暗纹东方TUN 同工酶生化表现型的研究

利用聚丙烯酰胺凝胶垂直平板电泳方法,对暗纹东方|TUN|的心、肝、肾、肌、性腺5种不同组织的7种同工酶(EST、LDH、POD、MDH、SOD、SDH、α-AMY)进行了研究, 讨论了各同工酶的基因表达谱式,观察到EST同工酶存在着多态现象;LDH同工酶有二个基因位点,但只表现3条带,A与B亚基的结合受阻; MDH同工酶存在性别差异,说明决定MDH同工酶表达的因素在不同性别中存在差异;SOD同工酶有3个基因位点。各同工酶酶谱稳定,有组织特异性,但EST、MDH、SOD、POD同工酶在各组织器官中又表现出较大的一致性,有利于物种的鉴定。α-AMY与SDH只在个别组织中有活性,可能与特定组织与器官的形态发生与机能分化有关。 Abstract:By means of polyacrylamid gel electrophoresis seven isozymes(EST.LDH,SOD,MDH,SDH, α-AMY)in heart,liver,kidney,muscle and gonad of Fugu obscurus were studied.The gene expression patterns of each isozyme were analyzed.The results indicated that polymorphism was detected in EST isozymes;LDH isozymes had two loci,but only three bands could be observed,the random association of two subunits(A and B)were restricted;Some MDH isozymes existed sexual differences in identical tissues.The suggested that the factors controlling the expression of MDH isozymes were different between sexes.All the isozyme phenotypes exhibited tissue-specificity and stability,but EST,MDH,SOD and POD isozymes showed relatively consistence in the five tissues.Their characteristic bands could be used in species determination.The activities of SDH andα-AMY isozymes could only be detected in some tissues and closely correlated to the morphological or functional differentiation of those tissues or organs.     

西方蜜蜂四个亚种酯酶同工酶型和苹果酸脱氢酶Ⅱ同工酶基因型的遗传差异

The esterase (EST) and the malate dehydrogenase Ⅱ (MDHⅡ) isozymes in A. mellifera carpatica (C), A. mellifera ssp.(D), “Zhejiang Agricultural University No.1” A. mellifera ligustica (Ea) and A. mellifera carnica (K), and the genotypes of MDHⅡ isozymes in four subspecies of A. mellifera were analysed by IEF PAGE. The results indicate that the four subspecies of A. mellifera showed the same EST zymogram, while A. cerana showed a different EST zymogram, which suggests that the genotypes of EST isozymes in A. mellifera are different from those in A. cerana. The genotypes of the MDHⅡ isozymes in four subspecies of A. mellifera were aa, ab, ac, bb, bc and cc. The C and Ea subspecies exhibited high homozyosity, while D and K displayed high heterozyosity. The allele b appeared to be the highest frequency in C, while the allele c had the highest frequency in Ea. The frequencies of the alleles a, b and c in sub species D were very similar. The alleles a and c were common, while allele b was rare in sub species K. There were highly significant differences in genotype frequencies, allele frequencies, homozyosity and heterozyosity among these four subspecies.     

Secretory/releasing proteome-based identification of plasma biomarkers in HBV-associated hepatocellular carcinoma

For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins. The enriched molecular functions and biological processes of the CM proteins, such as hydrolase activity and catabolic processes, were consistent with the liver being the most important metabolic organ. Moreover, 19% of the proteins were characterized as extracellular or membrane-bound. For validation, secretory proteins involved in transforming growth factor-β signaling pathways were validated in plasma samples. Alphafetoprotein (AFP), metalloproteinase (MMP)1, osteopontin (OPN), and pregnancy-specific beta-1-glycoprotein (PSG)9 were significantly increased in HCC patients. The overall performance of MMP1 and OPN in the diagnosis of HCC remained greater than that of AFP. In addition, this study represents the first report of MMP1 as a biomarker with a higher sensitivity and specificity than AFP. Thus, this study provides a valuable resource of the HCC secretome with the potential to investigate serological biomarkers. MMP1 and OPN could be used as novel biomarkers for the early detection of HCC and to improve the sensitivity of biomarkers compared with AFP.     

Nuclear proteome profile of C57BL/6J mouse liver

The liver proteome can serve as a reference to better understand both disease mechanisms and possible therapeutics,since the liver is an important organ in the body that performs a large number of tasks.Here we identify the organelle proteome of C57BL/6J mouse liver nuclei as a promising strategy to enrich low abundance proteins,in the sense that analysis of whole liver cells is rather complex for current techniques and may not be suitable for proteins with low abundance.Evaluation of nucleus integrity and purity was performed to demonstrate the effectiveness of the optimized isolation procedure.The extracted nuclear proteins were identified by 2-DE MS analyses,and a total of 748 proteins were identified.Bioinformatic analyses were performed to demonstrate the physicochemical properties,cellular locations and functions of the proteins.     

大鳞副泥鳅LDH同工酶的发育遗传学研究

采用聚丙烯酰胺凝胶电泳技术研究了大鳞副泥鳅早期发育过程(受精后0-227小时)和成体7种组织器官(脑、肾、骨骼肌、肠、心、肝和眼)中的LD H同工酶表达状况。结果表明,大鳞副泥鳅的LDH同工酶具有明显的组织特异性和发育阶段特异性。 Abstract:The pattern of expression of LDH isozymes was investigated in seven different adult tissues(brain,kidney,muscle,intestine,heart,liver and eye)and carly developmental stages of paramisgurnus dabryanus by polyacrylamide gel clectrophoresis.The results showed that the LDH isozymes of Paramisgurnus dabryanus exhibited a tissue-speeific distribution and an ontogenetic change clearly.     

Vegetative Storage Protein with Trypsin Inhibitor Activity Occurs in Sapindus mukorassi,a Sapindaceae Deciduous Tree

A vegetative storage protein （VSP） with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions： nitrogen storage and defense.     

Clinical application of specific antibody against glypican-3 for hepatocellular carcinoma diagnosis

Glypican-3(GPC3) is a promising tumor marker for hepatocellular carcinoma(HCC) diagnosis with high sensitivity and specificity.The aim of this study was to establish an immunohistochemical detection method for GPC3 using the 7D11 monoclonal antibody(7D11 mAb) and evaluate its application for HCC diagnosis.The feasibility of the 7D11 mAb was evaluated by immunohistochemistry performed on adjacent normal liver and intrahepatic cholangiocarcinoma(ICC) samples,Furthermore,the serum GPC3 levels were evaluated in 40 HCC patients,7 ICC patients and 50 healthy donors.The results showed that GPC3 was expressed in 85% of HCC tissues(34/40),but was undetectable in ICC tissues and adjacent normal tissues.GPC3 was significantly increased in the serum of HCC patients(17/40,42.5%) but was undetectable in the serum of ICC patients(0/7,0%) and healthy donors(0/50,0%).This prospective study evaluated the clinical usefulness of 7D11 mAb for GPC3 detection in HCC patients.In conclusion,the use of 7D11 mAb might be good for GPC3 large-scale applications for clinical diagnosis of HCC.     

鲇的消化能力与营养价值分析

采用常规方法测定了鲇(Silurusasotus)的消化道指数,其比肠长、比胃重、比肠重、比肝胰脏重和比内脏重分别为0726±0087、0027±0015、0013±0008、0022±0004和0081±0041。体重与体长的回归方程Y=00124X2.8451(r=09940,P<001);鲇的胃、前肠、中肠和后肠中消化液的pH值分别为48～62、66～70、69～75、72～77,且消化道各部分具有较高的蛋白酶和淀粉酶活性,蛋白酶的活性为前肠>胰脏>胃>中肠>后肠;而淀粉酶的活性则为胰脏>胃>前肠>中肠>后肠;肌肉中粗蛋白的含量1881%,粗脂肪含量152%;必需氨基酸含量占氨基酸总量的4451%,鲜味氨基酸含量占氨基酸总量的4598%,综合肌肉的常规营养成分和氨基酸分析的结果,鲇是一种营养价值较高的优质鱼类。     

兰州鲇染色体组型

本研究的目的在于认识兰州鲇(Silurus lanzhouensis)染色体组特征,为兰州鲇的细胞遗传学和人工育种研究提供基础参考资料。以兰州鲇培养肾细胞为材料,采用空气干燥法制备染色体玻片标本,分析了兰州鲇的染色体组型。兰州鲇的染色体数2n=58,核型公式为2n=20m+18sm+16st+4t,染色体总臂数(NF)为96。通过比较分析认为,兰州鲇在鲇属鱼类中属于较为原始的类群。     

Reproductive investment in the Silurus meridionalis

A comparison of pre- and postspawning Silurus meridionalis showed that 20·7% of body stored energy was utilized during spawning for a standard male (74·5 cm) and 23·8% for a standard female (85·3 cm). About one-third of the loss of the stored energy was released as eggs by females, and almost all of the energy loss for males and about two-thirds for females were expended in metabolism. Stored lipid as fuel for metabolism supplied 90·0% of energy in males and 95·2% in females, and protein supplied the rest of the energy. Models for predicting energy in released gametes ( G _g), deposited in the body as somatic growth ( G _s), utilized in spawning activity ( S _a), expended in maintenance ( M , including metabolism, faeces and excretion), and food energy ( C ) were developed, and annual energy budgets were compiled. The balanced budget for a male aged 4 was: 100 C =0·06 G _g+11·17 S _a+19·5 G _s+69·2 M , and for a female aged 5: 100 C =5·48 G _g+8·51 S _a+15·8 G _s+70·2 M .     

南方鲇幼鱼的热耐受特征

为了研究不同驯化温度对南方鲇（Silurus meridionalis Chen）幼鱼热耐受性特征的影响,在水温为10 ℃、20 ℃和30 ℃的条件下,对南方鲇幼鱼（（16.9±0.3）g）进行2周的驯化,以1 ℃·h^-1的变温速率连续观测实验鱼的耐受温度,各驯化温度下的最大临界温度（CT_max）、最大致死温度（LT_max）、最小临界温度（CT_min）和最小致死温度(LT_min）分别为3413 ℃、3822 ℃、39.41 ℃;34.84 ℃、38.63 ℃、39.53 ℃;4.88 ℃、5.90 ℃、9.80 ℃;4.12 ℃、5.03 ℃、8.29 ℃。结果表明：最大临界温度和最大致死温度随驯化温度的升高而增大,最小临界温度和最小致死温度随驯化温度的降低而减小;经计算,南方鲇幼鱼在各驯化温度下的温度耐受幅分别为29.25 ℃、32.32 ℃和29.61 ℃;在10 ℃～20 ℃的驯化温度范围内高、低温驯化反应率分别为0.41和0.12,而在20 ℃～30 ℃驯化温度范围内高、低温驯化反应率分别为0.10和0.39;热耐受区域面积为617.5 ℃²;证明南方鲇幼鱼的热耐受性明显有赖于驯化温度。     

Scatter-spawning of the catfish,Silurus asotus

Reproductive behaviour of the catfish,Silurus asotus was studied in temporary waters around paddy fields. Spawning occurred nocturnally during the first week from the initiation of irrigation. In reproductive activities, a male first energetically pursued a female with its head near to the female’s belly (chasing) and then began to cling to the female’s body from the side, bending its tail or head (clinging). Finally the male enfolded the female’s body, with its anus near to the female’s (enfolding). In some cases, 2–4 males pursued a single female and two males enfolded a female at the same time. Although no aggressive behaviour was evident between males, it was always the largest male that could most frequently approach and enfold the female. The mating pair moved a long distance in a ditch, paddy field and/or creek, performing reproductive activities. It is thought that the spawning site and period of spawning of the fish enable the larvae to avoid the danger of predation and to efficiently feed, firstly on plankton and later on larvae of other fishes which become abundant during the irrigation period. Although some eggs and larvae may die due to the drying out or high water temperatures of such unstable temporary waters, scattering eggs may reduce the incidence of the annihilation of the young.     

南方鲇

南方鲇是我国特有的大型肉食性名优鱼类。具有个体大,生长快,抗病力强等特点。其形态特征、生活习性、繁殖与生长、人工养殖等都具有自身的特殊性,也是一种很好的实验研究材料。     

鲶鱼染色体的显带研究

对鲶鱼染色进行Ａｇ－ＮＯＲｓ、Ｃ－带，ＣＭＡ３／ＤＡ／ＤＡＰＩ三重荧光染色昨帛 带的研究。结果发现，鲶鱼Ａｇ－ＮＯＲｓ定位在ｍ１ｑ的末端，上有一种特殊的形态和数目多态现象，即染色体上的ＮＯＲｓ发生串联重复。该种鱼只有部分染色体呈现阳性Ｃ带，，所显示的异染色质可分为三类，有丝粒异染色质、端粒异染色质和居间异染色质，其中ｍ１的整佧长臂都被深染，是Ｃ－带染色最深、染色面积最大的区域。ＣＭＡ３染色显示，Ｎ     

Development of microsatellite markers for southern catfish (Silurus meriaionalis) and cross‐species amplification in northern sheatfish (Silurus soldatovi)

Forty microsatellite markers were developed from a (CA)_n enrichment library created from the DNA of southern catfish (Silurus meriaionalis). Also, the population structure of northern sheatfish (Silurus soldatovi) was examined by 24 microsatellite loci. They are polymorphic in at least one of the two geographically distant populations sampled from the Heilongjiang River and the Songhuajiang River in North China, respectively. Unbiased expected heterozygosity levels varied from 0.435 to 0.946 and number of alleles per locus varied from three to 20. Results indicated that these microsatellite loci were highly polymorphic and could be used as genetic markers.     

Chromosomal and morphological abnormalities caused by oocyte ageing in Silurus glanis

The effects of ageing oocytes for 6 h before fertilization in the European catfish Silurus glanis are compared with the same experiment under normal conditions (stripping and fertilization immediately after ovulation). Fertilization rate and hatching rate are lower in the aged groups, while the percentages of malformed larvae and chromosomal abnormalities are significantly higher. Several kinds of abnormalities are described in detail.