Induction of hepatic cytochrome P4501A1/2B activity and disruption of thyroglobulin synthesis/secretion by mono-ortho polychlorinated biphenyl and its hydroxylated metabolites in rat cell lines

As the active metabolites of polychlorinated biphenyl (PCBs), hydroxylated polychlorinated biphenyls (OH-PCBs) are found in wildlife and human tissues. They have been proposed as main contributors for endocrine disruption of PCBs in living organisms. In this study, mono-ortho PCB 156 and its hydroxylated metabolites 4'-OH-PCB 159, 4'-OH-PCB 121, and 4'-OH-PCB 72 were selected to investigate the toxic effects on rat hepatoma H4IIE cell line and rat thyroid follicle FRTL-5 cell line at concentrations of 1, 10(2), 10(4) nM. 7-Ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) activities were determined with micro-EROD/PROD to indicate cytochrome P4501 A1 (CYP1A1) and cytochrome P4502B (CYP2B) induction in the H4IIE cell after exposure for 72 h. To assess thyroid disruption of these compounds, thyroglobulin concentrations also were detected inside FRTL-5 cell with immunocellularchemistry and in its medium with radioimmunoassay after exposure for 24 It. Significant inductions of EROD activity by PCB 156 at 102 and 104 nM (p < 0.05) were observed, but no effects by the three OH-PCBs in H4IIE cell line. 7-Pentoxyresorufin-O-dealkylase activities were induced only by 10(4) nM of PCB156 and the three OH-PCBs (p < 0.05). Meanwhile, significant increases of thyroglobulin concentrations were observed in the medium of FRTL-5 cell exposed to 4'-OH-PCB 121 and 4'-OH-PCB 72 at all of the test concentrations (p < 0.05), but not to the other compounds. The results demonstrated that mono-ortho PCBs mainly could be metabolized to hydroxylated metabolites through CYP1A1 instead of CYP2B. Moreover, after being metabolized, OH-PCBs still sustained the ability to induce PROD activity and did exhibit the disruption on thyroglobulin synthesis/excretion in rat cells.

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and co-immunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31-137 of EseC are required for EseC-EscA interaction, Mutation of EseC residues 31-137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31-137 of EseC increased the LD50 by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.

EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD

Edwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the 'translocon' of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by COILS. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of Delta escC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB-LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.

T型分叉流槽中前列环素和内皮素分泌的研究

动脉粥样硬化（atherosclerosis）的非随机分布与当地的血流动力环境有关，借助平行平板式平直流槽和以T型分叉流槽为代表的平行平板式异型流槽，可以模拟血管的主要形状特征，首,先在数值模拟的基础上分布了流型特征参数，确定了流的设计尺寸。然后，通过实验研究，探讨流型改变对内皮细胞血管活性物质分泌的影响，发现扩张效应流线偏转和驻点效应使得异型流槽前列环素和内定素的沁泌水平与相同入口雷诺数（Re）条件下的平直流槽分别有降低趋势和显著差异。为进一步研究流型对血管内皮细胞的影响提供了实验数据。

二脂酰甘油酰基转移酶基因(DGAT1)的沉默对烟草油脂合成的影响

二脂酰甘油酰基转移酶 (DGAT; EC 2.3.1.20) 是催化三脂酰甘油（TAG）合成的最后也是最关键步骤的酶。TAG是真核细胞中最重要的能量存储形式。在植物中，TAG主要在种子、花粉和许多物种的果实中积累。然而，DGAT1基因的转录本也存在于植物的其它器官中，这些器官包括根、茎、叶、花瓣、花粉囊、未成熟的角果、幼苗以及正在发芽的种子等。迄今为止，许多针对DGAT1基因的研究都集中于DGAT1基因的表达在对种子油脂的积累以及对种子中TAG的脂肪酸组成所起的作用上。在本研究中，我们通过构建烟草DGAT1基因带有内含子的发卡RNA（hpRNA）结构，使之在转基因烟草植株中表达双链RNA（dsRNA），利用RNAi原理达到使烟草内源DGAT1基因沉默的目的。转基因沉默烟草植株的获得将会为更好地研究DGAT1基因的功能奠定基础。本实验不仅研究分析了DGAT1基因的抑制对烟草种子油脂积累的影响，还对表现出沉默性状的转基因植株Sil7的不同器官中TAG的含量以及DGAT1的转录水平等进行了研究分析。此外，通过对转基因烟草不同株系种子中的主要贮藏物质——油脂、蛋白质和糖的含量测定，初步揭示出在烟草种子中三者生物合成代谢之间存在的相关性。主要研究结果如下： 采用烟草DGAT1基因的第615～1293碱基之间679bp的片段构建了能表达发卡RNA（hpRNA）结构的表达载体，并转化烟草（Nicotiana tabacum）Wisconsin 38。Northern杂交分析发现,与野生型对照烟草（WT）相比，在沉默植株的花和发育状态种子中DGAT1基因的转录水平有很大降低，这表明该发卡结构能够高效率地引起烟草DGAT1基因的沉默。此外，在对Sil1至Sil12共12株转基因烟草进行油脂含量分析的结果表明，其中有8株表现出油脂降低的性状，转基因沉默效率达到67％。这表明：利用RNAi的方法可对目标基因进行特异降解来研究基因的功能，因此是一个在研究基因的表达功能上十分有效的方法，而且已成为植物基因工程的有力工具。 为了研究DGAT1基因的沉默对转基因植株不同器官的影响，本实验分析了转基因植株Sil7的不同器官中TAG的含量和脂肪酸组成，并采用RT-PCR方法对野生型对照和转基因植株中DGAT1基因的转录水平进行了比较分析。研究发现，转基因植株不同器官中DGAT1基因转录水平的降低与各器官中TAG含量的减少呈正相关。由此看来，植物中DGAT1的表达水平与植物的各个器官内TAG的含量之间存在着一定的对应关系。此外，在转基因植株Sil7不同器官中依然能够产生TAG，这说明或者DGAT1酶活性丧失而由其它的酶（如DGAT2和PDAT）参与TAG的合成，或者DGAT1酶活性只是部分地受到影响。本实验还对Sil7的根、茎、叶、花瓣和种子中TAG的脂肪酸组成进行了分析，结果发现，与烟草野生型对照相比，在Sil7的这些器官中，除种子中TAG的脂肪酸组成无明显变化外，其余器官中TAG的18:3/18:2脂肪酸比例均有明显升高。 对其中8株转基因烟草种子进行油脂含量分析发现，在转基因烟草中由于DGAT1基因的沉默引起种子中TAG含量的减少，从而引起了种子平均千粒重的下降。而在TAG含量和种子平均千粒重下降的同时，种子中其它贮藏物质－蛋白质和糖类的含量却增加了。该实验结果表明：在烟草种子中TAG的生物合成与蛋白质和糖类物质的合成之间存在着负的相关性。

两栖类动物皮肤分泌物及其生物学适应意义--大蹼铃蟾皮肤分泌物蛋白质多肽组的启示

以大蹼铃蟾(Bombina maxima)为代表性物种,揭示了两栖类皮肤分泌物蛋白质多肽组丰富的分子和功能多样性.目前在大蹼铃蟾已发现的分子包括3类不同的抗菌肽、缓激肽、缓激肽增强肽和缓激肽拮抗肽、缓激肽基因相关肽、富含脯氨酸铃蟾肽及其基因相关肽、神经调节素U、Bv8肽、三叶型蛋白和蛋白酶抑制剂等.抗菌肽分子多样性及其形成机制、缓激肽及其基因相关肽功能和表达模式的多样性都较好地揭示了在多样的生态条件下,两栖类皮肤活性肽环境适应的分子基础及生物合成的调控机制.富含血红素辅基的白蛋白广泛分布在大蹼铃蟾皮肤外皮层细胞膜上和真皮海绵层内,表明它在皮肤的生理功能,如在呼吸、物质交换和渗透压调节中有重要作用.两栖类皮肤分泌物蛋白质多肽组,由于其分子结构的多样性、新颖性和哺乳类同系物的存在,在生物医学研究和天然药物开发中具有独特和不可替代的价值;同时,两栖类皮肤功能基因组具有多样性丰富、快速重组突变的特征,是探讨生物适应的基因基础、基因形成机制和进化特性等生物学基本问题的优秀模型.

A small trypsin inhibitor from the frog of Odorrana grahami

A novel peptide inhibitor (OGTI) of serine protease with a molecular weight of 1949.8, was purified from the skin secretion of the frog, Odorrana grahami. Of the tested serine proteases, OGTI only inhibited the hydrolysis activity of trypsin on synthetic chromogenic substrate. This precursor deduced from the cDNA sequence is composed of 70 amino acid residues. The mature OGTI contains 17 amino acid residues including a six-residue loop disulfided by two half-cysteines (AVNIPFKVHFRCKAAFC). In addition to its unique six-residue loop, the overall structure and precursor of OGTI are different from those of other serine protease inhibitors. It is also one of the smallest serine protease inhibitors ever found. (C) 2008 Elsevier Masson SAS. All rights reserved.

An immunoregulatory peptide from salivary glands of the horsefly, Hybomitra atriperoides

Horseflies are economically important blood-feeding arthropods and also a nuisance for humans, and vectors for filariasis. They rely heavily on the pharmacological propriety of their saliva to get blood meat and suppress immune reactions of hosts. Little information is available on horsefly immune suppressants. By high-performance liquid chromatography (HPLC) purification coupling with pharmacological testing, an immunoregulatory peptide named immunoregulin HA has been identified and characterized from salivary glands of the horsefly of Hybomitra atriperoides (Diptera, Tabanidae). Immunoregulin HA could inhibit the secretion of interferon-gamma (IFN-gamma) and monocyte chemoattractant protein (MCP-1) and increase the secretion of interteukin-10 (IL-10) induced by lipopolysaccharide (LIPS) in rat splenocytes. IL-10 is a suppressor cytokine of T-cell proliferative and cytokine responses. IL-10 can inhibit the elaboration of pro-inflammatory cytokines. Immunoregulin HA possibly unregulated the IL-10 production to inhibit IFN-gamma and MCP-1 secretion in the current experiments. This immunosuppression may facilitate the blood feeding of this horsefly. The current works will facilitate to understand the molecular mechanisms of the ectoparasite-host relationship. 2008 Elsevier Ltd. All rights reserved.