中国不同年代食品动物大肠杆菌耐药性调查研究

收集到20世纪70,80,90年代和2000年食品动物大肠杆菌共326株,对不同年代的大肠杆菌进行耐药性、整合子／基因盒测定,结果表明在过去的30多年里,食品动物大肠杆菌耐药性呈逐渐增长的趋势．从1970～2000年,对青霉素类的耐药率从19．0％增长到81．3％;对四环素类药的耐药率从61．9％～63．5％增长到89．5％～93．1％;对氨基糖甙类的耐药率从0-9．5％增长到5．0％～63．1％,对氟喹诺酮类的耐药率从0％增长到50.2％-53．0％;对磺胺类药的耐药率从11．7％～57．1％增长到77．7％～79．7％;对氯霉素的耐药率从39．7％增长到51．8％．整合子的携带率从20世纪70年代的17．5％增长到2000年的79．1％,其中大多数为I型整合子,也有少数Ⅱ型整合子（90年代为4．4％,2000年为11．8％）．在20世纪70和80年代,大多数I型整合子的基因盒编码aadAI基因,比例分别为72．7％和70．0％．20世纪90年代和2000年基因编码dfrA—aadA的占多数,分别为80．0％和74．7％．近4个世纪以来,细菌的多重耐药谱也逐渐增宽．本项研究给兽医从业者滥用抗菌药提出了警示,同时提出中国动物源细菌耐药性的监测和控制是必要的．     

微流控芯片监测绿色荧光蛋白在枯草芽孢杆菌中的表达

目前主要使用激光共聚焦扫描显微镜观察绿色荧光蛋白的表达,但需要昂贵的仪器并耗费大量时间。本研究开发了一种新型激光诱导的微流芯片检测系统来监测绿色荧光蛋白在枯草芽孢杆菌中的表达。该系统主要由激光装置、光路系统、微流控芯片、光电倍增管和计算机处理系统等5部分组成。对该系统的测试结果显示,随着诱导强度的增强监测信号峰也随之增强,并且与激光共聚焦显微镜观察的结果一致。利用该芯片系统能够快速准确地筛选和鉴定用绿色荧光蛋白作为标记的细胞克隆,可以替代PCR鉴定方法。但该系统仅仅能够监测表达强度,不能够满足蛋白定位等高水平研究,因此,该系统适合应用于环境的微生物监测、药物筛选和其他无需观察蛋白定位等研究。     

兼性肠球菌Enterococcus hirae AUH-HM195对黄豆苷原的开环转化

摘要：【目的】从褐马鸡粪样中分离对大豆异黄酮黄豆苷原具有转化作用的功能微生物菌株。【方法】在厌氧工作站内对褐马鸡新鲜粪样进行梯度稀释后涂板,从板上挑取单菌落与底物黄豆苷原厌氧混合培养,用高效液相色谱检测底物被转化情况。【结果】分离出一株对黄豆苷原具开环转化作用的革兰氏阳性兼性好氧菌株AUH-HM195（EU919863）,经BLAST比对,该菌株的16S rDNA基因全序与肠球菌属菌株Enterococcus hirae (DSM20160) 的相似性为100%。根据保留时间、代谢产物最大紫外吸图谱以及核     

Clear and independent associations of several HLA-DRB1 alleles with differential antibody responses to hepatitis B vaccination in youth

To confirm and refine associations of human leukocyte antigen (HLA) genotypes with variable antibody (Ab) responses to hepatitis B vaccination, we have analyzed 255 HIV-1 seropositive (HIV⁺) youth and 80 HIV-1 seronegatives (HIV^?) enrolled into prospective studies. In univariate analyses that focused on HLA-DRB1, -DQA1, and -DQB1 alleles and haplotypes, the DRB1*03 allele group and DRB1*0701 were negatively associated with the responder phenotype (serum Ab concentration ≥ 10 mIU/mL) (P = 0.026 and 0.043, respectively). Collectively, DRB1*03 and DRB1*0701 were found in 42 (53.8%) out of 78 non-responders (serum Ab <10 mIU/mL), 65 (40.6%) out of 160 medium responders (serum Ab 10–1,000 mIU/mL), and 27 (27.8%) out of 97 high responders (serum Ab >1,000 mIU/mL) (P < 0.001 for trend). Meanwhile, DRB1*08 was positively associated with the responder phenotype (P = 0.010), mostly due to DRB1*0804 (P = 0.008). These immunogenetic relationships were all independent of non-genetic factors, including HIV-1 infection status and immunodeficiency. Alternative analyses confined to HIV⁺ youth or Hispanic youth led to similar findings. In contrast, analyses of more than 80 non-coding, single nucleotide polymorphisms within and beyond the three HLA class II genes revealed no clear associations. Overall, several HLA-DRB1 alleles were major predictors of differential Ab responses to hepatitis B vaccination in youth, suggesting that T-helper cell-dependent pathways mediated through HLA class II antigen presentation are critical to effective immune response to recombinant vaccines.     

野化培训大熊猫采食和人为砍伐对拐棍竹无性系种群更新的影响

亚高山竹类占据着野生大熊猫(Ailuropoda melanoleuca)食物组分的99%,竹子的生命周期与大熊猫的生活史密切相关,竹子的更新和生长直接影响着大熊猫的生存与保护.为了弄清大熊猫的采食利用和人为砍伐是否促进或制约竹子的繁殖更新,应用样方法、定位观察法,连续研究了卧龙自然保护区大熊猫栖息地内野化培训大熊猫采食、人为砍伐和对照样方中拐棍竹无性系种群的更新动态.结果表明,在相同种群数量和环境条件的基础上(p>0.05),被大熊猫采食竹子的比例为67.07%,致死率29.07%;人为砍伐竹子的比例为65.67%,致死率46.68%,可见与大熊猫采食相比,砍伐更为影响拐棍竹种群的生存.从出笋数量来看,不同处理方式有利于拐棍竹无性系种群的更新(p<0.05),尤其是人为砍伐措施大大提高了竹子的出笋率,但人为砍伐样方的竹笋质量(地径和株高生长)却远低于大熊猫采食和对照样方,未能达到大熊猫觅食的选择利用标准.不同年份之间,各种处理方式下的拐棍竹出笋数量波动较大(p=0.006～0.035),并随着恢复时间的延长,逐渐趋于相似(2007,p=0 825).不同处理方式之间,拐棍竹无性系种群的年补充率,各年份均具有显著性差异(p<0.05,除2007年外),年死亡率仅2003年和2004年有明显的差异(p<0.05),2005～2007年均不显著(p>0.05).拐棍竹无性系种群的年补充率与年死亡率之间表现出年补充率﹥年死亡率的格局(p<0.05),唯有大熊猫采食样方的2004年和2005年、人为砍伐和对照样方的2005年的年死亡率略高于年补充率(p>0.05),这表明拐棍竹无性系种群对大熊猫采食和人为砍伐具有无性系整合的补偿效应.     

中国蛇类保护和养殖及展望

蛇类是中国一类重要的野生动物资源,对其利用由来已久,然而随着环境变化和社会发展,致使蛇类野生资源急剧下降,但从另一方面也促进了对蛇类人工驯养繁育的发展和探索。作者总结了中国蛇类资源概况、保护法律法规和措施以及上世纪80年代兴起的蛇类养殖及技术简况。     

Assessment of antifungal effects of a novel compound from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> against <Emphasis Type="Italic">Fusarium solani</Emphasis> by fluorescent staining

A novel compound CF66I produced by Burkholeria cepacia was investigated for its antifungal effects against Fusarium solani by three different fluorescent dyes. Dual staining with propidium iodide (PI) and fluorescein diacetate (FDA) demonstrated high doses of CF66I (120.0 μg ml⁻¹) killed the fungi by acting primarily on the cell membrane. However, at fungistatic concentration (20.0 μg ml⁻¹) of this compound, microscopic observations revealed swelling hyphae with abnormal chitin deposition, as determined by Calcofluor white (CFW) staining, which was indicative of the alterations in cell wall structure. In addition, inhibition of intracellular esterases activity was observed. These results led us to conclude that low doses of CF66I probably inhibited the fungal growth by interfering with the cell metabolic pathways.     

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.     

Gene cloning and heterologous expression of a serine protease from Streptomyces fradiae var.k11

The gene sfp1, which encodes a predicted serine proteinase designated SFP1, was isolated by the screening of a gene library of the feather-degrading strain Streptomyces fradiae var.k11. The open reading frame of sfp1 encodes a protein of 454 amino acids with a calculated molecular mass of 46.19 kDa. Sequence analysis reveals that SFP1 possesses a typical pre-pro-mature organization that consists of a signal sequence, an N-terminal propeptide region, and a mature proteinase domain. The pre-enzyme of SFP1 was expressed in Escherichia coli and consequently purified. The 25.6 kDa fraction with protease activity separated by gel filtration chromatography indicated that the mature enzyme of SFP1 was formed by autolysis of the propeptide after its expression. The purified SFP1 is active under a broad range of pH and temperature. SFP1 has pH and temperature optima of pH 8.5 and 65 degrees C for its caseinolytic activity and pH 9 and 62 degrees C for its keratinolytic activity. SFP1 was sharply inhibited by the serine proteinase inhibitor phenylmethyl sulfonyl fluoride and exhibited a good stability to solvents, detergents, and salts. Comparison of the protease activity of SFP1 with other commercial proteases indicates that SFP1 has a considerable caseinolytic and keratinolytic activity as does proteinase K.     

Effects of granulosa cell mitochondria transfer on the early development of bovine embryos in vitro

The objective of this study was to determine the effect of exogenous mitochondria obtained from granulosa cells on the development of bovine embryos in vitro. We classified cumulus oocyte complexes (COCs) as good (G)- and poor (P)-quality oocytes based on cytoplasmic appearance and cumulus characteristics, and assessed mtDNA copy numbers in the G and P oocytes with real-time polymerase chain reaction (PCR). The mitochondria were isolated by fractionation and suspended in mitochondria injection buffer (MIB). Part one of the experiment consisted of the following treatments: (1) G-oocytes + sperm, (2) P-oocytes + mitochondria + MIB + sperm, (3) P-oocytes + MIB + sperm, and (4) P-oocytes + sperm. In part 2, oocytes were parthenogenetically activated. The treatments were: (1) G-oocytes, (2) P-oocytes + mitochondria + MIB, (3) P-oocytes + MIB, and (4) P-oocytes alone. The results indicated a significant difference in mtDNA copy number between G (361 113 +/- 147 114) and P (198 293 +/- 174 178) oocytes (p < 0.01). The rates of morula, blastocyst, and hatched blastocysts derived from P-oocytes + mitochondria were similar to those of G-oocytes, but significantly higher than P-oocytes without exogenous mitochondria in both the ICSI and parthenogenetic activation experiments. We found no difference in blastomere numbers between G-oocytes and P-oocytes + mitochondria in either experiment, but blastomere numbers in these two groups were significantly higher than in P-oocyte groups without exogenous mitochondria. These data suggest that mtDNA content is very important for early embryo development. Furthermore, the transfer of mitochondria from the same breed may improve embryo quality during preimplantation development.