Oral administration recombinant porcine epidermal growth factor enhances the jejunal digestive enzyme genes expression and activity of early-weaned piglets

This study attempted to determine ingested porcine epidermal growth factor (pEGF) on the gastrointestinal tract development of early-weaned piglets. Thirty-two piglets (14-day weaned) were randomly allotted to supplemented with 0 (control), 0.5, 1.0, or 1.5 mg pEGF/kg diet. Each treatment consisted of four replicates with two pigs per pen for a 14 days experimental period. Piglets were sacrificed and gastrointestinal tract samples were collected to measure mucosa morphology, mRNA expression and activities of digestive enzymes in the gastrointestinal tract of piglets at the end of the experiment. Diets supplemented with pEGF failed to influence growth performance but tended to increase jejunal mucosa weight (p < 0.09) and protein content (p < 0.07). Piglets supplemental pEGF induced incrementally the gastric pepsin activity (p < 0.05) and stimulated jejunal alkaline phosphatase (ALP) and lactase activities accompanied with the increase of jejunal ALP and maltase mRNA expression. No effect of pEGF on the activities of all enzymes in ileum except the stimulation of ileal aminopeptide N mRNA expression. These results reveal that dietary pEGF supplementation might enhance gene expression and activities of digestive enzymes in the stomach and jejunum of piglets.     

表皮生长因子对葡聚糖硫酸钠诱导的结肠炎模型小鼠肠道损伤的修复研究

本文旨在讨论表皮生长因子(EGF)对葡聚糖硫酸钠(DSS)诱导的结肠炎模型小鼠肠道损伤的修复作用。选用24只6周龄BALB/c小鼠,随机分为3组,即:正常对照组、DSS模型对照组、DSS+EGF组。正常对照组小鼠饮用自来水;DSS模型对照组小鼠在试验第1~7天饮用5%DSS水溶液,第8~10天饮用自来水;DSS+EGF组小鼠按照DSS模型对照组处理,同时每天皮下注射EGF 2次,共注射10 d。结果表明:1)与正常对照组相比,DSS模型对照组小鼠结肠长度极显著降低(P0.01);与DSS模型对照组相比,DSS+EGF组小鼠结肠长度极显著增加(P0.01)。2)DSS模型对照组小鼠结肠可见典型溃疡,结肠损伤程度评分(CDS)极显著高于正常对照组(P0.01);DSS+EGF组小鼠结肠组织未见溃疡,与DSS模型对照组相比,CDS极显著降低(P0.01)。3)与正常对照组相比,DSS模型对照组小鼠结肠紧密连接蛋白(Occludin)浓度显著降低(P0.05);与DSS模型对照组相比,DSS+EGF组小鼠结肠Occludin浓度显著升高(P0.05)。4)与正常对照组相比,DSS模型对照组小鼠结肠白细胞介素-2(IL-2)和白细胞介素-4(IL-4)浓度极显著降低(P0.01),白细胞介素-10(IL-10)浓度显著降低(P0.05);与DSS模型对照组相比,DSS+EGF组结肠IL-2和IL-4浓度极显著增加(P0.01),IL-10浓度显著增加(P0.05)。由此可见,EGF可能通过提高肠道Occludin表达水平,调节肠道细胞因子浓度趋于正常水平,从而修复受损肠道组织,维持肠道黏膜屏障的完整性。     

Optimization of the protein fraction of microcapsules used in feeding of marine fish larvae using in vitro digestibility techniques

The development of artificial feeds for marine fish larvae is demanding and, taking into account the difficulties and costs associated with the rearing of the larvae of some fish, preliminary evaluation of feed ingredients using in vitro techniques may be an alternative to in vivo assays. Some proteins used in microfeeds for marine fish were tested in vitro for examination of their effect on the proteases of seabream larvae. Casein and cuttlefish meal did not affect protease activity of crude larval extracts, whereas ovalbumin produced a 60% inhibition. The use of a pH-stat for the assessment of the degree of hydrolysis (DH) of proteins confirmed the low nutritive value of ovalbumin, since a much lower value was obtained for microcapsules prepared using this protein source when compared with those prepared using casein or cuttlefish meal (3.2 vs. 7.3 and 7.6, respectively). Products resulting from such hydrolysis were analysed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Image analysis of such gels led to the formulation of an index named the coefficient of protein degradation (CPD) which expressed the extent to which the main protein fractions were hydrolysed by larval proteases in a given time. A significant correlation ( r ² = 0.98, P  < 0.05) was obtained between DH and CPD values measured for either protein sources or microcapsules. The combination of protease inhibition assays with measurements of DH and CPD is proposed as a preliminary evaluation of protein ingredients used in the formulation of artificial feeds for larval fish.     

小麦木瓜类半胱氨酸蛋白酶基因TaRD21的克隆及赤霉病菌诱导下的表达分析

木瓜类半胱氨酸蛋白酶（PLCPs）作为一类重要的蛋白水解酶,在植物生长发育以及胁迫应答过程中都发挥着重要作用。本研究从抗、感赤霉病小麦品种差异表达基因谱中获得1个注释为RD21 Cysteine proteases的EST（表达序列标签）,以此序列检索小麦最新基因组数据库并设计引物,从小麦中克隆到3个基因,分别命名为TaRD21-2A、TaRD21-2B和TaRD21-2D,属于PLCPs RD21家族。序列分析表明,3个基因的开放阅读框长度分别为1 410、1 428和1 419 bp,分别编码469、475和472个氨基酸。序列比对发现,3个基因的序列相似性为89.3％,所编码蛋白的氨基酸序列相似性为95.6％。系统进化分析表明,TaRD21-2A、TaRD21-2B和TaRD21-2D蛋白的同源性较高,且与乌拉尔图小麦TuRD21A蛋白聚为一类。qRT-PCR分析表明,3个TaRD21基因均受水杨酸(SA)、乙烯利(ETH)以及赤霉病菌诱导表达;感病品种中,TaRD21-2A对SA和赤霉病菌的响应更迅速,且表达量较高;抗病品种中,TaRD21-2B和TaRD21-2D基因对ETH的响应更迅速。     

Fluorescence localization of epidermal cathepsins L and B in the Japanese eel

Histochemical localization of proteolytic activities in the dorsal epidermis of Japanese eel was demonstrated by fluorescent microscopy utilizing 4-methoxy-2-naphthylamide (4M$\beta$NA) derivatives as substrates and 5-nitrosalicylaldehyde as a trapping agent. Carbobenzoxy-L-phenylalanyl-L-arginyl-4M$\beta$NA (Cbz-Phe-Arg-4M$\beta$NA) and Cbz-Arg-Arg-4M$\beta$NA were used for direct detection of cathepsins L and B activities, respectively, in fresh frozen sections and unfixed cells of the eel epidermis. The fluorescing areas, where Cbz-Phe-Arg-4M$\beta$NA was hydrolyzed by cathepsin L, were shown in mucus secretory cells and club cells and broadly around skin surface. The fluorescing areas due to Cbz-Arg-Arg-4M$\beta$NA hydrolysis by cathepsin B were localized similarly in these tissues. The fluorescing intensity for both catheptic activities in mucus secretory cells was higher than that in club cells, where small fluorescing granules were distributed. These results indicate that eel cathepsins L and B are stored in epidermal secretory cells at different levels and probably serve as defense factors before or after secretion by these cells. Abbreviations: Cbz – carbobenzoxy; 4M$\beta$NA – 4-methoxy-2-naphthylamide; NSA – 5-nitrosalicylaldehyde.     

鸭疫里默氏杆菌不同分离株生化特性及胞外蛋白酶的检测

研究测定了29株鸭疫里默氏杆菌不同分离株的生化指标,并采用底物平板法、底物-PAGE法检测了鸭疫里默氏杆菌胞外蛋白酶的产生情况。结果显示,所有分离株接触酶阳性,硝酸盐还原试验、M.R和V.P试验、尿素酶、精氨酸酶阴性,大部分菌株不发酵碳水化合物,能缓慢液化明胶,明胶液化阳性率为89.7%(26/29)。底物平板法和底物-PAGE法检测发现,89.7%(26/29)的鸭疫里默氏杆菌分离株能产生降解明胶和脱脂奶蛋白的胞外蛋白酶,且该酶类活性可被EDTA抑制。     

暗纹东方鲀冻藏品质劣化的原因解析

通过比较冰晶、内源蛋白酶以及氧化对冻藏暗纹东方鲀(Takifugu obscurus)品质的影响,确定冻藏暗纹东方鲀品质劣化的关键因素。实验通过液氮浸渍快速冻结抑制鱼肉组织中冰晶增长、碘乙酸浸泡前处理抑制鱼肉内源蛋白酶活性、复合抗氧化剂浸泡前处理抑制蛋白/脂质氧化作用的设计,区分冰晶、内源蛋白酶和氧化3种因素对冻藏鱼肉品质的影响;并以硬度、解冻流失率、蒸煮损失率、挥发性盐基氮含量、K值、Ca2+-ATPase活性、肌原纤维小片化指数和肌纤维长度作为鱼肉品质的评价指标。结果显示,冻结过程中控制冰晶增长对暗纹东方鲀冻藏品质的改善作用最大,冻藏24周后,速冻组的硬度比内源蛋白酶抑制组和氧化抑制组分别提高了26.8%和20.5%,解冻流失率分别降低了44.2%和44.8%,且该处理组TVB-N、K值和Ca2+-ATPase活性变化更慢,24周后分别为10.5 mg/100 g、6.8%和1.43 μmol Pi/mg/10 min。综合各项指标发现,冰晶、内源蛋白酶和氧化对冻藏暗纹东方鲀品质劣化均有影响;其中,冰晶作用是导致暗纹东方鲀在冻藏过程中品质下降的最主要影响因素,内源蛋白酶和氧化对冻藏暗纹东方鲀品质的影响次之,且二者对品质影响无显著性差异。     

Virucidal effects of various agents—including protease—against koi herpesvirus and viral haemorrhagic septicaemia virus

In a search for alternative, environmentally friendly and effective disinfecting agents, a commercially available protease—Neutrase^®—was tested in this work for inactivation of koi herpesvirus (KHV) and of viral haemorrhagic septicaemia virus (VHSV). For comparison, the stability of these viral pathogens in similar configurations at various pH values and concentrations of peracetic acid or quicklime, typically used for disinfection, was tested. Therefore, virus suspensions were incubated with various concentrations of different agents for 24 hr and the titre of the remaining infectious particles was determined by virus titration. Furthermore, the treatment of both viruses, with the agents at concentrations that were previously appointed as effective, was also examined in the presence of solid material (quartz sand). All procedures investigated in this study, including the protease treatment, were able to reduce the titre of KHV and VHSV below the detection limit of the titration. Although further studies are necessary, this is the first report of the application of a protease for the inactivation of the selected fish pathogens, demonstrating the great potential of the latter for disinfection.     

8种野生葱属植物的叶形态特征及其分类学意义研究

【目的】比较8种野生葱属植物的叶形态特征,为其分类学研究和生态适应性机制分析提供依据。【方法】以分布于新疆不同生境的8种野生葱属植物齿丝山韭、实葶葱、新疆蒜、健蒜、多籽蒜、奇台蒜、喀纳斯蒜和乌鲁木齐蒜为研究对象,采用田间观测和树胶制片法,测定其叶片数量、长度、宽度等指标,观察其解剖结构异同;通过相关性分析和聚类分析,研究叶片表皮性状之间的关系以及性状的分类学意义。【结果】8种葱属植物的叶形态和微形态存在差异,实葶葱和齿丝山韭叶直立,其余呈不同程度倒伏;实葶葱叶圆柱形,其余叶均为条形;实葶葱叶表面被蜡粉,其余均为光滑;实葶葱叶表皮细胞矩形,其余均为菱形;实葶葱垂周壁直线型,其余均为弧线形。8种植物叶片均为双面气孔型,气孔椭圆形,保卫细胞肾形,无副卫细胞,保卫细胞不同程度下陷。基于叶表皮结构性状的聚类分析结果显示,供试8种材料分为3类,齿丝山韭和实葶葱分别聚为一类,其余6种聚为一类。【结论】叶形态、表皮细胞形状、垂周壁形状、表皮细胞密度和气孔指数大小可为种的界定提供重要的形态分类学依据,具有分类学意义。叶片上表皮细胞比下表皮细胞可以提供更为全面的微形态特征。新疆8种野生葱属植物的叶片形态和微形态结构均呈现一定的抗旱和抗寒特征。     

合浦珠母贝表皮生长因子样(EGF-like)基因的克隆与表达分析

为探究表皮生长因子(epidermal growth factor,EGF)在合浦珠母贝幼体发育以及组织生长过程中的作用,实验通过RACE技术克隆了合浦珠母贝EGF-like基因并做了相应的表达分析。实验结果获得cDNA全长序列4 107 bp,命名为Pf-egf1,开放阅读框(ORF)702 bp,编码234个氨基酸,包含一个信号肽序列和一个跨膜结构域,功能结构域分析发现Pf-egf1具有一个EGF-like结构域,有6个半胱氨酸残基,由3个二硫键维持,是表皮生长因子家族及其相关蛋白的特征结构域,但其余序列与现有相关基因序列差异较大,推测可能是一个新的EGF-like基因。表达结果显示,Pf-egf1 mRNA在合浦珠母贝外套膜、闭壳肌、鳃、肝胰腺、珍珠囊、肠和性腺中均有表达,在肠中的表达显著高于其他组织;在合浦珠母贝幼体发育的担轮期、D型期、壳顶期、眼点期和变态期的表达呈现逐渐升高的趋势,并且其在变态期的表达量极显著地高于其他时期。上述结果表明,Pf-egf1基因可能在合浦珠母贝肠道修复和幼虫变态发育阶段起着重要作用,为进一步开展育珠与生长调控奠定了基础。