Volume regulation in glutathione-treated brook trout (Salvelinus fontinalis) erythrocytes

Brook trout erythrocytes that were washed with and suspended in Ringer's solution with reduced glutathione (1.0 mM) maintained steady state cell volume for up to 24h, while those without the thiol-protective agent steadily shrank. Changes in cell volume (measured as packed cell volume, PCV) were evoked by acidic media (Ringer's at pH 6.8), hypoosmotic solutions (or both) and intracellular K⁺ and Cl⁻ concentrations were monitored over 4h. Acid-swollen cells failed to volume regulate or release K⁺ but had significantly elevated intracellular Cl⁻ Osmotically-swollen cells at pH 7.8 but not at pH 6.8 underwent regulatory volume decrease (RVD) and returned to initial levels in 2h, accompanied by release of K⁺ and Cl⁻ In contrast, osmotically-shrunken cells did not show regulatory volume increase. The regulatory volume decrease and concomitant K⁺ release were dependent on Cl⁻ implying a direct or indirect coupling of K⁺ to Cl⁻ transport in volume regulation. RVD was partially blocked by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 0.1 mM), an anion exchange blocker, but was unaffected by amiloride (1.0 mM) which blocks Na⁺/H⁺ exchange. Amiloride and DIDS prevented the swelling response to low pH but had no effect on control cells, suggesting involvement of Na⁺/H⁺ and Cl⁻/HCO₃ ⁻ exchanges in acid-induced cell swelling. Quinine (1.0 mM) a known blocker of K⁺ channels, exacerbated the osmotically-induced swelling but had little effect on the subsequent RVD and release of KCl. The results suggest that low extracellular pH inhibits neutral C⁻-dependent K⁺ release and the resultant regulatory volume decrease in osmotically-swollen cells.     

斑节对虾细胞周期蛋白B慢病毒载体的构建及对Hela细胞增殖影响

细胞周期蛋白B（Cyclin B）是影响卵母细胞发育成熟的重要基因。文章构建了PmCyB慢病毒表达载体,并和慢病毒包装复合物在293T细胞中包装成慢病毒颗粒。利用包装好的慢病毒颗粒,感染干扰了Cyclin B1基因的Hela细胞,研究了PmCyB对Hela细胞增殖的影响。结果表明,通过siRNA干扰细胞自身的Cyclin B1后,转染对照组病毒液的Hela细胞增殖减缓;同时,干扰后转入含有PmCyB重组慢病毒表达载体,Hela细胞增殖速度明显要高于转染对照组病毒液的Hela细胞的增殖速度。这一结果表明,PmCyB基因的过表达能补偿Cyclin B1缺失导致的细胞增殖减缓的现象,说明PmCyB基因具有细胞周期调控功能,是细胞增殖与分化的重要调控基因,且功能可能与人类的Cyclin B1相似。人类的Cyclin B1是卵母细胞发育成熟的重要基因。由此可以推断,PmCyB基因可能对斑节对虾（Penaeus monodon）卵巢发育成熟起着重要作用。     

贝类配子细胞识别物的初步研究

利用薄层层析法快速检测了 11种经济贝类配子细胞所含的糖和脂肪酸的种类和活性。研究结果表明 ,各种贝精子和卵细胞含糖种类很相似 ,一般为 3～ 4种 5碳和 6碳糖 ;但不同贝类具体含糖种类和多少有差别 ,同一种类精子和卵细胞也有不同之处。脂肪酸种类差别明显 ,通常贝卵细胞比精子多一种到几种。这充分说明各种贝既有遗传上的相似性 ,又有不同种的特异性     

不同因子对花鲈胚胎干细胞增殖的影响

胚胎干细胞(ES细胞)是从动物早期发育胚胎中分离出来的具有发育全能性的一种未分化细胞系。维持花鲈胚胎干细胞(LJES1)的体外生长、增殖及未分化状态需要在培养基中添加一些生长因子。实验通过配制省略1种或多种因子的培养基PESM0～10，计数LJES1细胞在各培养基中增殖的数量，确定各种生长因子对LJES1细胞的增殖作用。同时，对LIF因子和bFGF因子的作用进行了重点的研究，发现LIF因子对早期的LJES1细胞增殖几乎没有作用，其主要作用是维持LJES1细胞的未分化状态，但对晚期LJES1细胞的增殖有一定的作用；bFGF因子对LJES1细胞有强烈的刺激增殖的作用；鲈鱼胚胎抽提液(PEE)以及鲈鱼血清(FS)也促进了LJES1细胞的增殖，2-巯基乙醇(2-ME)具有还原血清中的含硫化合物、防止过氧化物对LJES1细胞的损害及促进贴壁的作用，因而也促进了LJES1细胞的增殖。     

红鲫(♀)×鲤(♂)杂交鱼的胚胎染色体组倍性研究

红鲫属于鲤科、鲤亚科、鲫属,染色体数目为2n=100,核型为22m+34sm+22st+22t;鲤属于鲤科、鲤亚科、鲤属,染色体数目为2n=100,核型为22m+34sm+22st+22t。已有研究表明,红鲫(♀)×鲤(♂)杂交第一代(F₁)和第二代(F₂)为二倍体,F₂能产生染色体数不减数的配子,在第三代(F₃)中形成四倍体鱼。通对F₁和F₃进行胚胎染色体制片,在染色体水平上探讨红鲫和鲤远缘杂交的多倍体发生途径及F₂产生不减数配子的潜能。结果表明,F₁混合胚胎都为二倍性胚胎,没有发现单倍性和多倍性胚胎,但F₃混合胚胎中则有染色体数为100,150,200甚至300的染色体分裂相,推测F₂生殖细胞发生了一次或多次染色体数加倍,产生了二倍性和更高倍性的配子,它们结合形成了不同倍性的F₃胚胎。实验证明双亲基因组大小及核型相似的鲫鲤远缘杂交第一代不发生多倍化,但获得的二倍体杂交后代能产生不减数配子,在F₃中产生多倍体鱼。     

草鱼肾细胞中细胞色素P450 3A基因诱导表达及其酶活性分析

采用基因克隆技术获取草鱼(Ctenopharyngodon idellus)细胞色素P450 3A(CYP3A)cDNA序列长1 849 bp,包含完整开放阅读框(open reading frame,ORF)1 542 bp,1个终止密码和含polyA信号3'UTR; ORF编码514个氨基酸,含信号肽(29 aa),2个跨膜螺旋区(23 aa,23 aa),血红素结合域(21 aa)和6个底物识别位点(SRSl-6);与其他脊椎动物CYP3A氨基酸序列相似度达60％ ～ 92％.用实时定量PCR和分光光度计法分别研究了草鱼肾细胞系(Ctenopharyngodon idellus kidney cell line CIK)中利福平(rifampicin,RIF)诱导CYP3A基因表达和红霉素-N-脱甲基酶(ERND)活性,采用红霉素脱甲基酶活性法测定CYP3A的活性.结果显示,诱导组CIK细胞中CYP3A mRNA表达量在8最高,而CYP3A酶活在10h最高;CYP3A mRNA和酶活性与对照组均具显著差异性（P<0.05）.CYP3A转录水平先于相应酶活变化表达,且转录水平表达显著高于酶活性,这很可能反映了RIF对CYP3A的诱导主要作用于转录水平而不直接作用于酶活,本研究旨为药物对CYP3A转录调控和活性调节提供科学依据,并为渔药代谢酶的研究提供理论依据.     

长吻鮠人工催产及脑垂体促性腺激素细胞的超微结构的变化

通过对催产和未催产的长吻鮠脑垂体中腺垂体促性腺激素分泌细胞（ＧＴＨ 细胞） 的分泌活动分析,证实了用促黄体素释放激素类似物（ＬＲＨ－ Ａ） ５０μｇｋｇ 加ＤＯＭ５ｍｇｋｇ 混合注射催产长吻鮠,能有效地促使ＧＴＨ细胞分泌促性腺激素,诱导卵母细胞成熟和排卵,催产效果显著。超微结构的进一步观察,揭示了长吻鮠脑垂体ＧＴＨ 细胞中存在两种分泌颗粒,即分泌小球,直径１２００～２０００ｎｍ ,电子密度低;分泌颗粒直径３００ ～５００ｎｍ ,电子密度高。分泌小球释放与卵母细胞的发育成熟有关,分泌颗粒的释放则与排卵相关。     

Characterization of the pattern of cytokeratin proteins in the epidermal cells of loach,Misgurnus anguillicaudatus (Cyprinformes)

The pattern of cytokeratin proteins in the epidermal cells of loach was studied by immunotechniques and partial separation of the epidermal cells. Two monoclonal antibodies, namely 8F7 and 1C45, against the cytokeratin proteins of the loach epidermis were prepared. these two monoclonal antibodies exhibit distinctive results in immunohistochemical staining. The 8F7 monoclonal antibody stains mainly with the epithelial cells, while the 1C45 monoclonal antibody stains specifically with the club cells. The pattern of cytokeratin proteins in the club cells and the epithelial cells of various epidermal layers was further determined by partial separation of these cells. Immunoblotting analysis of the cell fractions confirms the cytokeratin proteins to be differentially expressed in the club cells and the epithelial cells. However, the cytokeratin proteins expressed in the epithelial cells of the basal, middle and outer layers are same. The results indicate that differentiation of the epithelial cells seems limited during their translocation from basal to upper layers, but in those cells that do differentiate into club cells, the cytokeratin pattern changes.     

Aquatic birnavirus induces apoptosis through activated caspase-8 and -3 in a zebrafish cell line

In this study, the possible influence of temperature on infectious pancreatic necrosis virus (IPNV)-induced apoptosis in a zebrafish liver epithelium (ZLE) cell line was investigated. At a lower temperature (18 degrees C), there was expression of viral proteins VP2 and VP3 at 4 h post-infection (p.i.). At this time no expression was found in the high temperature group at 28 degrees C. The cell survival ratio was 52 and 18% at 24 and 48 h p.i., respectively, during IPNV infection at 18 degrees C. In addition, we assayed for apoptosis in IPNV-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different dosages of virus. We found a ratio of apoptotic cells of 8 and 25% at 12 and 18 h p.i., respectively, in the multiplicity of infection (MOI) 1 group. The MOI 10 group had 20 and 45% apoptotic cells at 12 and 18 h, respectively. Furthermore, at 18 degrees C IPNV activated the caspase-8 and 3 from 1.5 to 2 times at 12 and 18 h p.i., respectively. Taken together, these findings suggest that successful virus replication occurs at the low temperature (18 degrees C) compared with the non-permissive temperature of 28 degrees C. Thus, IPNV replication is capable of activating caspase-8 and -3 and inducing host apoptosis.