Mg dependence and other properties of fructose-1, 6-diphosphatase and glucose-6-phosphatase in various organs of cattle]

The properties of fructose-1.6-disphosphatase in supernatant of homogenate of liver, kidneys, and adductor muscles of cattle were tested. EDTA was found to activate the enzyme up to concentrations of 10 mMol. The pH optimum was 7.5 in the presence of EDTA. Even lower concentrations of magnesium ions caused activation of the enzyme, but an activating effect was obtained as well from relatively high Mg concentrations. Fructose-1.6-diphosphatase could by activated also by 0.2 mMol Mn or Co ions or 1 mMol Zn ions. Inhibitive action was obtained from Cu, Cd, Pb, and Hg ions. Microsomal fractions of cattle liver and kidney caused high activity of glucose-6-phosphatase, but only low action was obtained be using microsomal fractions of mesenteric mucous membrane or brain. Mg ions, basically, failed to trigger activation, and higher concentrations even caused inhibition. The relatively high activity of both fructose-1.6-diphosphatase and glucose-6-phosphatase in kidney of cattle appeared to suggest an involvement of those enzymes in gluconeogenesis.     

受体相互作用蛋白1(RIP1)对BCG诱导小鼠巨噬细胞凋亡的调控作用

旨在通过构建受体相互作用蛋白1(RIP1)腺病毒干扰载体,研究其对BCG诱导的RAW264.7细胞凋亡相关指标的影响,以探讨其在BCG诱导RAW264.7凋亡过程中的调控作用。笔者构建RIP1腺病毒干扰载体,并转染感染BCG的小鼠RAW264.7细胞系,利用流式细胞仪检测各处理细胞凋亡率、细胞线粒体膜电位、细胞活性氧水平及细胞周期等指标,并用Western blot检测凋亡相关蛋白的表达水平。结果显示:BCG感染显著上调了RIP1的蛋白表达水平并提高了小鼠巨噬细胞RAW264.7的凋亡率,当RIP1被干扰后,BCG感染后的RAW264.7细胞凋亡率和活性氧水平显著降低,而促凋亡蛋白Bax表达量显著下调,线粒体膜电位和抑凋亡蛋白表达量上调。同时,BCG感染后细胞周期滞留于G_1期。BCG感染可有效上调RIP1表达量并诱导RAW264.7细胞凋亡。RIP1通过下调BCG感染后RAW264.7细胞的线粒体膜电位,上调活性氧含量并提高凋亡相关蛋白Bax/Bcl-2比值,使细胞周期阻滞于G_1期从而参与诱导细胞凋亡。     

STAT6介导的巨噬细胞极化对布鲁氏菌胞内存活的影响

旨在探讨STAT6介导的巨噬细胞极化对布鲁氏菌胞内生存的影响。本研究采用布鲁氏菌光滑株S2308（S2308）和粗糙型疫苗株RB51（RB51）侵染巨噬细胞。利用qRT-PCR检测M1型巨噬细胞标志因子p65、NOS2和IL-1β,M2型巨噬细胞标志因子STAT6、ARG1、IL-10的mRNA表达水平;流式细胞术检测M1型标记分子CD86和M2型标记分子CD206的表达;Western blot检测p-STAT6蛋白及抑制剂AS对蛋白的抑制作用;ELISA检测M1型细胞因子TNF-α、IL-12和M2型细胞因子IL-4、IL-10的表达量;最后对胞内菌落进行CFU计数。qRT-PCR结果显示,在感染8、12 h时可显著诱导M1型因子mRNA转录表达,72 h时低表达,而M2型因子在72 h时高表达;流式细胞术结果显示,S2308感染12 h可显著诱导CD86的表达,感染72 h可显著诱导CD206的表达,但RB51对二者无影响;Western blot结果显示,S2308菌株在感染72 h时激活STAT6信号通路,而RB51几乎不激活该通路,抑制剂AS在2 μmol·L^-1浓度时抑制效果最佳;ELISA结果显示,AS抑制剂可显著抑制IL-4、IL-10的释放,并促进TNF-α、IL-12的释放;CFU计数结果显示,S2308组的胞内菌呈先降低后显著上升趋势,加入AS抑制剂后可显著抑制布鲁氏菌胞内复制。布鲁氏菌S2308在感染后期能够通过STAT6诱导M1型巨噬细胞向M2型转化,并促进Th2型细胞因子的释放,从而有利于布鲁氏菌的胞内生存。而RB51几乎不激活该通路,不影响胞内生存。     

NRAMP1基因的研究进展

天然抗性相关巨噬蛋白(natural resistance associated macrophage protein 1,NRAMP1)与多种胞内菌如结核分枝杆菌、沙门氏杆菌和利什曼原虫等易感和抗性相关。综述了NRAMP1基因的结构特点、作用机理、表达调控及其与疾病的相关性,以期为应用于猪的抗病育种提供理论依据。     

Effect of E3 Ubiquitin Ligase Nrdp1 and SOCS-1 on Intracellular Survival and Macrophage Apoptosis Induced by Brucella

In order to investigate the effect of E3 ubiquitin ligase Nrdp1 and SOCS-1 genes on apoptosis during Brucella infection macrophages.The cell models of interference and over expression of Nrdp1 and SOCS-1 genes (pLL3.7-N1,pLL3.7-S1 and pLEX-Nrdp1,pLEX-SOCS-1) were constructed.Normal RAW264.7 cell,pLL3.7-N1,pLEX-Nrdp1,pLL3.7-S1 and pLEX-SOCS-1 group cells were infected with Brucella melitensis 16M (referred to 16M),the expression of Bax,Bcl-2,TNF-α genes were detected by qRT-PCR and apoptosis rate was detected by flow cytometry.pLL3.7-N1,pLEX-Nrdp1,pLL3.7-S1,pLEX-SOCS-1 cell model of the works best of interference and overexpression Nrdp1,SOCS-1 were successfully constructed and screened.After 16M infecting each group cells,compared with the control group,the expression of Bcl-2 and Bax mRNA in pLL3.7-N1,pLEX-Nrdp1,pLL3.7-S1 and pLEX-SOCS-1 groups were significantly different in different time periods (P < 0.05).The expression of TNF-α mRNA in pLL3.7-S1 group was significantly lower (P < 0.05),while the pLEX-SOCS-1 group was significantly higher (P < 0.05).Apoptosis rates in pLEX-Nrdp1,pLEX-SOCS-1 groups were significantly higher (P < 0.05),while pLL3.7-S1 group was lower (P < 0.05).The results showed that the Nrdp1 and SOCS-1 genes were closely related to apoptosis with 16M induction,the research laid the foundation for the study of 16M intracellular parasitism mechanisms.     

E3泛素连接酶Nrdp1和SOCS-1对布鲁氏菌诱导细胞凋亡的影响

为了探讨E3泛素连接酶Nrdp1、SOCS-1基因在布鲁氏菌侵染巨噬细胞过程中对细胞凋亡的影响,构建Nrdp1、SOCS-1基因的干扰和过表达细胞模型(简称为pLL3.7-N1、pLL3.7-S1和pLEX-Nrdp1、pLEX-SOCS-1);羊种布鲁氏菌16M(简称16M) 侵染正常细胞RAW264.7及pLL3.7-N1、pLEX-Nrdp1、pLL3.7-S1、pLEX-SOCS-1组细胞,qRT-PCR检测Bax、Bcl-2、TNF-α的mRNA表达量,并通过流式细胞仪术检测细胞凋亡率。结果显示,试验成功构建并筛选出干扰和过表达Nrdp1、SOCS-1效果最好的pLL3.7-N1、pLEX-Nrdp1、pLL3.7-S1、pLEX-SOCS-1细胞模型;16M侵染各组细胞,与对照组相比,在不同的时间段pLL3.7-N1、pLL3.7-S1、pLEX-Nrdp1、pLEX-SOCS-1组细胞的Bcl-2和Bax的mRNA表达量差异显著(P < 0.05);pLL3.7-S1组细胞的TNF-α mRNA显著降低(P < 0.05),而pLEX-SOCS-1组显著升高(P < 0.05);pLEX-Nrdp1、pLEX-SOCS-1组细胞凋亡率显著升高(P < 0.05),而pLL3.7-S1组显著降低(P < 0.05)。研究结果表明,Nrdp1、SOCS-1基因与16M诱导的细胞凋亡密切相关,为16M胞内寄生机制的研究奠定了基础。     

植物乳杆菌(Lactobacillus plantarum)8-6产细菌素发酵条件的优化

对植物乳杆菌(Lactobacillus plantarum)8-6产细菌素的发酵条件进行了优化,分别研究了培养时间、温度、接种量、培养基起始pH值、培养基碳源、氮源等因素对细菌素产生的影响,通过单因素水平试验和正交试验,确定产细菌素的最佳培养基组合和最佳发酵条件为葡萄糖3%,胰蛋白胨2%,蛋白胨1%,酵母膏1%,硫酸镁0.058%,吐温-80 0.2%,30℃培养24h,培养基起始pH值为6.5,接种量2%。乳杆菌8-6优化后效价为1825.56IU/mL,比优化前提高了373.15%。     

Pharmacokinetic behavior in sheep and cattle of 5-chloro-2-(methylthio)-6-(1-naphthyloxy)-1H-benzimidazole, a new fasciolicide agent

The physicochemical properties, p K _a, Log P and solubility of compound alpha, (5-chloro-2-(methylthio)-6-(1-naphthyloxy)-1 H -benzimidazole), a new fasciolicide agent, were characterized using conventional methods. Also, its pharmacokinetics was evaluated in sheep and cattle. In both species an oral dose of 12 mg/kg was administered. Blood samples were collected during 144 h and analyzed by using an HPLC assay. Results showed that compound alpha is a weak base with a p K _a value of 2.87 and log P of 1.44. The solubility was very low in aqueous solvents. Pharmacokinetic studies showed that in both species compound alpha could not be detected at any sampling time. The mean half-life ( t _½) values of alpha sulphoxide in sheep and cattle were 19.86 and 29.87 h, while the half-life values of alpha sulphone were 19.43 and 46.32 h respectively. C _max values of alpha sulphoxide did not differ between species while alpha sulphone values were higher in cattle. Plasma protein binding of alpha sulphoxide was between 82% and 86%. These results, combined with the previous efficacy studies, suggest that compound alpha could be a promising fasciolicide agent.     

Modulation of murine macrophages and T lymphocytes by lysozyme dimer

The effects of lysozyme dimer (2 and 20 microg/kg) administered i.p. once and four times to mice on the phagocytic and killing ability of peritoneal macrophages, interleukin-1 (IL-1) production by murine macrophages stimulated in vitro with lipopolisaccharide of E. coli and expression of thymocyte, splenocyte and mesenteric lymphonode cell CD3+, CD4+ and CD8+ markers were studied. It was found that lysozyme dimer administered once or four times at doses of 2 microg/kg and 20 microg/kg augments the phagocytic and killing activity of peritoneal macrophages. The strongest stimulating effect was noted after four injections of lysozyme dimer at a dose of 20 microg/kg. Moreover, lysozyme dimer is able to modulate the production of IL-1 by murine macrophages stimulated in vitro with LPS. Exposure to four doses of lysozyme dimer (20 microg/kg) enhances the synthesis and release of IL-1, but this drug administered once (2 microg/kg and 20 microg/kg) or four times (2 microg/kg) decreases IL-1 production by peritoneal macrophages. It was also found that administration of lysozyme dimer at a dose of 20 microg/kg, irrespective of the number of doses applied, increases the percentage of CD4+ thymocytes and splenocytes. Moreover, exposure to four doses of lysozyme dimer (2 and 20 microg/kg) increases the percentage of CD4+ and CD8+ mesenteric lymphonode cells.     

沙门菌sopB基因缺失诱导小鼠巨噬细胞死亡的研究

沙门菌是一种胞内寄生菌,能够引起人和多种动物疾病,是一种世界范围内的重要疾病。为了更好理解在沙门氏菌感染过程中效应蛋白SopB对巨噬细胞存活的影响,利用鼠伤寒沙门菌SL1344和sopB基因缺失菌株分别感染小鼠骨髓来源的巨噬细胞,对2株菌在巨噬细胞内存活情况进行统计。随后用Western blot检测DNA修复酶PARP的剪切情况,通过ELISA检测培养上清中细胞因子和趋化因子的分泌情况。结果发现sopB基因缺失之后,沙门菌在巨噬细胞内的存活显著降低;培养上清中细胞因子和趋化因子分泌增强,巨噬细胞坏死增加。说明sopB基因缺失可促进巨噬细胞坏死。     

Interleukin-1 stimulation of equine articular cells

Prostaglandin E2 (PGE2) and stromelysin are produced by equine chondrocytes and synovial cells in vitro in response to recombinant human (rh) interleukin-1 (IL-1) alpha and beta, and equine mononuclear cell supernatants (MCS) containing IL-1. However, culture conditions are important. PGE2 concentrations increase in proportion to the concentration of fetal calf serum (FCS) in the culture medium, whereas stromelysin concentrations are inversely proportional to the concentration of FCS. Equine MCS, containing a lower concentration of IL-1 than the concentration of rhIL-1 used in these experiments, stimulated production of much higher levels of PGE2 than rhIL-1. In addition, equine MCS induced the production of broadly similar levels of PGE2 by both chondrocytes and synovial cells, whereas rhIL-1 was more active on equine synovial cells than equine chondrocytes. Although equine MCS induced both stromelysin and PGE2 production by equine articular cells, on the whole rhIL-1 failed to induce stromelysin production. This supports previous observations of species restrictions in the activity of human IL-1 on equine cells. Therefore, experiments using mammalian cells and heterologous IL-1 should be interpreted with caution.     

Species restrictions demonstrated by the stimulation of equine cells with recombinant human interleukin-1

Equine thymocytes, which respond to equine monocyte supernatants, do not respond to stimulation with recombinant human interleukin-1 and β, and equine synovial fibroblasts show a limited response in the form of prostaglandin E₂ production without any evidence of neutral metalloproteinase production. Human interleukin-1β was about three to ten times as active on equine synovial cells as human interleukin-1 in terms of prostaglandin E₂ production. This preliminary evidence would suggest that there are qualitative and quantitative differences in the way recombinant human interleukin-1 stimulates human cells and the way in which it stimulates equine cells.     

The impact of staphylococcal mastitis on the level of milk IL-6, lysozyme and nitric oxide

Mammary gland secretions derived from secretory cows infected with coagulase +ve Staphylococcus spp. was examined for the expression of IL-6, production of lysozyme and NO(x). The examined cows reflected 25 cases of subclinical mastitis and 15 cases of clinically mastitic animals. The IL-6 concentration in the subclinical animals was significantly higher (30.8 ng/ml) than the clinically manifested animals (18.0 ng/ml) and the normal cows (5.2n g/ml). On the other hand the level of lysozyme although significantly higher than the normal cows (6.9 microg/ml) yet its level in the subclinical animals (11.2 microg/ml) was lower than that estimated in the clinical animals (15.6 microg/ml). Similarly, the level of NO(x) in the normal animals was found to be 5.6 microM/ml to increase to 6.2 microM/ml in the subclinical mastitic animals and to significantly increase further to 11.5 microM/ml in the clinically affected cows. These results suggest the promising use of whey IL-6, lysozyme or/and NO concentration variabilities as prognostic parameters on the degree of the commencement of mastitis in cows.     

vMDV感染雏鸡Mφ活性及IL-1体外诱生活性的变化

本研究以AA肉用雏鸡为试验动物,采用MTT法研究了马立克氏病(vMDV)感染雏鸡后巨噬细胞(Mφ)活性和白细胞介素1(IL-1)体外诱生活性的变化。结果表明:1日龄雏鸡vMDV强毒感染后,巨噬细胞活性和白细胞介素1体外诱生活性显著降低(P<0.05),提示雏鸡受vMDV攻击后,Mφ功能受损,IL-1生成减少,与马立克氏病(MD)及马立克氏病肿瘤的发生和发展有关。     

Diagnosis of equid herpesviruses -1 and -4 by polymerase chain reaction.

The polymerase chain reaction (PCR) is a sensitive technique used to detect DNA of viral pathogens. We have applied the technique to the detection of Equid herpesviruses-1 and -4 (EHV-1 and EHV-4) DNA within nasopharyngeal swab samples from horses. Ninety-eight samples from suspected field cases and in-contact horses were analysed. The assays were conducted blind and later decoded and compared with virus isolation data. Our results indicate that PCR is a sensitive and rapid technique for the diagnosis of EHV-1 and EHV-4 infection.     

Upregulation of mRNA of interleukin-1 and -6 in subchondral cystic lesions of four horses

This study investigated the potential association of interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in subchondral cystic lesions (SCL) in horses. With the technique of in situ hybridisation in paraffin sections of fibrous tissue of SCL and quantitative real-time PCR in fresh frozen fibrous tissue and undecalcified bone sections of SCL embedded in acrylic resin, upregulation of mRNA of both cytokines could be demonstrated. mRNA of IL-1beta was upregulated at the periphery of the cystic lesion adjacent to normal bone, whereas IL-6 mRNA was upregulated within the fibrous tissue found within the centre of the SCL. It was concluded that both cytokines are associated in pathological bone resorption observed in SCL and, in combination with increased production of prostaglandin E2, may be responsible for the slow healing, maintenance or further expansion of the cystic lesions.     

猪舍细颗粒物促进猪原代肺泡巨噬细胞向M1极化

旨在研究猪舍细颗粒物(PM2.5)对猪原代肺泡巨噬细胞(PAMs)精氨酸代谢、炎症因子表达和极化标志物表达的影响.利用支气管肺泡灌洗方法,体外分离PAMs,细胞纯化后,利用Diff-quik染色和F4/80标记鉴定PAMs,并测定细胞活力.将纯化培养的PAMs分为对照组和PM2.5处理组(50 μg·mL-1),继续培...     

Anticoccidial activity of 9-(2-chloro-6-fluorobenzyl)-6-methylaminopurine and 9-(2-chloro-6-fluorobenzyl)-6-dimethylaminopurine against Eimeria tenella, E. acervulina, and E. maxima in battery trials

In a series of battery experiments utilizing 9-day-old White Leghorn male chicks, 9-(2-chloro-6-fluorobenzyl)-6-methylaminopurine (VM 6387) and 9-(2-chloro-6-fluorobenzyl)-6-dimethylaminopurine (VM 6736) showed remarkable anticoccidial activity against Eimeria tenella, E. acervulina, and E. maxima. The effective dose range was estimated from the results of the efficacy test against E. tenella on the basis of improvement of body weight gain and clinical signs of infection. The tests included dietary levels of 60-100 ppm of VM 6387 and 70-110 ppm of VM 6736. Both compounds gave good protection against E. acervulina and E. maxima at the same dose range. The sporulation of oocysts obtained from cecal contents of birds fed lower levels of VM 6387 was inhibited. Studies of the effects of VM 6387 on stages of the E. tenella life cycle demonstrated that the compound possesses remarkable activity at 1-5 days post-infection corresponding to the period of schizogony, as well as prolonged activity thereafter, when the parasite was undergoing gametogenesis.