Capacity for digestive hydrolysis and amino acid absorption in Atlantic salmon (Salmo salar) fed diets with soybean meal or inulin with or without addition of antibiotics

This experiment was done to study the effects of dietary soybean meal (SBM) and inulin (a prebiotic) on the capacity for digestive hydrolysis and amino acid absorption by Atlantic salmon, and how a dietary supplement of the broad-spectrum antibiotic oxytetracycline (OTC) modulated these responses. A control diet (FM) was made from fish meal, fish oil and extruded wheat. Two similar diets were made with 250 g soybean meal (SBM) or 75 g inulin kg^− 1. Each diet was made with or without a supplement of 3 g OTC kg^− 1. All six diets contained yttrium oxide for estimation of apparent nutrient absorption. Each diet was fed to two groups of 172 g salmon kept in 1 m² tanks with 9 °C saltwater for 3 weeks. Intestinal organs were then sampled and weighed. Gastrointestinal tracts (GIT) were sectioned for analyses of brush border alkaline phosphatase (ALP) and leucine aminopeptidase (LAP) activities. Tissue from the distal intestine (DI) was also fixed for histological examination. Digesta from the different sections were freeze dried for estimation of trypsin and amylase activities, and of apparent absorption of amino acids (AA), nitrogen (N), and sulphur (S). About 85% of the trypsin activity, 70% of the amylase activity, 85% of the ALP activity, and 82% of the LAP activity were found in the proximal (PI) and mid (MI) intestine of fish with functional DI, and the absorption of AA, N, and S was quantitatively completed in the MI. Dietary OTC resulted in lower relative liver weight, but apart from increased ALP and LAP activities in DI when feeding OTC in combination with inulin, OTC did not modify the responses to dietary SBM or inulin. Dietary SBM resulted in lower relative liver weight, and induced pathomorphological changes in the DI mucosa, thus lower the ALP and LAP activities in the DI. SBM also stimulated absorption of AA, N, and S in the PI, but at the same time increased the activities of trypsin and amylase in the DI, indicating reduced re-absorption and increased faecal losses of these endogenous enzymes. Dietary inulin did not damage the DI, and stimulated intestinal growth and higher relative mass of the GIT. Inulin without OTC did not affect the hydrolytic and absorptive capacity of the salmon GIT.     

核酸疫苗SjCA/pc和SjMf1/pc在小鼠体内的表达

为了观察核酸疫苗SjCA／pc和SjMfl／pc在小鼠体内的表达，取昆明鼠，于其后腿外侧肌注免疫SjCA／pc和SjMfl／pc，2d后剖杀动物．取注射部位肌肉作石蜡切片，免疫组化分析，荧光显微镜下镜检。结果表明，SjCA／pc和SjMfl／pc在小鼠注射部位肌肉可见黄绿色荧光，而对照组无此现象。可见，SjCA／pc和SjMfl／pc可在小鼠体内表达。     

Changes of urinary exosomal leucine aminopeptidase and dipeptidyl peptidase 4 in diabetic nephropathy

AIM: To investigate the changes of urinary exosomal enzymes and the correlation with diabetic nephropathy.METHODS: Thirty-four healthy volunteers and 127 patients of type 2 diabetes mellitus (T2DM) were included in the study. The healthy volunteers served as control. The patients with T2DM were divided into 3 groups based on their 24 h urinary albumin/creatinine ratio (UACR): 50 patients with microalbuminuria in early DN group (DN1), 34 patients with macroalbuminuria in overt DN group (DN2) and 43 patients without albuminuria in DM group. The levels of urine exosomal leucine aminopeptidase(exosome-LAP) and exosomal dipeptidyl peptidase 4(exosome-DPP4) were determined by enzyme-linked immunosorbent assay (ELISA). The following methods were used to determine the biochemical parameters: liquid chromatography for glycated hemoglobin (HbA1c), chemical modification method for cholesterol (CH), Jaffe-kinetic assay for creatinine (CR) and urease-GLDH method for blood urea nitrogen (BUN). Multiple stepwise linear regressions were used to analyze the relationship of exosome-LAP or exosome-DPP4 with HbA1c, CH, UACR, CR and BUN. RESULTS: The levels of exosome-LAP and exosome-DPP4 in DM, DN1 and DN2 groups were significantly higher than those in control group (P<0.01). The exosome-LAP in DN2 group was significantly higher than that in DM group. Correlation analysis showed that the levels of urinary exosome-LAP and exosome-DPP4 were positively correlated with HbA1c, CH, UACR, CR and BUN. Multiple stepwise linear regression analysis showed that CH and UACR were independent determinants for exosome-LAP (P<0.01), and UACR and HbA1c were independent determinants for exosome-DPP4 (P<0.01). CONCLUSION: Urine exosome-LAP and exosome-DPP4 are correlated with the severity of diabetic nephropathy. These parameters may serve as clinical markers for the diagnosis and prognosis evaluation of diabetic nephropathy.     

昆虫氨肽酶N的研究进展

氨肽酶N(aminopeptidase N,APN)是一种偏好水解蛋白或寡肽N端中性氨基酸的酶,广泛分布于动植物中。在昆虫中,APN主要分布于中肠刷状缘膜上,涉及食物蛋白的消化以及Bt毒素对靶标害虫的作用。从APN的结构特征和分类地位、APN的分离纯化及其酶活性位点与结合位点的相关性、APN同工酶的区分特征及系统发育关系、APN同工酶的基因功能、不同APN同工酶在昆虫体内的分布及表达量、APN基因功能最新研究方法和结果以及APN与昆虫抗药性的关系等方面概述了昆虫APN的最新研究进展。鉴于昆虫体内受体的多样性、复杂性及当前研究手段的局限性,对APN功能的全面了解还有赖于研究方法的改进和提高而逐步深入。     

红曲菌氨肽酶产酶条件的优化

通过单因子筛选试验和正交试验对红曲菌产氨肽酶的液体发酵培养基进行优化,结果表明,最佳液体发酵培养基组成为90.0g/L蛋白胨、25.0g/L可溶性淀粉、0.5g/L硫酸镁。稳定性试验验证证明,以最佳培养基发酵,氨肽酶酶活力高,平均酶活力为376.9U/mL。     

棉铃虫氨肽酶N基因片段克隆、表达和内源蛋白检测

氨肽酶N（APN）是苏云金芽孢杆菌杀虫毒素Cry在昆虫中肠中的一个重要受体。研究氨肽酶N在昆虫中肠中的分布特征对于阐明Cry毒素的杀虫机理和昆虫对Cry毒素的抗性机理具有重要的意义。通过RT-PCR的方法从棉铃虫中肠上皮细胞中克隆得到氨肽酶N的基因片段APN1551,并诱导表达纯化得到其重组蛋白APN517。以此蛋白为抗原,制备其抗血清。用该抗血清能检测到棉铃虫中肠上皮细胞中的APN蛋白。为研究Cry毒素的作用机理奠定基础。     

美国白蛾hcapn3基因的克隆及HcAPN3蛋白与Cry1Ac结合特性分析

通过比对分析已知昆虫氨肽酶N(aminopeptidase N,APN)氨基酸序列并结合本实验室的美国白蛾(Hyphantria cunea)中肠iTRAQ结果,设计了hcapn3基因特异引物,获得hcapn3基因片段。通过RACE-PCR技术获得美国白蛾中肠氨肽酶N基因(hcapn3)全长序列(GenBank登录号为KJ013598)。Blastp分析表明,获得的氨肽酶属于氨肽酶N3家族,命名为HcAPN3。序列分析显示HcAPN3包括952个氨基酸残基,具有典型的谷氨酸锌化氨肽酶(Gluzincin)结构域和羧基端(ERAP1_C)结构域。利用Bac to Bac表达系统在昆虫细胞中表达108kDa的HcAPN3蛋白。在原核表达系统中表达58kDa的Gluzincin结构域和49kDa ERAP1_C的结构域蛋白。Ligand Blot分析结果显示,HcAPN3蛋白及Gluzincin结构域可与Cry1Ac蛋白特异性结合,但ERAP1_C结构域未能与Cry1Ac结合。本研究首次克隆了美国白蛾氨肽酶基因并分析了HcAPN3与Cry1Ac的结合特性,为下一步功能研究提供基础。     

Novel insecticide polymer chemistry to reduce the enzymatic digestion of a protein pesticide, trypsin modulating oostatic factor (TMOF)

A limiting factor in the use of proteins as insecticides, especially when the site of action is in the insect hemocoel, is protease degradation in the digestive system and hemolymph and movement across the midgut ventriculus. Trypsin modulating oostatic factor (TMOF) is a per os mosquito peptidic larvacide which moves across the digestive system and binds to receptors on the hemolymph side of the gut where the hormone inhibits protease synthesis and food utilization ultimately causing death. In the current study, the in vitro degradation of TMOF by the digestive enzyme, leucine aminopeptidase, was inhibited by conjugation of TMOF-K with aliphatic polyethylene glycol (PEG) polymers. Structure activity studies demonstrated a correlation between the molecular weight of the PEG polymer and resistance to digestion and show proof of concept that aliphatic-PEG protein polymerization can be used to prevent protease degradation of a protein insecticide.     

氨肽酶N(APN)与鳞翅目昆虫对Bt抗性的关系

使用Bt Cry毒素防治农业害虫是作物生产上的一个革命性的进步,受体与Bt杀虫蛋白结合能力的改变可能是昆虫对Bt产生抗性的主要原因。氨肽酶N(aminopeptidase N,APN)是一类存在于昆虫中肠内的Bt毒素受体蛋白,通过讨论APN与Bt毒素的结合作用,综述了APN基因变异与鳞翅目昆虫Bt抗性相关的分子机理,并介绍了(Bt)Cry毒素与APN相关的作用方式新模型。     

Early Morphological and Physiological Events Occurring During Germination of Maize Seeds

The early morphological and physiological events occurring during maize （Zea mays cv. Nongda 108） seed imbibition and germination were studied. Water uptake of seeds exhibited a triphasic pattern with a marked increase during the initial phase of imbibition, and then a slow increase, followed by a second substantial increase. Imbibition time for 10 and 50% of seed germination was about 26 and 46 h at 30℃, respectively. The relative conductivity of maize seeds dramatically decreased during the initial phase of imbibition, followed by a substantial increase. Respiratory rate of seeds gradually increased with imbibition. Length of root cap cells decreased during the initial phase and then increased; those of meristematic zone cells increased during the initial phase and then decreased; and those of elongation zone cells and of the whole elongation zone of the radicle gradually increased during germination. The contents of soluble sugars and starch in embryos gradually decreased as the activities of α- and β-amylase strikingly increased with imbibition. In the meantime, protein contents of embryos gradually decreased and free amino acid content increased. The activities of aminopeptidase and endopeptidase increased until 12 h of imbibition and then decreased. It is concluded that germination of maize seeds is mainly completed by extension of cells in the elongation zone of the radicle, and that mobilization of stored reserves in the embryo during the initial phase of imbibition is also an early event during seed germination.