Therapy of domestic animals affected by alkylphosphates]

Sheep were studied for the possibility of treatment after parenteral (intramuscular) intoxication with EDMM (methylthiophosphorous acid O-ethyl-S-2-dimethylamino-ethylester) and with EDIM (methylthiophosphorous acid O-ethyl-S-2-diisopropyl-aminoethylester). In both cases of intoxication, the therapy was based on a system of an anticholinergic and cholinesterase reactivator administered singly at a time of the maximum development of the clinical signs of poisoning and maximum inhibition of both erythrocytic (AChE, E.C.3.1.1.7.) and plasma (BChE, E.C.3.1.1.8.) cholinesterase. The optimum therapeutic system requires the administration of 20.0 mg atropine s. c. pro toto and 10.0 mg trimedoxim per kg 1. w. i. v. In both cases of poisoning with doses = LD50 in i. m. administration, the mentioned system was actually positive. In a single administration irrespective of the doses of the used drugs, the system does not guarantee survival after ingestion of anticholinesterasic doses above LD50.     

桑椹利口酒挥发性成分的气相色谱-质谱分析

桑椹利口酒中的挥发性成分是构成桑椹酒的主体气味因子。采用液-液萃取法提取桑椹利口酒中的挥发性成分,经气相色谱-质谱联机分析(GC-MS),共检测出39个特征峰,鉴定出35种挥发性化合物,占总峰面积的99.3%。其中,质量分数排在前7位的挥发性化合物分别是邻苯二甲酸二丁酯(19.81%)、二十五烷(12.66%)、二十一烷(12.35%)、邻苯二甲酸二异丁酯(10.77%)、4-(1,1-二甲基乙基)-2-甲基苯硫酚(7.26%)、十八炔酸甲酯(5.01%)和十五碳酸(4.65%)。桑椹利口酒挥发性成分中含量较高的酯类化合物形成了酒香的主体气味:呈优雅浓郁果香,且甜中带有淡淡酸果味。     

猪乳风味物质的SDE-GC-MS和SPME-GC-MS分析鉴定

研究采用同时蒸馏-气相色谱-质谱(SDE-GC-MS)和固相微萃取-气相色谱-质谱(SPME-GC-MS)法对猪乳风味物质进行分析鉴定。结果表明:同时蒸馏提取鉴定出分子量较小的挥发性物质21种,其中顺-1-甲基-4-(1-甲基乙基)-环己烷、萜烯醇和反-1-甲基-4-(1-甲基乙基)-环己烷为3种主要成分;而固相微萃取提取鉴定出分子量相对较大的挥发性物质22种,其中芥酸、环己基甲酸十二醇酯和十六烷基环己基甲酸酯为主要的3种;同时蒸馏提取的风味物质出峰时间均早于固相微萃取,综合两者分析显示,猪乳中主要风味成分是酯、脂肪酸、烷烃、醛、醇、酮等化合物。研究结果对模拟猪乳乳香风味,开发仔猪用饲料香味剂具有一定的参考辅助作用。     

果香味剂对奶牛乳腺上皮细胞培养液中乳成分含量及风味影响的研究

试验旨在研究果香味剂对奶牛乳腺上皮细胞培养液中乳成分含量及培养液风味的影响。试验选用自制的奶牛乳腺上皮细胞,试验设5个处理组,果香味剂的添加剂量分别为0、5、10、15和20μg/ml。结果表明:①当培养基中果香味剂添加量为15μg/ml时,乳腺上皮细胞培养液中总蛋白水平、甘油三酯及乳糖含量均达到最高,其中对脂肪含量影响显著(P<0.05),而对蛋白和乳糖含量影响不显著(P>0.05),之后呈下降趋势,说明果香味剂可能会诱导奶牛乳腺上皮细胞乳蛋白、乳脂和乳糖分泌水平的升高;②运用PT/GC-MS分析乳腺培养液中的风味成分,与对照组相比,各试验组中除具有原有乳腺培养液的风味外,还有乙酸异丁酯、乙酸异戊酯、丙酸异戊酯、丙酸戊酯、丁酸异丁酯、丁酸戊酯、丁酸乙酯、邻苯二甲酸二异丁酯等物质成分,而这些物质成分正好是果香味剂中进入到乳腺培养液中的风味物质,且当添加剂量为15μg/ml时,各物质成分含量为最高(P<0.05),之后呈下降趋势。果香味剂能显著影响奶牛乳腺上皮培养液中乳成分含量和风味。     

果香味剂对奶牛乳腺上皮细胞培养液中乳成分含量及风味影响的研究

试验旨在研究果香味剂对奶牛乳腺上皮细胞培养液中乳成分含量及培养液风味的影响。试验选用自制的奶牛乳腺上皮细胞,设5个处理组,果香味剂的添加剂量分别为0、5、10、15和20μg/mL。结果表明：①当培养基中果香味剂添加量为15μg/mL时,乳腺上皮细胞培养液中总蛋白水平、甘油三酯及乳糖含量均达到最高,其中对脂肪含量影响显著（P〈0.05）,而对蛋白和乳糖含量影响不显著（P〉0.05）,之后呈下降趋势,说明果香味剂可能会诱导奶牛乳腺上皮细胞乳蛋白、乳脂和乳糖分泌水平的升高;②运用PT/GC-MS分析乳腺培养液中的风味成分,与对照组相比,各试验组中除具有原有乳腺培养液的风味外,还有乙酸异丁酯、乙酸异戊酯、丙酸异戊酯、丙酸戊酯、丁酸异丁酯、丁酸戊酯、丁酸乙酯、邻苯二甲酸二异丁酯等物质成分,而这些物质成分正好是果香味剂中进入到乳腺培养液中的风味物质,且当添加剂量为15μg/mL时,各物质成分含量为最高（P〈0.05）,之后呈下降趋势。果香味剂能显著影响奶牛乳腺上皮培养液中乳成分含量和风味。     

家蚕体内叶酸的存在形态和转换代谢

采用高效液相色谱 (high performanceliquidchromatography ,HPLC)技术 ,探讨了家蚕体内叶酸化合物的存在形态和转换代谢途径。用添加蝶酰谷氨酸 (pteroylglutamicacid ,PteGlu)的人工合成饲料饲育 5龄幼虫 ,其体内叶酸的主要存在型 ,血淋巴为PteGlu ;血淋巴以外的组织为四氢叶酸 (H4PteGlu)。脂肪体是H4PteGlu的主要储存场所。叶酸转换代谢酶的检索分析结果也证实家蚕缺乏哺乳动物体内的 5 ,10 亚甲四氢叶酸 ( 5 ,10 CH2 H4PteGlu)→ 5 甲基四氢叶酸 ( 5 CH3 H4PteGlu)→H4PteGlu转换途径。家蚕体内叶酸代谢方式简单 ,推测主要和核酸合成系、丝氨酸 /甘氨酸转换系相联系     

Direct measurements of methane emissions from grazing and feedlot cattle.

Methane (CH4) emissions from animals represent a significant contribution to anthropogenically produced radiatively active trace gases. Global and national CH4 budgets currently use predictive models based on emission data from laboratory experiments to estimate the magnitude of the animal source. This paper presents a method for measuring CH4 from animals under undisturbed field conditions and examines the performance of common models used to simulate field conditions. A micrometeorological mass difference technique was developed to measure CH4 production by cattle in pasture and feedlot conditions. Measurements were made continuously under field conditions, semiautomatically for several days, and the technique was virtually nonintrusive. The method permits a relatively large number of cattle to be sampled. Limitations include light winds (less than approximately 2 m/s), rapid wind direction changes, and high-precision CH4 gas concentration measurement. Methane production showed a marked periodicity, with greater emissions during periods of rumination as opposed to grazing. When the cattle were grazed on pasture, they produced .23 kg CH4 x animal(-1) x d(-1), which corresponded to the conversion of 7.7 to 8.4% of gross energy into CH4. When the same cattle were fed a highly digestible, high-grain diet, they produced .07 kg CH4 x animal(-1) x d(-1), corresponding to a conversion of only 1.9 to 2.2% of the feed energy to CH4. These measurements clearly document higher CH4 production (about four times) for cattle receiving low-quality, high-fiber diets than for cattle fed high-grain diets. The mass difference method provides a useful tool for "undisturbed" measurements on the influence of feedstuffs and nutritional management practices on CH4 production from animals and for developing improved management practice for enhanced environmental quality.     

Liver metabolomic analysis in broiler chicks: Profiling the metabolites after oral administration of l-citrulline

Hypothermia is directly linked to metabolism; however, it is still unknown how the overall metabolism is altered by oral administration of hypothermic agent, l -citrulline (l -Cit). The present study aimed to determine the characteristics of liver metabolites of chicks orally administered l -Cit to provide a greater understanding of its metabolism. Capillary electrophoresis–time-of-flight mass spectrometry (CE-TOFMS) and liquid chromatography–time-of-flight mass spectrometry (LC-TOFMS) were conducted on liver samples after oral administration of l -Cit. A total of 361 liver metabolites were identified. Although a small number of samples were used for each group, a principal component analysis and heatmap patterns confirmed that the composition of metabolites could be segregated from each other. Of the 361 compounds detected in the liver, 41 compounds, including amino acids related to the Cit-arginine (Arg) cycle, argininosuccinic acid, Arg, ornithine, and Cit, as well as gamma aminobutyric acid, glycine, histidine, and nicotinamide adenine dinucleotide were abundant in l -Cit-treated livers. In contrast, 24 compounds containing fatty acids, amino acids, and cyclic adenosine monophosphate were lower in the l -Cit group. These data imply that the active Cit-Arg cycle, TCA cycle metabolism, and a low activity in fatty acid metabolism occur in l -Cit-treated broiler chicks.     

倍他米松ELISA检测方法的建立

为建立倍他米松(BET)ELISA检测方法,先使BET与琥珀酸酐反应,再采用活化酯法与牛血清白蛋白(BSA)偶联制备免疫原;其次,利用免疫动物获得多克隆抗体,经Sephrose 4B-proteinA方法对抗体进行纯化;最后,建立倍他米松ELISA检测方法。结果表明,免疫抗原偶联成功,获得了亲和力较高的多克隆抗血清,间接竞争ELISA测定其滴度为102 400、IC15为6.60×10-5 ng/mL和IC50为0.97ng/mL。该方法适用于残留在动物性食品中的BET现场大批量检测。     

Identification of lidocaine and its metabolites in post-administration equine urine by ELISA and MS/MS

Lidocaine is a local anesthetic drug that is widely used in equine medicine. It has the advantage of giving good local anesthesia and a longer duration of action than procaine. Although approved for use in horses in training by the American Association of Equine Practitioners (AAEP), lidocaine is also an Association of Racing Commissioners International (ARCI) Class 2 drug and its detection in forensic samples can result in significant penalties. Lidocaine was observed as a monoprotonated ion at m/z 235 by ESI+ MS/MS (electrospray ionization-positive ion mode) analysis. The base peak ion at m/z 86, representing the postulated methylenediethylamino fragment [CH2N(CH2CH3)2]+, was characteristic of lidocaine and 3-hydroxylidocaine in both ESI+ and EI (electron impact-positive ion mode) mass spectrometry. In addition, we identified an ion at m/z 427 as the principal parent ion of the ion at m/z 86, consistent with the presence of a protonated analog of 3-hydroxylidocaine-glucuronide. We also sought to establish post-administration ELISA-based 'detection times' for lidocaine and lidocaine-related compounds in urine following single subcutaneous injections of various doses (10, 40, 400 mg). Our findings suggest relatively long ELISA based 'detection times' for lidocaine following higher doses of this drug.     

Cloning, expression and bioassay of canine CTLA4Ig

Blockade of the B7:CD28 costimulatory pathway has been shown to inhibit humoral immunity, graft rejection, graft versus host disease and ameliorate autoimmune diseases. A soluble chimeric fusion protein, CTLA4Ig, binds to B7 with greater affinity than CD28 and blocks the binding of CD28 to B7. We describe the cloning and expression of canine CTLA4Ig, a recombinant chimeric fusion protein composed of the extracellular domain of canine CTLA-4 and the CH2-CH3 domains of canine immunoglobulin alpha constant region (IGHA) genes, linked via an immunologically inert flexible peptide. The recombinant CTLA4Ig protein of approximately 45kDa molecular weight was expressed mainly as insoluble inclusion bodies in Escherichia coli. The protein was solubilized in denaturing buffer and purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography followed by refolding. The yield was about 6mg of recombinant CTLA4Ig per liter of culture. The purified protein was biologically active in one-way mixed lymphocyte reactions, demonstrating immunosuppressive activities in a dose-dependent manner. The findings suggest that recombinant canine CTLA4Ig protein could be valuable in assessing the function of CTLA-4 in the canine immune system and may be effective in autoimmune disease therapy.     

猪流行性腹泻病毒M基因克隆与分子特征分析

为更好地了解近年来中国暴发的猪流行性腹泻（porcine epidemic diarrhea,PED）疫情,本研究采集荣昌及其周边地区疑似患有PED的猪小肠上皮组织进行RNA的提取,扩增猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）M基因序列,进行序列分析及M蛋白分子特性研究。结果显示,M基因开放阅读框（ORF）长为681 bp,编码226个氨基酸。通过DNAMAN软件及系统进化树分析发现,本试验中PEDV与GenBank中参考毒株的M基因氨基酸同源性在96.0%以上,其中与广东毒株CH/SD-M/2012株亲缘关系最为相近,同源性高达96.9%,表明M基因相对比较保守。通过M蛋白同源建模及功能预测发现,M蛋白晶体模型同SARS冠状病毒nsp14-nsp10复合体5c8s.1.A及NAD激酶Ⅰ2i1w.1.B模型序列相似性为27.69%、15.07%;其高级结构中在第110-113、118-125、132-136、140-145、147-152、154-157、160-173位氨基酸处存在7个α螺旋,在第98-100、102-107、135-137、139-145、149-154氨基酸存在5个锌指结构配体,M蛋白为PEDV病毒基因组复制酶的多聚蛋白。     

Cloning and Molecular Characterization Analysis of M Gene of Porcine Epidemic Diarrhea Virus

In order to study the porcine epidemic diarrhea (PED) epidemic situation recently,small intestine tissue from piglets suspected porcine epidemic diarrhea virus (PEDV) were collected in this study. The PEDV M gene was amplified using RT-PCR method and the molecular characterization were analysed. The results showed that the ORF of M gene was 681 bp which encoded 226 amino acids. The percent identity of M gene amino acid between PEDV in this study and reference strains in GenBank were higher than 96.0%,and that with CH/SD-M/2012 strain was highest (96.9%).The phylogenetic tree based on the Neighbor-Joining (NJ) and Minimum-Evolution (ME) showed that the PEDV in this study was closely related to CH/SD-M/2012 strain which were belonged to the same evolutionary branch. The phylogenetic tree analysis illustrated that M gene of PEDV was relatively conservative. The results of homology modeling analysis found that crystal model of M protein was similar to coronavirus nsp14-nsp10 complex 5c8s.1 and NAD kinase Ⅰ 2i1w.1.B which shared 27.69% and 15.07% sequence similarity,respectively. The protein structure prediction analysis found seven α-helix structures located in 110 to 113,118 to 125,132 to 136,140 to 145,147 to 152,154 to 157 and 160 to 173 amino acid regions,and five ligand structures of zinc finger located in 98 to 100,102 to 107,135 to 137, 139 to 145 and 149 to 154, amino acid regions. The protein structure prediction analysis indicated that the M protein might be a polyprotein of viral genome replication enzyme.     

Protective effects of thiol compounds on chromate-induced cytotoxicity in HeLa cells.

The effects of several thiol compounds on the cytotoxicity induced by chromate (potassium dichromate) were examined. HeLa cells were incubated in Eagle's minimum essential medium (MEM) with or without the chromate alone, or with both chromate and any one of L-cysteine ethyl ester (LCysEE), L-cysteine methyl ester (LCysME), N-acetyl-L-(+)-cysteine, 2,3-dimercaptosuccinic acid (DMSA), 2, 3-dimercapto-1-propanesulfonic acid (DMPS), or dithiothreitol. After a given period of incubation, the number of viable cells was counted using the trypan blue exclusion test and the chromium content of the cells was estimated by atomic absorption spectrophotometry. The results obtained were as follows. 1) Chromate-induced cytotoxicity evaluated by inhibition of cell growth at 3 days of incubation was diminished by all of the thiol compounds tested when the cells were incubated in MEM with 2.5 to 10.0 microM chromate and 25 to 100 microM thiol compounds. 2) All of the thiol compounds produced a concentration-dependent reduction of chromate when a solution of the thiol compound (12.5 to 100 microM) was mixed with a solution of chromate (10 microM) in distilled water. 3) When cells were incubated in MEM with both 10 microM chromate and 25 to 100 microM thiol compounds, the chromium content of the cells at 6 hr of incubation was decreased in a concentration-dependent manner. 4) When these thiol compounds were added to MEM 1 hr before or after chromate, no or little protective effects of these thiol compounds against chromate-induced cytotoxicity and chromium uptake by the cells were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma and Follicular Fluid Fatty Acid Profiles in Dairy Cows

Composition of follicular fluid to which the preovulatory follicle is exposed may be one of the major factors determining subsequent fertility, as fatty acids are a precursor of hormones involved in dominance, ovulation and atresia mechanisms. The objective of this paper is to observe fatty acid profiles in various lipid classes according to estrogenic activity of follicles. For each of the 18 cows, we analysed plasma and follicular fluid fatty acid profiles of phospholipids, non-esterified fatty acid (NEFA), cholesteryl esters and triglycerides fractions. Follicles were classified as active (ratio oestrogen to progesterone E2/P4 > 1) and inactive (E2/P4 < 1). For seven cows, we get both types of follicles, six had only one active follicle and five cows had only one inactive follicle. The NEFA profile for palmitic acid, stearic acid, oleic acid, linoleic acid, C20:3n6, arachidonic acid and docosapentaenoic acid (DPA; p < 0.001) is different between inactive and active follicles and plasma. Compared with active follicular fluid and plasma, follicular fluid of inactive follicles showed lower stearic acid, higher oleic acid, arachidonic acid and DPA (p < 0.05) in phospholipids. No significant differences were observed in the cholesteryl ester fraction, which is composted mainly of linoleic acid. Triglyceride concentrations were too low to get reliable results. This study suggests that follicles have a specific fatty acid metabolism depending on oestrogen activity.     

Characterization of bioactive recombinant woodchuck interleukin-2 amplified by RLM-RACE and produced in eukaryotic expression system

Woodchucks (Marmota monax) infected with woodchuck hepatitis virus (WHV) represent a highly valuable laboratory model of hepatitis B virus (HBV) infection, in which molecular, immunological and pathological events occurring in infected humans are adequately reflected. To advance studies on T cell immune responses and propagation of hepadnavirus in T lymphocytes in this animal model, we determined the complete sequence of woodchuck interleukin-2 (wIL-2) cDNA by utilizing RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) reaction. The wIL-2 sequence revealed a single open reading frame encoding for the predicted precursor protein comprised of a signal peptide and a 134 amino acid-long mature protein. The mature wIL-2 protein produced in the Escherichia coli expression system, designated as ec-rwIL-2, was found to be immunogenic but not biologically active. In contrast, precursor wIL-2 protein cloned into baculovirus transfer vector and expressed in Sf9 cells, designated as bac-rwIL-2, demonstrated functional competence. Further, bac-rwIL-2 was able to stimulate proliferation and to induce multiple daughter cell generations in woodchuck T cells, as well as facilitated the survival of standard IL-2-dependent mouse CTLL-2 cells in culture. Western blot analysis of bac-rwIL-2 using antibodies generated against ec-rwIL-2 revealed a single protein band of 15.5kDa. The availability of biologically active recombinant wIL-2 should facilitate ex vivo studies on functional competence of woodchuck T lymphocytes derived from different stages of hepadnaviral hepatitis and assist in recognizing their contribution to the pathogenesis of liver injury in the woodchuck model of hepatitis B.     

Peroral Application of Water-soluble Derivative of Propolis (WSDP) and Its Related Polyphenolic Compounds and Their Influence on Immunological and Antitumour Activity

Polyphenolic compounds are widely distributed in the plant kingdom and display a variety of biological activities, including chemoprevention and growth inhibition of tumours. Propolis contains a conglomerate of polyphenolic compounds. We investigated the effect of propolis and polyphenolic compounds, components of propolis, on the growth and metastatic potential of a transplantable mammary carcinoma (MCa) of the mouse. Metastases in the lung were generated by 2 x 10 tumour cells injected intravenously (i.v.). A water-soluble derivative of propolis (WSDP) and the polyphenolic compounds (caffeic acid (CA) and caffeic acid phenethyl ester (CAPE)) were given to mice perorally before or after tumour cell inoculation. WSDP, CA and CAPE reduced the number of metastases in the lung. This implies that the antitumour activities of the compounds used in these studies are mostly related to the immunomodulatory properties of the compounds, their cytotoxicity to tumour cells, and their ability to induce apoptosis and/or necrosis.     

新型含香豆素基哌嗪衍生物的超声辅助合成与表征

以水杨醛和丙二酸二乙酯为起始原料,经Knoevenagel缩合反应合成香豆素-3-羧酸乙酯,再经水解、酸化得到香豆素-3-羧酸,然后与新蒸的亚硫酰氯反应生成香豆素-3-甲酰氯,超声辅助KI催化,再与单取代哌嗪反应,合成了含有香豆素基和哌嗪两种生物活性基团的新化合物,并对中间体和目标产物的结构进行了表征。结果表明,设计的技术路线合理,反应条件温和,目标产物收率高,成本低,有较好的开发应用前景。     

连作烤烟根际土壤自毒物质成分分析

本研究以连作烤烟12年的土壤为材料,分析烤烟连作条件下烤烟根际土壤自毒潜力及土壤物质成分差异,为深入揭示烟草连作障碍提供依据。结果表明,连作条件下,烤烟根际土壤对受体植物莴苣（Lactuca sativa）、烟草（Nicotiana tabacum）具有较强的化感自毒潜力。物质成分分析结果显示,未种植烤烟的对照土壤中存在7种糖类物质、14种酸类物质、16种酯类物质、1种其他类物质;而连作烤烟根际土壤中存在2种糖类物质、3种酸类物质、8种酯类物质、12种其他类物质。与对照相比,连作烤烟根际土壤中存在特有的8种与自毒作用相关的物质,如香草醛、洋地黄毒素、雪松醇、植酮、诱虫烯、β 谷甾醇、胆甾烷醇、胆甾烷 3 酮。     

苦马豆生物碱成分的初步分析

为进一步筛选苦马豆的主要毒性成分和生物碱类活性物质,对苦马豆生物碱成分进行初步分析。采用生物碱预试、薄层色谱和气相色谱质谱联用等方法对宁夏采集的苦马豆生物碱成分进行定性分析。经薄层色谱分析,苦马豆生物碱出现了Rf值与苦参碱相近的斑点;气相色谱质谱联用分析确定苦马豆生物碱出现了与苦参碱色谱保留时间相近的峰,且该峰对应的离子碎片质谱图与苦参碱离子碎片质谱图一致。但通过薄层色谱和气相色谱质谱联用分析均未从苦马豆中检出吲哚里西啶生物碱——苦马豆素。结果表明,宁夏采集的苦马豆中含有苦参碱,不含苦马豆素或所含苦马豆素低于检测限。