氯霉素在牙鲆体内的药代动力学及残留消除规律研究

采用高效液相色谱法测定牙鲆体内氯霉素的药物浓度,非房室模型统计矩原理分析药动学数据.结果表明:1)单次口服剂量为80 mg·kg-1的氯霉素,药物在牙鲆体内的药-时曲线呈明显双峰现象:第一次达峰时间Tmax(1)出现在2.00 h,鳃、肝、肾、血、肌的第一次达峰浓度Cmax(1)依次为15.01、11.80、10.35、8.56、5.21μg·mL-1;第二次达峰浓度Cmax(2)小于第一次的浓度Cmax(1),第二次达峰的时间Tmax(2)出现在8.00 h,药-时曲线下面积(AUC):肾、鳃、肝、血、肌分别为176.87、133.77、118.77、65.33、50.36μg·mL-1·h.消除半衰期(T1/2)为4.89-10.39 h,平均滞留时间(MRT)为8.67-17.05 h,说明氯霉素在牙鲆体内吸收较迅速,但滞留时间较长.2)连续5 d口服剂量为40 mg·kg-1的氯霉素,药物消除半衰期(T1/2)为39.40-115.50 h,说明口服氯霉素在牙鲆体内消除缓慢,残留较严重,其中以肾脏和肝脏组织中残留最明显.     

以mRNA差异显示法分离牙鲆白化相关基因的初步研究

以正常和白化牙鲆表皮组织总RNA为模板,用Oligo d(T)13M(M分别为A、C、G)3种锚定引物和26种差异显示随机引物组合进行差异显示,PAGE凝胶电泳后,用新型高灵敏度核酸荧光染料SYBR GREEN I显示差异DNA条带,得到区分度较好的差异表达图谱,共分离49条牙鲆白化相关DNA片段,片段长度260～800bp左右,经克隆测序后将可作为新的ESTs进行同源序列比较.     

不同饵料系列对漠斑牙鲆苗种培育效果的影响

报道了不同饵料系列对漠斑牙鲆苗种成活率和生长率的影响.32.0万粒的受精卵经57 d培育,共获得全长2.7～5.5 cm的鱼苗16.6万尾.在仔鱼开口后自始至终加投配合饵料的饵料系列组(B组)的成活率比在35日龄才开始驯化配合饵料的饵料系列组(A组)的成活率高.初孵仔鱼至变态伏底时A、B组的成活率分别为58.9%和88.7%,从变态伏底始至出苗时全长平均为3.84 cm的A、B组幼鱼的成活率分别为79.0%和83.6%,从初孵仔鱼至出苗时的A、B组的总成活率分别为46.5%和72.4%,而两组的生长率差异不大.     

溶解氧水平对褐牙鲆幼鱼能量代谢和氧化应激的影响研究

【目的】查明溶解氧含量下降对褐牙鲆(Paralichthys olivaceus)幼鱼能量代谢及氧化应激的影响。【方法】测定褐牙鲆幼鱼在溶解氧由7.4mg/L下降至0.9mg/L过程中耗氧率、排氨率、氧氮比以及氧化应激指标的变化。【结果】随着溶解氧含量(DO)降低,褐牙鲆幼鱼耗氧率呈"U"型变化,氧氮比呈倒"U"型变化,褐牙鲆幼鱼肌肉中丙二醛(MDA)含量增加,尽管肌肉和肝脏中过氧化物歧化酶(SOD)酶活性有增强的趋势,但由于体内产生过多的活性氧自由基,使得脂质过氧化反应增强,脂质过氧化物增多,机体仍表现出氧化损伤。【结论】褐牙鲆幼鱼的能量代谢指标和部分氧化应激指标对溶解氧含量下降有明显反应,可作为判断溶解氧含量是否对褐牙鲆产生胁迫及调控养殖水体溶解氧含量的依据。     

Occurrence, Development and Histochemical Characteristics of Lymphocystis in Cultured Japanese Flounder （Paralichthys olivaceus）

Occurrence and development of lymphocystis cells in flounder Paralichthys olivaceus, and some his-tochemical features are studied using light and electron microscopy and histochemical methods. Fibrob-lasts engulf virus particles forming phagocytosis vacuole in the cytoplasm. The infected cells proliferate and turn round and hypertrophied; basophilic inclusion bodies, Feulgen-positive, are present in the cyto-plasm, and a hyaline capsule, containing acid ploysaccharide and protein, appears outside cell mem-brane. The mature lymphocystis cells possess numerous virus particles (200～220nm in diameter with a central core 120～140nm) and high electron-dense inclusions with budding virious at the surface in the cytoplasm, and show margination of chromatin. Moderate electron density particles (70～80nm in diame-ter) fill out perinuclear cistema and arrange in crystalloid arrays in nucleus, liberated into the cytoplasm by rupture of nuclear membrane. No virus particles are observed in nucleus. Therefore, inclusion bodies are the sites of virus assembly and maturity, while replication processes of lymphocystis virus are involved in both nuclear and cytoplasmic phases. Besides, strongly basophilic link-like structures are present be-tween adjacent cells or through connective tissue, and virus particles are observed in somewhere of the capsule, thus where may be the spread route of virus from one cell to another or to the environment. The senile lymphocystis cells release virus bv the runture of cells.     

Isolation and characterization of a new species of Vibrio-Vibrio olivaceus sp.nov. from diseased flounder （Paralichthys olivaceus temminck et schlegel）

One kind of pathogenic bacteria was isolated from diseased (or dead) flounder (Paralichthys olivaceus L.) which expressed bacterial septicaemia. The phenotyic information of the 10 pure cultures was studied extensively, including morphological characteristics, colony characteristics in different media, physiological and biochemical characteristics, and the mol%G C ratio of the DNA for representative strain. The results showed that the isolates belonged to a new species of Vibrio. In addition, the representative strains have been re-checked by China Center for Type Culture Collection (CCTCC), the results were the same as ours, the examined strains were also regarded as a new species of Vibrio, and designated as Vibrio olivaceus sp.nov. by its isolated fish (Paralichthys olivaceus) based on its biological properties following Rules of International Code of Nomenclature of Bacteria. In the same time, we conducted studies on the serum homology of isolates, pathogenicity of isolates by experimental infection. The results showed that all the isolates were of serologic similarity and the isolates have strong pathogenicity to flounder. Inspection to pure cultures and isolates from liver of moribund fish from infection experiment by fluorescent antibody technique showed the feasibility of fluorescent antibody technique as an auxiliary method of examination.     

牙鲆fibrinogen β基因的克隆及表达分析

 通过mRNA差异显示发现一个鳗弧菌刺激后在牙鲆肝脏中表达量显著增加的片段,结合RACE技术得到了该差异片段1 856 bp的全长cDNA,包含一个1 479 bp的开放阅读框,编码的蛋白质含有fibrinogen β的C末端签名序列。同源性分析表明其氨基酸序列与鱼类以及哺乳类的fibrinogen β都具有高度的相似性,因此可断定此基因为牙鲆的fibrinogen β基因(GenBank登录号为EF581895)。RT-PCR分析表明,在注射鳗弧菌后,该基因在肝脏、肾脏、脾脏、腮、肠、心脏中的表达量呈现逐渐增加的趋势。推测其可能在鳗弧菌侵染中,起到了促进伤口愈合的作用,或通过和其他细胞因子结合在防御细菌的侵染中发挥免疫调节作用。     

Subcellular localization and inductive expression of the dsRNA-dependent protein kinase PKR from Japanese flounder, Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) belongs to the eIF2α kinase family and plays a critical role in interferon (IFN)-mediated antiviral response. Recently, in Japanese flounder (Paralichthys olivaceus), a PKR gene has been identified. In this study, we showed that PoPKR localized to the cytoplasm, and the dsRNA-binding motifs (dsRBMs) played a determinative role in protein localization. In cultured FEC cells, PoPKR was detected at a low level of constitutive expression but was highly induced after treatment with UV-inactivated grass carp hemorrhagic virus, active SMRV and Poly I:C although with different expression kinetics. In flounder, PoPKR was ubiquitously distributed in all tested tissues, and SMRV infection resulted in significant upregulation at mRNA and protein levels. In order to reveal the role of PoPKR in host antiviral response, its expression upon exposure to various inducers was characterized and further compared with that of PoHRI, which is another eIF2α kinase of flounder. Interestingly, expression comparison revealed that all inducers stimulated upregulation of PoHRI in cultured flounder embryonic cells and fish, with a similar kinetics to PoPKR but to a less extent. These results suggest that, during antiviral immune response, both flounder eIF2α kinases might play similar roles and that PoPKR is the predominant kinase.     

牙鲆幼鱼营养需要的研究

在实验条件下,当饲料中蛋白质含量为52.78 %、脂肪含量8.78 %、碳水化合物含量15.80 %时,牙鲆增重率最高;在饲料中不含EPA和DHA时,牙鲆幼鱼生长缓慢,肝体指数升高,当EPA和DHA在饲料中含0.5 %和1.0 %时,牙鲆生长最好;EPA和DHA的比率对于鱼体生长和脂肪酸的组成影响较大,摄入EPA/DHA比率为1/2的饲料,鱼的增重率都显著高于其他各组,饲料中脂肪酸的组成对于鱼体肌肉和肝脏中极性和非极性脂肪酸组成都无显著影响,而且发现摄食不同饲料,鱼体肌肉和肝脏极性脂中EPA/DHA的比率都接近于1/2.牙鲆幼鱼饲料中,各微量元素的适宜需要量分别为铁374 mg/kg,锌119.2 mg/kg,锰19.24 mg/kg～29.15 mg/kg,铜5.62 mg/kg～7.24 mg/kg,钴2.20 mg/kg,硒1.94～2.19 mg/kg.对维生素的需要量为VA8780 IU/kg, VD 1880 IU, VE 186 mg/kg,VK 11.7 mg/kg, VB1 8.2 mg/kg,VB2 14.7 mg/kg,水溶性VB3 60 mg/kg,水不溶VB3 45 mg/kg,VB5 106 mg/kg,VB6 25.4 mg/kg,生物素1.91 mg/kg,叶酸9.4 mg/kg,VC(LAPP)400 mg/kg,肌醇800～1200 mg/kg,胆碱2000 mg/kg.      

亚非马蜂（Polistes olivaceus）粗毒的生物学活性

本文报道一种新的蜂毒采集方法，测定了亚非马蜂（Ｐｏｌｉｓｔｅｓ　ｏｌｉｖａｃｅｕｓ）粗毒的部分物理性状和生物学活性．该粗毒含有透明质酸酶、胆碱酯酶、蛋白水解酶、ＤＮＡ酶、酸性磷酸酶、碱性磷酸酶，并具有很强的溶血活性（其半溶血量ＨＵ_（５０）＝１７μｇ／ｍｌ），但肝素对其溶血作用有一定的抑制作用．其对小鼠ＬＤ_（５０）为５．５４ｍｇ／ｋｇ，对美洲蜚蠊ＬＤ_（５０）为７．４５μｇ／ｇ．药理学实验表明该粗毒对小鼠的膈神经－膈肌接头的神经冲动的传递以及膈肌的收缩都有不可逆的阻断作用．该粗毒能明显抑制蟾蜍离体心肌的收缩，但其抑制作用可被阿托品所解除。