小鼠胎脑组织活体转基因技术的建立

目的:建立实用的小鼠活体脑组织基因转染技术。方法:将EGFP质粒(CAGS启动子)注射到胎龄16 d(E16d)的胎鼠侧脑室,用镊形电极隔着子宫壁夹住胎鼠头部,在45 V电压下给予5次电脉冲刺激,每次刺激50ms,间隔1 s;转染后不同天数将胎鼠脑组织完整取出,以4%PFA固定后冰冻切片,进行激光共聚焦照相。结果:EGFP质粒被转入小鼠活体脑组织细胞中并获得表达,动物存活率为90%,GFP阳性率高于80%。结论:通过对麻醉剂、电脉冲刺激、质粒浓度、术中术后处理等多种实验条件的摸索,建立了实用的小鼠胎脑组织活体转基因技术。     

Cloning and Expression of a Chitinase Gene from Sanguibacter sp. C4

The chitinase Chi58 is an extracellular chitinase produced by Sanguibacter sp.strain C4. The gene-specific PCR primers were used to detect the presence of the chiA gene in strain C4. A chiA fragment (chiA-F) was amplified from the C4 genomic DNA and was used to blast-search the related sequences from the GenBank dadabase. By alignment and selection of the highly conserved regions of the homologous sequences, two pairs of primers were designed to amplify the open reading frame (ORF) of the chitinase from strain C4 by nested PCR. The results revealed that the Chi58 ORF consisted of 1 692 nucleotides encoding a protein of 563 amino acid residues. The molecular weight of the mature protein was predicted to be 58.544 kDa. The Chi58 ORF was a modular enzyme composed of a signal peptide sequence, a polycystic kidney disease I domain, and a glycosyl hydrolase family 18 domain. The chitinase of C4 exhibited a high level of similarity to the chitinase A of Serratia (88.9%-99.6%) at the amino acid sequence level. The Chi58 gene was cloned into the expression vector pET32a to construct the recombinant plasmid pChi58 and was expressed in E. coli BL-21 (DE3) cells with IPTG induction. The molecular weight of the Trx-Chi58 fusion protein was estimated to be 81.1 kDa by SDS-PAGE.     

Effect of arginine vasopressin in the nucleus raphe magnus on antinociception in the rat

Previous work has shown that arginine vasopressin (AVP) regulates antinociception through brain nuclei rather than the spinal cord and peripheral organs. The present study investigated the nociceptive effect of AVP in the nucleus raphe magnus (NRM) of the rat. Microinjection of AVP into the NRM increased pain threshold in a dose-dependent manner, while local administration of AVP-receptor antagonist-d(CH2)5Tyr(Et)DAVP decreased the pain threshold. Pain stimulation elevated AVP concentration in the NRM perfuse liquid. NRM pretreatment with AVP-receptor antagonist completely reversed AVP's effect on pain threshold in the NRM. The data suggest that AVP in the NRM is involved in antinociception.     

白粉藤的木脂素和三萜成分

从白粉藤（Cissus repens Lank）地上部分分离得到5个木脂素和8个三萜,其中一个木脂素是新化合物,它的结构通过波谱分析和碱水解的方法鉴定为：（＋）-异落叶松树脂醇-9′-（2-对-香豆酰）-O-β-D-吡喃木糖苷（1）。其余化合物分别是：（＋）-异落叶松树脂醇-9′-O-β-D-吡喃木糖苷（2）,（＋）-Lyoniside（3）,（—）-开环异落叶松树脂醇-9-O-β-D-吡喃木糖苷（4）,（7′R,8′S）-4′-hydroxy-3′,5-dimethoxy-7,8′-dihydrobenzofuran-1-propanolneolignan-9′-O-β-D-xylopyranoside（5）,木栓酮（6）,表木栓醇（7）,蒲公英赛醇乙酸酯（8）,熊果酸（9）,2α-羟基乌索酸（10）,积雪草酸（11）,Niga-ichigoside F1（12）,羽扇豆醇（13）。这些化合物都是首次从该植物中分离得到。     

A systematic analysis of disease-associated variants in the 3′ regulatory regions of human protein-coding genes I: general principles and overview

The 3' regulatory regions (3' RRs) of human genes play an important role in regulating mRNA 3' end formation, stability/degradation, nuclear export, subcellular localization and translation and are consequently rich in regulatory elements. Although 3' RRs contain only approximately 0.2% of known disease-associated mutations, this is likely to represent a rather conservative estimate of their actual prevalence. In an attempt to catalogue 3' RR-mediated disease and also to gain a greater understanding of the functional role of regulatory elements within 3' RRs, we have performed a systematic analysis of disease-associated 3' RR variants; 121 3' RR variants in 94 human genes were collated. These included 17 mutations in the upstream core polyadenylation signal sequence (UCPAS), 81 in the upstream sequence (USS) between the translational termination codon and the UCPAS, 6 in the left arm of the 'spacer' sequence (LAS) between the UCPAS and the pre-mRNA cleavage site (CS), 3 in the right arm of the 'spacer' sequence (RAS) or downstream core polyadenylation signal sequence (DCPAS) and 7 in the downstream sequence (DSS) of the 3'-flanking region, with 7 further mutations being treated as isolated examples. All the UCPAS mutations and the rather unusual cases of the DMPK, SCA8, FCMD and GLA mutations exert a significant effect on the mRNA phenotype and are usually associated with monogenic disease. By contrast, most of the remaining variants are polymorphisms that exert a comparatively minor influence on mRNA expression, but which may nevertheless predispose to or otherwise modify complex clinical phenotypes. Considerable efforts have been made to validate/elucidate the mechanisms through which the 3' untranslated region (3' UTR) variants affect gene expression. It is hoped that the integrative approach employed here in the study of naturally occurring variants of actual or potential pathological significance will serve to complement ongoing efforts to identify all functional regulatory elements in the human genome.     

Expression of recombinant kringle 1-5 domains of human plasminogen by a prokaryote expression system

Kringle1-5 (K1-5), a proteolytic fragment containing five kringle domains of human plasminogen generated by plasmin-mediated proteolysis, has been already identified by Cao et al. with relation to anti-angiogenesis and proliferation of endothelial cells. To investigate anti-angiogenesis activity of recombinant human K1-5 (rhK1-5) expressed in Escherichia coli BL21, the cDNA of human K1-5 obtained from cloning vector pUC57-K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E. coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody and an anti-hexahistidine tag antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed, purified, and refolded to a purity of 96% as estimated by capillary electrophoresis and the final purification yield of K1-5 in E. coli system was approximately 5.8 mg/L. Purified K1-5 protein was tested on chicken embryo chorioallantoic membranes (CAMs), and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, the rhK1-5 potently inhibited endothelial cell proliferation but not non-endothelial cells. For the first time, these findings demonstrate that the rhK1-5 produced by a prokaryote expression system effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells. This fact derived from the present study further suggests the rhK1-5 can be used for anti-angiogenesis therapy of cancer.     

昆明地区鲜切花产区朱砂叶螨抗药性监测

采用玻片浸渍法测定了昆明地区花卉朱砂叶螨Tetranychus cinnabarinus(Boisduval)对阿维菌素、甲氨基阿维菌素苯甲酸盐、溴虫腈、丁醚脲、炔螨特和哒螨灵的抗性。结果表明,昆明北郊和呈贡地区玫瑰上的朱砂叶螨雌成螨对阿维菌素与甲氨基阿维菌素苯甲酸盐产生了极高的抗药性,阿维菌素对2个地区的朱砂叶螨的LC50分别为40.25mg·L-1和19.67mg·L-1,相对毒力指数分别为敏感品系的2441.08倍和1192.86倍;甲氨基阿维菌素苯甲酸盐对其LC50分别为118.18mg·L-1和9.24mg·L-1,相对毒力指数是敏感品系的2805.73倍和219.35倍。昆明北郊的朱砂叶螨对溴虫腈的相对毒力指数是敏感品系的2371.40倍,呈贡和晋宁分别为162.01倍和173.38倍。丁醚脲对北郊、呈贡和晋宁朱砂叶螨的LC50分别为244.58mg·L-1、385.41mg·L-1和54.93mg·L-1,相对毒力指数在3.01倍～21.10倍之间。北郊、呈贡和晋宁的朱砂叶螨种群对炔螨特和哒螨灵的LC50分别为155.39mg·L-1、424.49mg·L-1和62.70mg·L-1,其相对毒力指数是敏感品系的6.45倍、17.63倍和2.60倍。朱砂叶螨对药剂抗药性水平趋势从高到低为:阿维菌素、甲氨基阿维菌素苯甲酸盐>溴虫腈、丁醚脲>炔螨特、哒螨灵,抗性最高的地区为昆明北郊,晋宁相对较低。     

Monitoring pesticide resistance of Tetranychus cinnabarinus in Kunming' s flower regions

采用玻片浸渍法测定了昆明地区花卉朱砂叶螨Tetranychus cinnabarinus (Boisduval)对阿维菌素、甲氨基阿维菌素苯甲酸盐、溴虫腈、丁醚脲、炔螨特和哒螨灵的抗性.结果表明,昆明北郊和呈贡地区玫瑰上的朱砂叶螨雌成螨对阿维菌素与甲氨基阿维菌素苯甲酸盐产生了极高的抗药性,阿维菌素对2个地区的朱砂叶螨的LC50分别为40.25 mg·L -1和19.67 mg·L-1,相对毒力指数分别为敏感品系的2 441.08倍和1 192.86倍;甲氨基阿维菌素苯甲酸盐对其LC50分别为118.18 mg·L-1和9.24 mg·L-1,相对毒力指数是敏感品系的2 805.73倍和219.35倍.昆明北郊的朱砂叶螨对溴虫腈的相对毒力指数是敏感品系的2 371.40倍,呈贡和晋宁分别为162.01倍和173.38倍.丁醚脲对北郊、呈贡和晋宁朱砂叶螨的LC50分别为244.58 mg·L-1、385.41 mg·L-1和54.93 mg·L-1,相对毒力指数在3.01倍～21.10倍之间.北郊、呈贡和晋宁的朱砂叶螨种群对炔螨特和哒螨灵的LC50分别为155.39 mg·L-1、424.49 mg·L-1和62.70 mg·L-1,其相对毒力指数是敏感品系的6.45倍、17.63倍和2.60倍.朱砂叶螨对药剂抗药性水平趋势从高到低为:阿维菌素、甲氨基阿维菌素苯甲酸盐＞溴虫腈、丁醚脲＞炔螨特、哒螨灵,抗性最高的地区为昆明北郊,晋宁相对较低.     

粪便类固醇激素检测准确性的影响因素

近年来,采用非损伤性取样方法监测野生动物的生理状况越来越受到重视。本文对检测过程中影响动物粪便类固醇激素检测准确性的因素进行了分析总结,包括样品的新鲜程度、样品量和保存方法;激素代谢的日节律和季节性变化;动物的年龄、性别和繁殖状态等,以期为粪便类固醇激素检测技术在野生动物中的准确应用提供借鉴。     

Paraoxonase 1 polymorphisms L55M and Q192R were not risk factors for Parkinson's disease: a HuGE review and meta-analysis

Purpose

The Paraoxonase 1 (PON1) has been studied as a potential candidate gene for Parkinson's disease risk, but direct evidence from genetic association studies remains inconclusive. We performed a meta-analysis pooling data from all relevant studies in order to determine the effects of two PON 1 polymorphisms (L55M and Q192R) on Parkinson's disease.

Methods

We applied a random effects to combine odds ratio (OR) and 95% confidence intervals. Q statistic was used to evaluate the homogeneity, and Egger's test and Funnel plot were used to assess publication bias. In secondary analyses, we examined dominant and recessive models as well.

Results

Concerning the PON1 L55M polymorphism, we identified 9 eligible studies (a total of 2582 cases and 3997 controls). The random effects pooled OR was OR = 1.29, (0.90, 1.84). Concerning the Q192R polymorphism, we identified 7 eligible studies (a total of 2582 cases and 3997 controls). The random effects pooled OR was OR = 1.08(0.81, 1.43). Analysis with dominant and recessive genetic models yielded the same inferences as genotype-based comparisons for both of the two polymorphisms.

Conclusion

The results of this meta-analysis suggested that both PON1 L55M and Q192R were not responsible for PD.