Cathepsins B, H and L in Peritoneal Macrophages and Hepatopancreas of Carp Cyprinus carpio

Cathepsin B was purified from the crude extract of carp hepatopancreas by a modified method, and the specific activity increased about 3,400-fold with a 17% recovery. The purified enzyme gave a single protein band on Native-PAGE, whereas two bands with molecular weights of 30,000 (single chain) and 26,000 (heavy chain) migrated on SDS-PAGE. The enzyme potently hydrolyzed Z-Arg-Arg-MCA and Z-Phe-Arg-MCA but lacked the ability to hydrolyze most of the other MCA substrates. The kinetic constants of the enzyme with two Z-blocked substrates revealed that V_max and K_cat values of Z-Phe-Arg-MCA were much higher than Z-Arg-Arg-MCA, while their K_m values were approximate. The optimum hydrolysis pH and temperature of the enzyme for these two substrates were determined to be pH 6.0 and 45°C, respectively. A variety of protease inhibitors such as E-64, DTNB, antipain, leupeptin, chymostatin, TLCK and TPCK and metal compounds such as CuCl₂, CdCl₂, HgCl₂, and Zn(CH₃COO)₂ were able to significantly inactivate the enzyme. In contrast, cysteine and 2-mercaptoethanol activated the enzyme, with cysteine being more effective for activation than 2-mercaptoethanol over the tested concentrations.     

Biochemical properties of purified cathepsin B from Schistosoma mansoni

A previously described “major acidic proteinase” of adult Schistosoma mansoni, believed to play a key role in the parasite's metabolism, has been identified as a cathepsin B (Sm31). Purified Sm cathepsin B was not recognized by anti-Sm32 or anticathepsin L antibodies. The enzyme hydrolyzes the synthetic protease substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC as well as protein substrates. Its pH optimum is 3.0 with serum albumin, 4.0–5.0 with globin and 5.5–6.0 with the synthetic substrates. The enzyme was inactivated by cysteine proteinase inhibitors. Its activity against protein substrates would support the hypothesis that it plays a role in schistosome nutrition.     

Genetic characterization of common carp (Cyprinus carpio) populations from Greece using mitochondrial DNA sequences

Wild common carp from two lakes and two rivers in Greece were genetically characterized with sequencing analysis of two mitochondrial DNA segments: cytochrome b (1119 bp) and D-loop (646 bp). A total of 9 variable singleton sites and 7 unique haplotypes were detected. A common haplotype was found in three out of the four populations examined, which seems to be the ancestral one and represents the European origin of common carp from Greece. This haplotype could be also justified by the introductions reported with individuals belonging to the Central European race, into many natural habitats in Greece. Limited genetic variation — in Evros and Aliakmonas populations — could be due to bottleneck effects and small effective population sizes, whereas the different haplotypes found in Lake Volvi could represent different common carp stocks. Values of sequence divergence among Greek haplotypes ranged from 0.0006 to 0.0023. The Neighbour-Joining (NJ) phylogenetic tree constructed based on the combined sequences, reveals that the populations of common carp from Greece belong to the European group of populations — which is highly divergent from the South East-Asia cluster — and to the subspecies Cyprinus carpio carpio.     

Quantitative genetic estimates of growth-related traits in the common carp (Cyprinus carpio L.): A review

The common carp (Cyrpinus carpio L.) is the oldest cultured and the most domesticated fish species, as well as one of the most important freshwater fishes in the world. However, scientific studies on evaluating the growth-related quantitative traits in this fish are limited. Heritability, the most important parameter in selective breeding programs, was extensively studied for the growth-related traits. The values varied widely among the experiments and methods used because of the existence of common environmental, dominance and maternal effects. However, correlations in phenotypic and genetic levels first evaluated several years ago were limited. On the other hand, heterosis was widely reported and easily obtained for growth-related traits in the common carp. Meanwhile, genotype environment interaction and prediction of breeding values have been studied recently, and are very important in conducting selective breeding programs. The developmental quantitative genetics of growth-related traits was first analyzed in the common carp for reasonable selection during ontogeny. It is expected that genetic improvement will be achieved by carrying out direct selective breeding in the common carp.     

Purification and some properties of buffalo spleen cathepsin B

Purification of cathepsin B from buffalo-spleen, a hitherto unstudied system has been achieved by a simple procedure developed by incorporating suitable modifications in the existing methods for isolation of the enzyme from other sources. The purified enzyme has a molecular weight of 25 KDa and its Stokes radius was found to be 2·24 nm. Effects of several reducing agents, urea and thiol-protease inhibitors such as leupeptin and antipain, have been studied and the data unequivocally support the contention that the buffalo-enzyme is similar to cathepsin B from other tissues with respect to these properties.     

Cloning, characterization, and expression analysis of Toll-like receptor-7 cDNA from common carp, Cyprinus carpio L.

The Toll-like receptor 7 (TLR7) is activated by single strand RNA and RNA-like compounds (imidazoquinoline), and it induces interferon production. We identified and described carp TLR7 cDNA and its mRNA expression. The full-length cDNA of carp TLR7 gene is 3427 bp, encoding 1049 amino acids (AB553573). The similarities of carp TLR7 with zebrafish, rainbow trout, fugu, and human TLR7 were 89.6, 83.4, 80.6 and 74.6%, respectively, at the amino acid sequence level. Furthermore, the expression of TLR7 mRNA was investigated in normal tissues of carp by semi-quantitative RT-PCR analysis. Carp TLR7 expression was exhibited in healthy tissues (kidney, brain, spleen, skin, intestine, muscle, liver, gills and heart) and though the expression level in each tissue varied among healthy fish. Carp TLR7 expression was significantly increased in head kidney stimulated with TLR7 agonist, imiquimod, at 8, 24 and 48 h in vitro when compared to expression in the control group. Moreover, carp head kidney leukocytes produced elevated levels of pro-inflammatory and type 1 interferon cytokine mRNA in response to imiquimod stimulation.     

The effects of water temperature and ration size on growth and body composition of fry of common carp, Cyprinus carpio

Experiment was conducted with the aim of determining the effect of varying water temperature and ration size on growth and body composition of fry of the common carp, Cyprinus carpio. Common carp fry with an initial body weight (BW) of 0.86 g were fed a diet (34.9% protein, 18.3 KJ/g diet) at four ration sizes 4%, 5%, 6% and 7% of their body weight per day and reared at two water temperatures 28 and 32 °C for 60 days. Fry fed with 6% ration showed the highest mean final body weight at 28 °C. Final body weight was significantly (P<0.05) affected by ration and temperature. Cyprinus carpio fry raised at 28 °C had higher feed efficiency (FE) (44.36%) than the fry reared at 32 °C (40.98%) with 4% ration. Further, feed efficiency decreased with increase in ration levels in both temperatures. Protein efficiency ratio (PER) was higher (1.26) at 28 °C than at 32 °C (1.17). At 6% ration, common carp fry showed highest specific growth rate (SGR) (3.82%/day) at 28 °C as compared with at 32 °C (3.57%/day). A linear increase in protein and lipid contents was evident with increasing ration levels up to 6% body weight at both temperatures 28 and 32 °C. Second-order polynomial regression analysis of weight gain and SGR indicated the breakpoints at ration level 6.04% and 6.08% body weight per day at 28 and 32 °C. Hepatosomatic index (HSI) not affected by temperature and ration size while, viscerosomatic index (VSI) influenced (P<0.05) by ration size and temperature. Based on the above results, it may be concluded that 6% BW/day ration is optimal for growth of Cyprinus carpio fry at both the temperatures 28 and 32 °C.     

Vitellogenesis-related ovary cathepsin D from Xenopus laevis: Purification and properties in comparison with liver cathepsin D

Cathepsin D was purified from ovaries of Xenopus laevis by both QAE-cellulose and pepstatin-Sepharose chromatography and then characterized and compared with Xenopus liver cathepsin D. Ovary cathepsin D appeared predominantly as a 43-kilodalton (kDa) molecular mass, as revealed by SDS-polyacrylamide gel electrophoresis, whereas the liver enzyme was obtained exclusively as a 36-kDa protein. The purified 43-kDa ovary enzyme cleaved vitellogenin limitedly to produce yolk proteins at pH 5.6. The specific activity of ovary cathepsin D was five to six times lower than that of the liver enzyme, as measured by hemoglobin-hydrolysis at pH 3, but the ovary enzyme was shown to be superior to the liver enzyme in terms of vitellogenin-cleaving activity, as examined at pH 5.6. Ovarian enzyme preparations contained variable amounts of 36-kDa species; this form was considered to be an autolytic product of the 43-kDa form arising during purification, because it was not detected in oocyte extracts but was generated by incubation of the purified 43-kDa enzyme alone in an acid solution. The conversion of the 43-kDa form by hepatic factors was accompanied by a marked increase in hemoglobin-hydrolytic activity.     

Protective effects of propolis on cryopreservation of common carp (Cyprinus carpio) sperm

Cryopreservation of sperm is common procedures in aquaculture, particularly used for routine in artificial insemination. However, these application cause damages and adversely affected sperm motility, viability and consequently lower hatching rates. The objective of this study is to determine whether propolis has an effect on cryopreservation and fertilization ability and to investigate the potential protective effect of propolis on spermatozoa of Cyprinus carpio. Many studies have been done in cryopreservation offish spermatozoa, but none of them contain propolis in extender composition. The extenders were prepared by using modified Kurokura Solution to which 10% Me₂SO added with different levels of propolis (0.2, 0.4, 0.6, 0.8 and 1 mg ml⁻¹) and 10% egg yolk (as a control without propolis). The pooled semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. The percentage and duration of motility and fertilization tests of cryopreserved sperm samples have been done immediately after thawing and compared with control and fresh semen. The extenders containing propolis exhibited higher percentage motility and motility duration than control group (P < 0.05). Especially the group IV (0.8 mg ml⁻¹ propolis) and the group V (1 mg ml⁻¹ propolis) showed significant positive effects on both post thaw motility and hatching ability. The propolis maintained the integrity of the spermatozoa during the cryopreservation process. Evaluating with its contents, it has been shown that propolis is an appropriate cryoprotective agent in fish semen.     

元江鲤种群遗传多样性

选择12对微卫星标记检测了于2011年采集自元江(红河上游中国江段)5个样点192尾鲤的群体遗传多样性.共检测到201个等位基因,每个位点等位基因2-27个.各群体各位点平均等位基因(NA)12.25-14.67个,平均有效等位基因(NE)8.28-9.73个,平均观察杂合度(Ho)o.7765-0.8037,平均期望杂合度(HE)0.7761-0.8080,平均多态信息含量(PIC)0.7534-0.7843.元江鲤种群192个个体各位点NA、NE、Ho、HE、PIC分别为16.50、11.26、0.7927、0.8049、0.7966,种群遗传多样性水平高.元江鲤群体之间遗传分化小,可作为一个种群管理单元进行管理.增殖放流要防止遗传多样性丧失.     

从大豆中纯化棉铃虫组织蛋白酶B抑制因子HCB-SoyI

棉铃虫组织蛋白酶B（Helicoverpa armigera cathepsin B,HCB） 在胚胎发育过程中降解卵黄蛋白为氨基酸,供给胚胎发育必需的养料,是棉铃虫胚胎正常发育的重要因素。许多植物种子中存在蛋白酶抑制因子,如大豆和向日葵种子。用棉铃虫组织蛋白酶B为酶源,通过硫酸铵沉淀、排阻层析和离子交换层析等方法,从大豆中分离纯化到了一种对HCB有抑制活性的蛋白酶抑制因子,命名为HCB-SoyI。用牛血清白蛋白为底物进一步证明该抑制因子对HCB具有抑制作用。该抑制因子的纯化为进一步克隆其基因奠定了基础,为转基因抗虫作物研究提供了新的候选靶标。     

小浪底水库排沙对黄河鲤鱼的急性胁迫

为探讨黄河小浪底水库排沙过程中出现"流鱼"现象的原因,在水库排沙开始前和过程中以黄河鲤鱼为对象进行了生理生态观测。结果显示,相对于对照组,排沙组的黄河鲤鱼消化道充塞系数显著下降(P<0.001),消化道内容物含沙质量比率显著上升(P<0.001);肝脏总抗氧化能力显著上升(P=0.036),而脾脏总抗氧化能力显著下降(P=0.017);肝过氧化氢酶CAT活性显著上升(P=0.035);肾脏超氧化物歧化酶SOD活性显著下降(P=0.038);肝脏、脾脏丙二醛MDA含量显著下降(肝脏:P=0.039;脾脏:P=0.027);鳃丝钠钾ATP酶活性显著下降(P=0.001);血清乳酸LD含量显著上升(P=0.019);肝脏乳酸脱氢酶LDH活性显著下降(P=0.001);推测高含沙水流对黄河鲤鱼造成的急性缺氧应激是导致小浪底水库排沙过程中"流鱼"现象的主要原因之一。     

建鲤leptin两种原核表达载体的构建和表达

使用SignalP-4.0软件预测信号肽剪切位点,设计引物扩增建鲤(Cyprinus carpio var.jian)leptin基因(jlLEP-A1,jlLEP-B)不含信号肽的ORF区域。扩增产物分别连接到pET-32a(+)和pGex-4T-1原核表达载体,构建重组质粒,测序验证插入位点的正确性。将重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导表达重组蛋白pET-32a(+)/jlLEP-A1,pET-32a(+)/jlLEP-B和pGex-4T-1/jlLEP-A1,pGex-4T-1/jlLEP-B。结果表明,在37℃和1 mmol/L IPTG的条件下诱导5 h,重组蛋白表达量最大。SDS-PAGE电泳显示重组蛋白主要以可溶蛋白形式存在。这一结果为后续的建鲤leptin基因功能研究奠定了基础。     

Generalization and discrimination of positive and negative acoustic stimuli in the common carp (Cyprinus carpio)

Groups of common carp (Cyprinus carpio) were trained for 33 days to discriminate between two acoustic signals differing in frequency and temporal pattern. One signal (positive stimulus) was reinforced by food, while the other (negative stimulus) was not rewarded. When exposed to 3 positive and 3 negative stimuli per day (training Paradigm 1) the fish responded similarly to the two signals. When daily negative stimuli outnumbered positive stimuli (3 positive and 33 negative, training Paradigm 2) the fish responded similarly to the two signals for the first 2 weeks and then began to discriminate between them. Discriminatory response was statistically significant from Day 21 of training. After 33 days, training was switched from Paradigm 2 to Paradigm 1 and the fish maintained their discriminatory capability. The results are interpreted in terms of anticipatory frustration and risk management.     

温度和饥饿对鲤鱼幼鱼静止代谢率及自发运动的影响

分别在15和25℃条件下对鲤鱼(Cyprinus carpio)幼鱼[体重(10.36±0.22)g,n=88]进行2周的饥饿处理(饥饿组)或持续投喂(对照组),然后对实验鱼的静止代谢率(resting metabolic rate,RMR)进行了测定,同时以活动期的胸鳍﹑尾鳍摆动频率,观察期(10 min)胸鳍﹑尾鳍平均摆动次数,和运动时间比为指标对实验鱼的自发运动进行了观察。研究发现:温度和饥饿均显著影响鲤鱼幼鱼的静止代谢率(P0.05);在15与25℃条件下饥饿分别导致静止代谢率下降了19%和20%;温度由15℃上升至25℃,对照组与饥饿组静止代谢率分别增加了83%和80%。在15与25℃条件下,饥饿对鲤鱼幼鱼的自发运动具有显著不同的影响(P0.05),且胸鳍和尾鳍的结果十分相似;在15℃饥饿组与对照组的胸鳍、尾鳍摆动频率(活动期,以下类同)均无显著差异,而饥饿组运动时间比显著低于对照组,导致饥饿组的胸鳍、尾鳍平均摆动次数(观察期,以下类同)均显著低于对照组(P0.05);而在25℃饥饿组的胸鳍、尾鳍摆动频率均显著高于对照组,运动时间比无显著差异,导致饥饿组的胸鳍、尾鳍平均摆动次数均显著高于对照组(P0.05)。研究结果提示,在15℃下鲤鱼幼鱼通过降低自发运动以降低能量消耗来适应饥饿,而在25℃条件下,鲤鱼幼鱼通过增加自发运动来增大获取食物的机率以应对饥饿,这可能与野外环境中不同温度下食物丰富度及鲤鱼幼鱼的生理状况有关,但静止代谢率并未呈现与自发运动相关联的变化。     

Purification and some properties of a carboxypeptidase B from dogfish Scyliorhinus canicula

A carboxypeptidase B (CPB) has been purified from dogfish (Scyliorhinus canicula) pancreas and partially characterized. The purification procedure included acetone precipitation, ion-exchange chromatography on a CM-cellulose column and gel filtration on Sephadex G-75. The purified enzyme migrates as a single band both on PAGE and SDS-PAGE. Its molecular mass is estimated to be about 32 kDa. The optimum of activity is obtained at pH 7.5–8.2. The enzyme is inhibited by typical metal-chelating agents (EDTA and o-phenanthroline) and by Hg²⁺. It is activated by Co²⁺, l-cysteine and by heat treatment at 40° and 50°C. Kinetic parameters, K_m and k_cat, of native enzyme, Co²⁺-activated CPB and heat-treated CPB have been determined     

Evaluation of Beauveria bassiana and B. brongniartii strains and four wild-type fungal species against adults of Bactrocera oleae and Ceratitis capitata

The virulence of two isolates of the hyphomycete fungi, Beauveria bassianaand B. brongniartii, and additional fungal species isolated from diseased Bactrocera oleae pupae and Sesamia nonagrioideslarvae were assessed against adults of the olive fruit fly B. oleae and the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae). Contact and oral bioassays revealed that moderate to high mortality rates for the olive fruit fly occurred when the adults were exposed to conidia of Mucor hiemalis, Penicillium aurantiogriseum, P. chrysogenum and B. bassianaisolates. A strain of M. hiemalis isolated from S. nonagrioides larvae was the most toxic resulting in 85.2% mortality to the olive fruit fly adults. B. brongniartiiand B. bassiana were the most pathogenic to the C. capitataadults causing 97.4 and 85.6% mortality. Metabolites collected from the M. hiemalis and P. chrysogenum isolates were toxic to adults of both species.     

黑曲霉产木聚糖酶的分离纯化与鉴定研究

木聚糖酶的分离纯化是对其进行酶学研究和分子改良研究的基础。利用实验室选育的黑曲霉菌株Aspergillus niger SM24/a进行木聚糖酶发酵,粗酶液经过(NH_4)_2SO_4分级沉淀Bio-Gel P6除盐、UNO sphere Q阴离子交换和Enrich SEC70凝胶色谱层析四个步骤的分离纯化,成功获得了3种木聚糖酶蛋白定义为X-Ⅰ、X-Ⅱ和X-Ⅲ。随着纯化步骤的增加,各组分酶比活力得到显著提高,其数值分别为37.41、34.56和53.96 U/mg,纯化倍数分别为3.96、3.66和5.72。经质谱分析和蛋白氨基酸序列比对,初步认定X-Ⅰ属于糖基水解酶第十家族内切-β-1,4-木聚糖酶,X-Ⅱ和X-Ⅲ均属于糖基水解酶第十一家族木聚糖酶。     

Purification and properties of the xylanases from the termite Macrotermes bellicosus and its symbiotic fungus Termitomyces sp.

Four xylanases were purified, two from the termite Macrotermes bellicosus workers (X1T and X2T) and two from its symbiotic fungus Termitomyces sp. (X1Mc and X2Mc). The analysis of the step required for the purification of X1T and X1Mc and the comparison of their different properties suggested that xylanases X1T and X1Mc were the same enzyme, X1. The determination of the reducing sugars by TLC revealed that X1 was an endoxylanase (EC 3.2.1.8) and X2T and X2Mc were exoxylanases (EC 3.2.1.37). The apparent molecular weights of the three xylanases, determined by SDS-polyacrylamide gel electrophoresis, were 36 kDa for X1, 56 kDa for X2T and 22.5 kDa for X2Mc. The optimal pH of the three xylanases was 5.5, and K_m values determined with birchwood xylan as substrate were 0.2% for X1, 0.1% for X2T and 0.3% for X2Mc, showing a high affinity for this substrate. The three enzymes differed also by their thermal stability.