An improved protocol for electrotransformation of Corynebacterium pseudotuberculosis

We developed an improved protocol for the electrotransformation of Corynebacterium pseudotuberculosis, testing variations of parameters in the procedures that are routinely used for the preparation of electrocompetent cells of this species, including (i) culture conditions, (ii) cell growth phase, (iii) electroporation solutions and (iv) quantity of plasmid DNA. We obtained the greatest efficiency of transformation when the cells were grown until the stationary phase and then washed with 10% glycerol electroporation solution. The transformation efficiency was inversely proportional to the quantity of plasmid DNA. The transformation efficiency reached 10(5) colony-forming units (cfu)/mug plasmid DNA. This protocol would be useful for genetic studies of C. pseudotuberculosis.     

Immunohistochemical Colocalization of Serotonin, Substance P and Met-enkephalin-Arg6-Gly7-Leu8 in the Endocrine Cells of the Ruminant Duodenum

The duodenum of various ruminants was examined by immunohistochetnical staining for serotonin, substance P and Metenkephalin-Arg⁶-Gly⁷-Leu⁸ (MENK8). Serotonin- and substance P-immunoreactive endocrine cells were detected in samples from cow, calf, sheep, goat, and barbary sheep. MENK8-immunoreactive endocrine cells were detected exclusively in samples from cow and calf. Substance P and MENK8 immunoreactivity was found in serotonin-immunoreactive endocrine cells of cattle. Substance P- and MENK8-immunoreactive nervous elements were detected in all ruminants examined. The present results suggest that the expression and/or the mechanism that controls the expression of each peptide might differ between endocrine cells and nerve cells and might also depend on animal species.     

The glucocorticoid sparing efficacy of PhytopicaTM in the management of canine atopic dermatitis

This double-blind randomized placebo-controlled trial indicates that Phytopica™ can be an effective glucocorticoid sparing agent in canine atopic dermatitis (AD). Twenty-two dogs with perennial AD [Canine Atopic Dermatitis with Severity Index (CADESI-03) ≥ 60] were given 200 mg/kg Phytopica™ or an identical placebo in food once daily for 56 days. All dogs were initially given 0.4 mg/kg methyl-prednisolone once daily, which was then adjusted according to the daily pruritus score (0–100 mm visual analogue scale). The cumulative dose and pruritus score were lower in the Phytopica™ than the placebo group. There were statistically significant time and treatment effects for the methyl-prednisolone dose and pruritus score, but there were no significant differences between the Phytopica™ and placebo groups in the proportion of dogs that achieved a > 50% reduction in dose or pruritus scores at day 56; the mean CADESI-03 scores at days 0, 28 and 56; the numbers achieving >50% reduction in CADESI-03 at days 28 and 56; or in the owners' global efficacy score at days 28 and 56. Adverse events included diarrhoea (three Phytopica™ and one placebo treated dog), polyuria/polydipsia (three dogs in each group), and polyphagia, intermittent anorexia and panting (one dog each in the placebo group). None of these by themselves required withdrawal of treatment.     

Role of Ca2+ in corticosterone-induced muscle growth retardation

The increased plasma glucocorticoid concentration in stressful conditions stimulates muscle protein degradation, and results in growth retardation in animals. However, the mechanism is still to be clarified. The present study was undertaken to examine the participation of Ca²⁺ in the glucocorticoid action, using nifedipine (NIF), a Ca²⁺ channel antagonist. The effects of NIF on growth, differentiation, and protein degradation were examined in glucocorticoid-treated primary cultured chick muscle cells. Muscle cell growth and cell differentiation were assessed by protein content and creatine kinase (CK) activity, respectively, and the rate of myofiblillar protein degradation was estimated by the release of N ^τ-methylhistidine (MeHis). Creatine kinase activity was increased by corticosterone (CTC) and this effect was minimized by NIF. Protein content was decreased by CTC and normalized by NIF. N ^τ-methylhistidine release was significantly increased by CTC and tended to be minimized by NIF. The present results indicate that CTC increases skeletal muscle proteolysis followed by muscle growth retardation partially because of enhanced Ca²⁺ influx through the NIF-sensitive Ca²⁺ channel. Enhanced muscle differentiation by CTC is mediated also by the NIF-sensitive Ca²⁺ channel.     

高效毛细管电泳法同时检测饲料中七种防腐剂

建立酸性条件下同时分离测定山梨酸(SA)、苯甲酸(BA)、脱氢乙酸(DHA)、对羟基苯甲酸甲酯(MP)、对羟基苯甲酸乙酯(EP)、对羟基苯甲酸丙酯(PP)、对羟基苯甲酸丁酯(BP)七种防腐剂的高效毛细管电泳法。本试验用甲醇:水=1:1(体积比)的混合液作为提取剂提取饲料样品中防腐剂;以40 mmol/l磷酸二氢钾(KH2PO4)和100 mmol/l十二烷基硫酸钠(SDS)的1:1体积比混合液(pH=4.00)作为缓冲液,采用高效毛细管胶束电动色谱法测定样品中七种防腐剂。该方法在19 min内实现了七种防腐剂的分离;SA、BA的线性范围分别为1～500μg/ml和3～500μg/ml,DHA、MP、PP、BP、PP的线性范围均为5～500μg/ml,线性相关系数≥0.999 5。SA、BA、DHA和四种对羟基苯甲酸酯类防腐剂的检测限分别为0.5、1.5、3.0、2.5μg/ml;样品平均回收率为80.8%～107.0%,相对标准偏差≤5.0%。该方法高效快速分离了多种防腐剂,并且可以应用到各种饲料样品的检测。     

高效毛细管电泳用于饲料中5种氟喹诺酮类药物的同时测定

本实验利用高效毛细管电泳建立了同时检测雏鸡、哺乳猪等饲料中5种氟喹诺酮类药物的方法.研究了缓冲液的类型、离子浓度、pH、分离电压和分离温度等务件对分离的影响,通过正交实验设计得到了最佳的电泳条件.在274 mn处,分离电压为18 kV,分离温度为22℃,在80mmol/L柠檬酸-柠檬酸钠pH 6.5的运行缓冲液下,5种药物可在10 min内完全分离.5种药物的浓度与峰面积的相关系数r=0.9999～0.9994,方法精密度(RSD)为1.36%～1.69%(迁移时间)和2.09%～5.89%(峰面积).同时建立了饲料的前处理方法,方法平均回收率为84.42%～107.5%.该方法操作简便、准确、快速.