大鼠运动性肥大心脏心肌血管内皮生长因子及其基因表达的研究

目的和方法：血管内皮生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）是新近确定的一种特异作用于血管内皮细胞的活性肽。最近发现正常心肌细胞可表达ＶＥＧＦ,高血压肥大心脏心肌ＶＥＧＦ及基因表达增强,但对运动性心肌肥大时的变化尚不清楚。本实验采用免疫组化和分子杂交方法,对游泳运动１０周大鼠稳定期肥大心脏心肌ＶＥＧＦ及其基因表达进行研究。结果：ＷｉｓｔａｒＫｙｏｔｏ（ＷＫＹ）大鼠、自发性高血压大鼠（ｓｐｏｎｔａｎｅｏｕｓｌｙｈｙｐｅｒｔｅｎｓｉｖｅｒａｔｓ,ＳＨＲ）和运动大鼠心肌细胞浆内均有特异性ＶＥＧＦ染色颗粒,但运动大鼠心肌细胞胞浆内染色颗粒增加最明显。Ｎｏｒｔｈｅｒｎ分子杂交结果表明三组大鼠心肌均有ＶＥＧＦｍＲＮＡ表达,其中ＳＨＲ表达最强,运动大鼠比ＷＫＹ大鼠增强,但低于ＳＨＲ。结论：目前对这一结果的生理意义还不清楚,推测可能与心肌肥大时细胞间质血管增生有关。     

RNAi介导的OsBTF3基因沉默及其对水稻籽粒相关性状的影响

为了创制OsBTF3基因沉默的水稻植株、验证该基因在水稻籽粒相关性状中的功能、评价其在水稻遗传改良中潜在的应用价值,设计和合成OsBTF3基因序列的引物、扩增部分基因片段,构建RNAi基因沉默载体、通过农杆菌介导转化愈伤组织、植株再生、潮霉素抗性筛选和PCR验证、定量分析OsBTF3基因表达量,测定转基因水稻籽粒相关性状。结果表明,成功地获得了20个T1代OsBTF3-RNAi转基因株系,OsBTF3基因表达量得到显著的抑制和干扰,抑制效果平均达到85%;与野生型对照株相比,5个所测定RNAi转基因株系的穗长、穗粒数、千粒重和穗粒重等籽粒相关性状明显地减小或降低。因此,RNAi介导的基因沉默导致了OsBTF3基因表达水平抑制以及在籽粒性状中的功能缺失;OsBTF3可能是一个调控水稻籽粒相关性状重要的功能基因。     

Generation of Hepatitis B virus PreS2-S antigen in Hansenula polymorpha

Despite the long availability of a traditional prophylactic vaccine containing the HBV surface antigen(HBsA g) and aluminum adjuvant, nearly 10% of the population remains unable to generate an effective immune response. Previous studies have indicated that hepatitis B virus(HBV) PreS 2-S is abundant in T/B cell epitopes, which induces a stronger immune response than HBsA g, particularly in terms of cytotoxic T lymphocyte(CTL) reaction. In the current study, the HBV PreS 2-S gene encoding an extra26 amino acids(PreS 2 C-terminus) located at the N-terminus of HBsA g was cloned into the pV CH1300 expression vector. Pre S2-S expressed in the methylotrophic yeast, Hansenula polymorpha, was produced at a yield of up to 250 mg/L. Subsequent purification steps involved hydrophobic adsorption to colloidal silica, ion-exchange chromatography and density ultracentrifugation. The final product was obtained with a total yield of ~15% and purity of ~99%. In keeping with previous studies, ~22 nm viruslike particles were detected using electron microscopy. The generated PreS 2-S antigen will be further studied for efficacy and safty in animals.     

Multiplex real-time PCR assay for detection of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and screening for non-O157 Shiga toxin-producing <Emphasis Type="Italic">E. coli</Emphasis>

Background

Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency.

Methods

The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s.

Results

The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella enterica, Shigella strains, or any other pathogenic strains tested.

Conclusions

A multiplex real-time PCR assay that can rapidly and simultaneously detect E. coli O157:H7 and screen for non-O157 STEC strains has been developed and assessed for efficacy. The inclusivity and exclusivity tests demonstrated high sensitivity and specificity of the multiplex real-time PCR assay. In addition, this multiplex assay was shown to be effective for the detection of E. coli O157:H7 from two common food matrices, beef and spinach, and may be applied for detection of E. coli O157:H7 and screening for non-O157 STEC strains from other food matrices as well.

     

Regulatory perspective of antibiotic biosynthesis in Streptomyces

<正>Streptomycetes are Gram-positive bacteria with high GC DNA content. They produce the most abundant secondary metabolites including over two-thirds of the clinically used antibiotics of natural origin (Barka et al., 2016), for example,the important broad-spectrum antimicrobials oxytetracycline(OTC) and chlortetracycline, which are the tetracycline antibiotics, produced by Streptomyces rimosus and Strepto-     

重组α-银环蛇毒素的纯化及活性分析

以表达重组α-银环蛇毒素的高效表达菌株BL21(PDZ04)为材料,研究重组α-银环蛇毒素(α-bungarotoxin,α-BgTx)的分离纯化。采取亲和色谱和离子交换色谱的方法都得到了重组α-银环蛇毒素的纯品。参考文献报道的方法从天然的银环蛇毒干粉中分离纯化得到了天然α-银环蛇毒素的纯品。然后以天然α-银环蛇毒素为对照来检测重组α-银环蛇毒素的抗原性和毒性。ELISA结果显示其具有与天然α-银环蛇毒素相似的抗原性,以小鼠为动物模型,纯化的重组α-银环蛇毒素与天然α-银环蛇毒素相比,其腹腔注射的LD50也基本一致,约为0.22μg/g。结果表明利用基因工程的方法生产蛇神经毒素是可行的。     

Binding of the maize cytosolic Hsp70 to calmodulin, and identification of calmodulin-binding site in Hsp70

Using a gel-overlay technique of biotinylated calmodulin (CaM), we showed that maize cytosolic Hsp70 protein could bind to CaM in the presence of 1 mM CaCl2. The purified maize cytosolic Hsp70 inhibited the activity of CaM-dependent NADK in a concentration-dependent manner. A synthetic peptide, which possesses the 21 amino acid sequence, PRALRRLRTACERAKRTLSST, at positions 261-281 in maize cytosolic Hsp70, could associate with CaM in the presence of 1 mM calcium. The synthetic peptide inhibited CaM-dependent NADK activity and PDE activity. This indicates that the 21-amino acid sequence at positions 261-281 is the CaM-binding site. The binding of CaM to Hsp70 inhibited the ATPase activity of Hsp70. The possible regulator function of Hsp70 in cell signaling events in response to heat stress is discussed.     

1%蛇床子素粉剂对三种储粮害虫的防效

研究1%蛇床子素粉剂在5种处理浓度下对3种储粮害虫谷蠹Rhizopertha dominica Fabricius、玉米象Sitophilus zeamai Motschulsky和赤拟谷盗Tribolium castaneum Herbst的防治效果。室内毒力测定结果表明,按有效成分0.5mg/kg(蛇床子素:粮食)浓度处理粮食,7d后,粮食中谷蠹、玉米象和赤拟谷盗的校正死亡率分别为97.78%、100%、86.70%,防治效果优于对照药剂防虫磷和谷虫净。将药剂处理4个月后的粮食进行接虫试验,15d后谷蠹和玉米象的防治效果仍可达100%,达到储粮害虫防治要求。     

Further Daphniphyllum Alkaloids with Insecticidal Activity from the Bark of Daphniphyllum macropodum Miq.

Two new Daphniphyllum alkaloids, macropodumines J and K ( 1 and 2 , resp.), together with six known structurally related alkaloids, 3 – 8 , were isolated from the bark of Daphniphyllum macropodum Miq . The structures of the new compounds 1 and 2 were elucidated on the basis of a comprehensive analysis of their spectroscopic and chemical data. Macropodumine J ( 1 ) contains a CN group which is relatively rare in naturally occurring alkaloids. All isolated compounds were tested for their insecticidal activities against a number of insect species. Daphtenidine C ( 5 ) is the most active compound against Plutella xylostella. This is the first report of insecticidal properties of Daphniphyllum alkaloids.     

Review of enucleation methods and procedures used in animal cloning: state of the art

Enucleation of a recipient oocyte is a crucially important process for nuclear transfer efficiency. Several procedures have been developed and used in the production of nuclear transfer embryos. Although the use of excitable fluorochromes and ultraviolet (UV) light are commonly used for complete enucleation, they also pose the risk of damaging the maternal cytoplast. Telophase and chemically assisted enucleation have also been used for cloning, but the quality and quantity of the recipient cytoplasm varies with the procedure used. This paper reviews various methods used for enucleation, and discusses their benefits and limitations with respect to cloning efficiency.