovine blood neutrophils were determined in the presence of glycolytic (NaF) and cytoskeletal (colchicine, cytochalasin B, and prostaglandin E1) inhibitors. Phagocytosis and post-phagocytic oxidative metabolic activity, measured by nitroblue tetrazolium reduction, were determined using zymosan, Escherichia coli, Staphylococcus aureus, or Streptococcus agalactiae. Sodium fluoride (1.25 microM to 1.25 mM concentrations) did not significantly (P greater than 0.05) inhibit phagocytosis of S aureus and Str agalactiae, whereas phagocytosis of zymosan and E coli was significantly (P less than 0.05) inhibited only at 1.25 mM concentration. Colchicine at 1.25 nM to 1.25 microM concentrations significantly inhibited phagocytosis of zymosan and E coli, but not of S aureus and Str agalactiae. Cytochalasin B at 1.25 nM to 1.25 microM concentrations significantly inhibited phagocytosis of zymosan and all 3 bacteria, whereas prostaglandin E1 was noninhibitory at similar concentrations. Nitroblue tetrazolium reduction, in general, was not significantly affected by NaF and cytoskeletal inhibitors.