tsRNA在急性心肌梗死早期诊断中的价值

目的: 转运tRNA衍生片段（tRNA-derived small RNA,tsRNA）是近期发现的一类来源于tRNA的新型小RNA,尽管其研究尚处于起步阶段,但其在多种肿瘤疾病和神经系统疾病中的重要调控作用已引起研究者的关注。但tsRNA在心血管疾病尤其是心梗发生发展中是否存在差异表达及其功能尚完全不清楚。本文通过tiRNA&tRFs测序初步探索tsRNA表达谱在心梗前后的差异,并通过后续实验验证了tsRNA在急性心肌梗死早期诊断中的价值及其作为急性心梗诊断标志物的潜能。方法:选用8-10周龄c57雄性小鼠构建急性心梗模型,24小时后取左室心肌组织提取RNA,经去修饰预处理,连接人工化接头后进行反转扩增,筛选对应于15～40nt长度的小RNA大小范围的扩增产物构建文库进行测序,测序结果与GtRNAdb数据库成熟的tRNA和tRNA前体序列进行比对,获得在心肌中心梗前后发生表达差异的tsRNA谱。生物信息学分析同一密码子对应的tRNA在心梗前后剪切方式的改变。依据tsRNA心梗前后表达谱差异,获得在心梗后特异性高丰度表达的tsRNA,并在心梗心肌和血浆中进行验证,探索tsRNA作为心梗诊断标志物的潜能。结果：tsRNA表达谱在组内具有较好的重复性,组间具有较大的区分性。在心梗前后多种转运tRNA包括tRNA Asn-GTT、Glu-TTC、Gly-ACC、Gly-GCC、His-GTG、Ile-AAT、Ile-GAT、Pro-TGG、Ser-AGA和Trp-CCA在心肌中剪切方式发生改变,生成具有明显差异的tsRNA表达谱。统计分析发现与Sham组相比,心梗组有268个tsRNA表达显著上调,1228个tsRNA表达发生下调,且有35个tsRNA特异性存在心梗组。进一步通过验证发现,tRF-Gly-CCC-2-31、tiRNA-Val-CAC-1-32、tiRNA-Val-AAC-2-32、tiRNA-Glu-TTC-2-32、和tiRNA-Lys-TTT-1-34均在心梗1天心肌中存在特异性表达,且其中tiRNA-Val-AAC-2-32和tiRNA-Lys-TTT-1-34在心梗小鼠血浆中呈特异性高丰度存在,并随心梗时间呈动态变化。结论：tsRNA表达谱在心梗前后发生显著差异,特异性表达在小鼠心梗心肌和血浆的tiRNA-Val-AAC-2-32和tiRNA-Lys-TTT-1-34具有心梗诊断标志物潜能,可能在心梗修复过程中发挥着重要作用。 关键词：转运tRNA衍生片段、急性心梗、生物信息学、诊断标志物     

PRODUCTION 1N PICHIA PA STORIS AND CHARACTERIZATION OF GENETIC ENGINEERED CHIMERIC HBV／HEV VIRUS-LIKE PARTICLES

Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV)on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). Methods The gene fi-agment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in flame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichiapastoris under the control of a methanol-induced alcohol oxidase 1 (A OXI) promoter and expressed intraceUularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCI density gradient analysis, and electron microscopic visualization. Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichiapastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological, physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg. Conclusion The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.     

Assignment and conformation of neurotensin in aqueous solution by 1H NMR

A complete assignment of exchangeable and unexchangeable proton resonances of neurotensin 1–13 in aqueous solution has been carried out with the help of its 1–8 and 8–13 fragments. To detect formation of a secondary structure, the effects of peptide fragmentation, temperature decrease, pH changes and addition of denaturing agents on the neurotensin ¹H NMR spectrum were investigated. The small changes observed in all cases support the conclusion that neurotensin exists mainly as a flexible random coiled polypeptidic chain in aqueous solution in agreement with previous CD studies.     

Multiple forms of pituitary prolactin,a glycosylated form of porcine prolactin with enhanced biological activity

Three forms of prolactin differing in molecular weight (M_r 23 000, 25 000 and 50 000, respectively) and electrophoretic mobility have been isolated and purified from fresh-frozen porcine pituitary glands. The prolactin form with M_r 25 000 is identified as a glycoprotein having an affinity to concanavalin A. The carbohydrate unit containing GlcN Ac₃, GalNAc₁, Man₃, Fuc₀₅, Gal₀₄ is linked to asparagine at the 31 position. The amino acid composition and partial primary structure of the glycosylated prolactin are identical to those of the major prolactin form (M_r 23 000). Based on isoelectric focusing and non-denaturing disc-electrophoresis, the glycosylated prolactin appears more acidic than the major form. The glycosylated form of the hormone has 140% of the activity of the non-glycosylated prolactin when measured by the pigeon crop sac assay. The new form accounts for 30–40% of the total monomeric prolactin in the porcine pituitary. The yield was 200 mg purified glycosylated prolactin from 1000g pituitary gland. The third form of hormone (M_r 50 000) was shown to be a disulphide linkéed prolactin dimer.     

Monoclonal antibody A7 tumor localization enhancement by its F(ab')2 fragments to colon carcinoma xenografts in nude mice

The monoclonal antibody A7 (MoAb A7), which belongs to IgG1, was digested with pepsin to yield F(ab')2 fragments. The maximum binding to the human colon cancer cell line, SW1116, was 27% with 125-I labeled whole MoAb A7 and 24% with 125-I labeled F(ab')2 fragments using an in vitro binding assay. The results showed that the binding activity of F(ab')2 to SW1116 was practically the same as that of whole MoAb A7. The preferred localization of the fragments to tumor tissue, compared with normal mouse tissue, was demonstrated in mice carrying SW1116 xenografts. The tumor:blood ratio three days after injection was 2.64:18.5 for whole MoAb A7:F(ab')2, respectively. The tissue:blood ratios for the F(ab')2 fragments showed a value of 18.5 in tumors, whereas its was a value less than 1.0 in normal organs. The tumor accumulation of F(ab')2 fragments was also dependent on the antigenic expression of each tumor among xenografts of colon carcinoma SW1116 and WiDr, and squamous cell carcinoma KB. In kinetic experiments with whole MoAb A7 and its F(ab')2 fragments, whole MoAb A7 was lost, with a half-life of 4 days, in both blood and tumors, whereas F(ab')2 fragments were rapidly lost with a half-life of 1.5 days. These results suggested that the F(ab')2 fragments were cleared from the blood faster than was whole MoAb A7.     

苣荬菜、苦苣菜和苦荬菜茎叶中脂肪酸含量分析

目的:分析苣荬菜、苦苣菜和苦荬菜茎叶中脂肪酸成分及含量.方法:采用气相色谱/质谱(碎片质谱法),以改良的脂肪酸甲酯化法制样,测定苣荬菜、苦苣菜和苦荬菜茎叶中脂肪酸含量.结果:从苣荬菜、苦苣菜和苦荬菜茎叶中分别检出10、9和9种脂肪酸,其中不饱和脂肪酸分别为4、3和2种,总脂肪酸含量分别为88.64 %、93.67 %和86.21 %,三者都以亚油酸含量最高.结论:此研究结果为苣荬菜、苦苣菜和苦荬菜的药用提供科学依据.     

Vasostatins,N-terminal products of chromogranin A,are released from the stimulated calf spleen in vitro

Vasostatins are the N-terminal chromogranin A peptides 7 ? 22 kDa. They have been shown to be present in several endocrine tissues and exhibit vasoinhibitory activity in vitro. In a first series of experiments, we investigated the presence and subcellular localization of vasostatins in the bovine splenic nerve. Experimental results, obtained using gradient centrifugation, showed that noradrenaline was enriched 25-fold in the large dense core vesicle fraction, compared with the original homogenate. In the latter fraction, the 7 and 18 kDa peptides were observed following immunodetection with antiserum to chromogranin A_{1, 4o} and laser densitometric scanning revealed these two fragments as the major N-terminal fragments. Subsequently, we examined the release of the 7 and 18 kDa peptides from perfused calf spleen during veratridine (20 μM) or 1,1-dimethyl-4-phenylpiperazinium iodide (20 μM) stimulation. In the prestimulatio samples, we were not able to detect these peptides, however, following stimulation, the 7 and 18 kDa chromogranin A fragments became apparent. The vasostatin-immunoreactivity, in both bovine chromaffin granule lysate and calf spleen perfusate, elutes at the same retention time on reversed-phase high performance liquid chromatography. The present study demonstrated that vasostatins are present in the large dense core vesicles of sympathetic axons and are released from the nerve terminals in response to stimulation. The release of vasostatins from sympathetic nerves in the spleen suggest an in vivo function for N-terminal chromogranin A products of neuronal origin.     

Intracerebroventricular galanin and N-terminal galanin fragment enhance the morphine-induced analgesia in the rat

Summary Behavioral effect of galanin and its fragments, galanin_1–15 and galanin_16–29 (200 ng, 1 and 5 g), after intracerebroventricular (i.c.v.) administration was studied in rats. The number of crossings and pippings and the time of locomotion (an open field test) showed a similar sedative action of galanin and galanin_16–29, with no significant effect of galanin_1–15. Galanin and its fragments, injected in doses of 200 ng, 1 and 5 g, did not affect nociception, as measured by a tail-flick and paw pressure test. Galanin and galanin_1–15, but not galanin_16–29 (5 g i.c.v.), injected together with morphine (2.5 g i.c.v.), significantly potentiated the analgetic effect of morphine assessed by a paw pressure test; a similar tendency was also observed in a tail-flick test. Galanin and its two fragments injected in doses of 200 ng, 1 and 5 g, did not change the effect of morphine given in a dose of 1 g. These data suggest that galanin, having no effect when given alone, potentiate the analgetic effect of morphine. The fact that the N-terminal fragment of galanin acts like a natural peptide suggests a receptor mediated action.In conclusion, the analgesic effect of morphine was potentiated by galanin and its N-terminal fragment galanin_1–15. On the other hand, behavioral study showed a similar sedative action of galanin and C-terminal fragment galanin_16–29. This suggests that the N- and C-terminal fragments of galanin are differentially involved in behavioral effects of the peptide.