Expression and immunogenicity analysis of two iron-regulated outer membrane proteins of Vibrio parahaemolyticus

Genes of two iron-regulated outer membrane proteins of Vibrio parahaemolyticus zj2003, a pathogenic strain isolated from large yellow croaker （Pseudosciaena crocea）, psuA and pvuA, were cloned and expressed as N-terminal His6-tagged proteins in Escherichia coli BL21（DE3）. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of the proteins, groups of large yellow croaker were immunized with the purified recombinant psuA, pvuA or both, by intraperitoneal injection. Antibody response was assessed by enzyme-linked immunosorbent assay. Titers to the recombinant proteins increased from log2 3.25 to log29.80, 4-8 weeks following immunization. The relative percent survival of the groups vaccinated with psuA, pvuA, or a combination of the two, reached 50%, 62.5% and 75%, respectively. Western blot analysis was carried out with the serum from unvaccinated survival fish after infection. Both recombinant proteins were detected, indicating that these two proteins of V. parahaemolyticus zj2003 were immunogenic and could produce synergistic effects during in vivo infection, and they might be considered as important components for developing an aquaculture vaccine against this pathogen.     

Cloning, expression and immunogenicty analysis of five outer membrane proteins of Vibrio parahaemolyticus zj2003

Genes of five outer membrane proteins of Vibrio parahaemolyticus zj2003, including OmpW, OmpV, OmpK, OmpU and TolC, were cloned and expressed as N-terminal His(6)-tagged proteins in Escherichia coli. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of these proteins, large yellow croaker (Pseudosciaena crocea) were immunized by intraperitoneal injection. Antibody response was assessed by method of enzyme-linked immunosorbent assay. Titres to all five recombinant proteins increased during 4 to 8 weeks post immunization, within the range of log 2 values of 5.75 to 10.8. Recorded relative survival percent (RPS) of the vaccinated groups varied from 80% to 90%, while 10 fish in control group all died. Western blot tests were undertaken with the serum of survival fish after experimental infection. Except for recombinant TolC, the other four recombinant proteins were recognized by the serum. It is indicated that four outer membrane proteins of V. parahaemolyticus zj2003, including OmpW, OmpV, OmpU and OmpK, are immunogenic during in vivo infection, which would be of some significance in developing efficient vaccine in aquaculture. This is the first report of successful vaccination against V. parahaemolyticus with purified recombinant outer membrane proteins.     

术中胆道镜诊治肝胆管结石的应用（附64例报告）

术中应用胆道镜治疗胆道疾病６４例,使残余结石发生率由１１％下降至２％左右,认为纤维胆道镜对明确诊断减少残余结石的发生有重要价值。     

漳州市蝶类名录初报

１９９７－１９９９年在福建漳州调查蝶类,共计１０科、１１０属、１７６种。     

福州地区对虾白斑病病毒的超微结构

在福州地区分离到一种高致病性的白斑病病毒 ,该病毒仅存在于细胞质中 ,完整的病毒粒子有囊膜 ,一端略圆 ,一端稍尖 ,直径约为 80～ 10 0nm ,囊膜与核衣壳之间的间隙约为 2 0～ 2 5nm。该病毒在细胞内不形成包含体 ,但有些可形成封入体。负染观察到的病毒核衣壳呈直杆状 ,但长度和直径相差较大 ,最长的病毒超过 6 0 0nm。这些特征与其它已报道的白斑病病毒有所不同 ,因此暂将它称为“对虾白斑病病毒福州分离株”。     

Development of triptolide-nanoemulsion gels for percutaneous administration: physicochemical,transport, pharmacokinetic and pharmacodynamic characteristics

Background

This work aimed to provide useful information on the use of nanoemulsions for the percutaneous administration of triptolide. Lipid nanosystems have great potential for transdermal drug delivery. Nanoemulsions and nanoemulsion gels were prepared to enhance percutaneous permeation. Microstructure and in vitro/in vivo percutaneous delivery characteristics of triptolide (TPL)-nanoemulsions and TPL-nanoemulsion gels were compared. The integrity of the nanoemulsions and nanoemulsion gels during transdermal delivery and its effects on the surface of skin were also investigated. The penetration mechanisms of nanoemulsions and nanoemulsion gels were investigated by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR). The transport characteristics of fluorescence-labelled nanoemulsions were probed using laser scanning confocal microscopy. A chronic dermatitis/eczema model in mice ears and the pharmacodynamic of the TPL-nanoemulsion gels were also investigated.

Results

Compared to TPL gels, significantly greater cumulative amounts of TPL-nanoemulsion gels and TPL-nanoemulsions penetrated rat skin in vitro. The in vivo microdialysis showed the concentration–time curve AUC_0–t for TPL-NPs is bigger than the TPL-gels. At the same time, TPL-NPs had a larger effect on the surface of skin. By hydrating keratin and changing the structure of both the stratum corneum lipids and keratin, nanoemulsions and nanoemulsion gels influence skin to promote percutaneous drug penetration. Both hairfollicles and the stratum corneum are also important in this transdermal drug delivery system. Moderate and high dosages of the TPL-nanoemulsion gels can significantly improve the symptoms of dermatitis/eczema inflammation and edema erythematic in mice ears and can reduce the expression of IFN-γ and IL-4. Moreover, the TPL-nanoemulsion gels cause less gastrointestinal damage than that of the Tripterygium wilfordii oral tablet does.

Conclusions

Nanoemulsions could be suitable for transdermal stably releasing drugs and maintaining the effective drug concentration. The TPL-nanoemulsion gels provided higher percutaneous amounts than other carriers did. These findings suggest that nanoemulsion gels could be promising percutaneous carriers for TPL. The TPL-nanoemulsion gels have a significant treatment effect on dermatitis/eczema in the mice model and is expected to provide a new, low-toxicity and long-term preparation for the clinical treatment of dermatitis/eczema in transdermal drug delivery systems.

     

核酸对肝纤维化大鼠肝组织中M1型胆碱能受体表达的影响

目的探讨核酸在CCl4诱导的肝纤维化模型中对肝脏中M1型乙酰胆碱受体的影响及其抗肝纤维化的可能机制。方法应用四氯化碳（CCl4）诱导大鼠慢性肝纤维化模型。将55只Wistar大鼠分为4组：正常对照（N）组10只、肝复乐胶囊对照（G）组15只、核酸（Z）组15只及模型（M）组15只。采用光镜观察肝组织病理学改变;运用免疫组织SP法、逆转录聚合酶链反应（RT-PCR）和Western-blot方法检测各组肝组织M1型胆碱能受体（M1-AChR）不同水平的表达情况。结果核酸组、肝复乐胶囊对照组病理改变较模型组明显改善;免疫组织化学、RT-PCR、Western-blot三种不同层次水平的表达结果均表明核酸组和肝复乐胶囊对照组M1-AChR的表达较模型组显著降低（P〈0．05）。结论核酸可降低肝纤维化肝组织中M1型乙酰胆碱受体的含量,可能主要通过调节肝纤维化大鼠肝脏中的M1型乙酰胆碱受体的含量而达到抗肝纤维化的作用。     

Endogenous HMGB1 contributes to ischemia-reperfusion-induced myocardial apoptosis by potentiating the effect of TNF-α/JNK

High-mobility group box 1 (HMGB1) is a nuclear protein that has been implicated in the myocardial inflammation and injury induced by ischemia-reperfusion (I/R). The purpose of the present study was to assess the role of HMGB1 in myocardial apoptosis induced by I/R. In vivo, myocardial I/R induced an increase in myocardial HMGB1 expression and apoptosis. Inhibition of HMGB1 (A-box) ameliorated the I/R-induced myocardial apoptosis. In vitro, isolated cardiac myocytes were challenged with anoxia-reoxygenation (A/R; in vitro correlate to I/R). A/R-challenged myocytes also generated HMGB1 and underwent apoptosis. Inhibition of HMGB1 attenuated the A/R-induced myocyte apoptosis. Exogenous HMGB1 had no effect on myocyte apoptosis. However, inhibition of HMGB1 attenuated myocyte TNF-α production after the A/R was challenged; surprisingly, HMGB1 itself did not induce myocyte TNF-α production. Exogenous TNF-α induced a moderate proapoptotic effect on the myocytes, an effect substantially potentiated by coadministration of HMGB1. It is generally accepted that apoptosis induced by TNF-α is regulated by the balance of activation of c-Jun NH(2)-terminal kinase (JNK) and NF-κB. Indeed, in the present study, TNF-α increased the phosphorylation status of JNK and p65, a subunit of NF-κB; HMGB1 greatly potentiated TNF-α-induced JNK phosphorylation. Furthermore, inhibition of JNK (SP-600125) prevented the myocyte apoptosis induced by a TNF-α/HMGB1 cocktail. Finally, A/R increased HMGB1 production in both wild-type and toll-like receptor 4-deficient myocytes; however, deficiency in toll-like receptor 4 diminished A/R-induced myocyte apoptosis, TNF-α, and JNK activation. Our results indicate that myocyte-derived HMGB1 and TNF-α work in concert to promote I/R-induced myocardial apoptosis through JNK activation.     

安西极旱荒漠国家级自然保护区重引入普氏野马繁殖成效与种群生存力分析

安西自然保护区放归的普氏野马是一个孤立封闭的小种群,种群数量增长极其缓慢,同时,面临着严峻的捕食压力和近交衰退。因此,对这个物种制定长期的自然保护计划是当务之急。本研究通过对重引入普氏野马种群繁殖力和种群生存力进行分析,结果表明：（1）安西保护区野马种群数量增长缓慢是由生育率（44.3%）低和幼驹死亡率（43.4%）高造成的,天敌捕食（35.3%）和疾病（26.5%）成为影响野马繁殖存活率的主要因素;普氏野马的繁殖具有明显的季节性特征,80.9%的幼驹出生在5月和6月;（2）种群生存力分析（PVA）预测本放归野马种群未来100年的灭绝概率是60%,野马种群数量达200匹以上才能保证100年后90%的存活率;（3）参数敏感度分析显示,外部因素--狼袭对保护区野马种群的灭绝和种群数量最具影响力,其次是决定生殖成功的内部因素--生育率、最大繁殖年龄;（4）保护区野马小种群效应明显,疾病和幼驹羸弱而死亡的比例高（分别为26.5%和8.8%）,合理的补充新个体是降低种群灭绝风险长期的管理对策。同时,减小狼对野马的捕食压力,加强幼驹的保护,是目前改善本区内普氏野马小种群生存状态的有效手段。     

重组人成骨生长肽的表达、纯化和活性的研究

将人工合成的人成骨生长肽基因 (hOGPgene) ,与质粒pTYB2 重组 ,转化大肠杆菌E .coliBL2 1 (DE3) .经异丙基硫代 β D 半乳糖苷 (IPTG)诱导 ,融合蛋白在E .coli中得到表达 .菌体经超声破碎 ,一步亲和层析纯化得到重组人成骨生长肽 (recombinanthumanosteogenicgrowthpeptide ,rhOGP) .体外实验证明 :rhOGP对成纤维细胞 (NIH3T3)的增殖有促进作用 .体内实验证明 :rhOGP加速兔移植骨成活 ,使血清中碱性磷酸酶活性增高 ,骨钙素增高 .重组人成骨生长肽促进成骨 ,有可能成为治疗骨移植及骨折愈合的有效药物