Action Selection and Flexible Switching Controlled by the Intralaminar Thalamic Neurons

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Plasmodium falciparum Rhoptry Proteins of 140/130/110 kd (Rhop-H) Are Located in an Electron Lucent Compartment in the Neck of the Rhoptries

ABSTRACT. To investigate in more detail the structure of the high molecular weight rhoptry protein complex of Plasmodium falciparum, Rhop-H (140/130/110 kd), the complex was affinity purified from parasite extracts using rhoptry protein specific antisera prepared against Rhop-H proteins bound to and eluted from Balb/c mouse erythrocytes, using 0.5 M NaCl. The individual proteins (140 kd/Rhop-1, 130 kd/Rhop-2, and 110 kd/Rhop-3) were separated, electroeluted, and monospecific polyclonal antisera prepared against the individual proteins, and against the affinity purified complex. Immunofluorescence assays and immunoelectron microscopic studies were performed to verify the subcellular localization of the Rhop-H epitopes. Immunoblotting and immunoprecipitation assays were also performed. We report novel findings regarding the localization of the rhoptry proteins to an electron lucent compartment in the neck of the rhoptries. Analysis of the amino acid composition of the individually purified Rhop-H proteins demonstrated a predominance of negatively charged (E, D) as well as hydrophobic residues (L, A, P, S) in the three proteins. The percentage of negatively charged residues was high for all three proteins. Similarities in amino acid composition for the three proteins supports the previous data demonstrating shared properties such as erythrocyte and liposome binding, for the three proteins. Results of antibody characterizations using rhoptry protein specific antisera demonstrate the immunodominance of the Rhop-H complex.     

Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon

Abstract A plasmid-based food-grade vector system was developed for Lactococcus lactis by exploiting the genes for lactose metabolism. L. lactis MGS267 is a plasmid-free strain containing the entire lactose operon as a chromosomal insertion. The lacF gene was deleted from this strain by a double cross-over homologous recombination event. The lacF -deficient strain produced a Lac⁻ phenotype on indicator agar. A cloned copy of the lacF gene expressed on a plasmid was capable of complementing the lacF -deficient strain resulting in a Lac⁺ phenotype. This stably maintained system fits the requirements of a self-selecting vector system and has the potential to be exploited in the food industry.     

pFC1 to pFC7: A novel family of combinatorial cloning vectors

We have designed and constructed a series of plasmid vectors based on pBlueScript, where additional restriction sites have been incorporated into theSstI andKpn I sites. These sites, of enzymes that cut only rarely, permit expression cassettes constructs to be easily excised and multimerized with others.     

细胞因子基因导入人肿瘤细胞的方法及鉴定

从人外周血单核细胞中抽提总RNA为模板,分别用5’含EcoRⅠ、3’含BamHⅠ限制性酶切位点的细胞因子基因特异引物合成合信号肽全长IL－2、γ－IFN及α－TNFcDNA．然后将这些细胞因子cDNA分别重组入含筛选标记基因Neo的逆转录病毒载体LXSN中．采用磷酸钙共沉淀法转染逆转录病毒包装细胞系PA317．分别收集到含IFN－γ、TNF－α和IL－2基因的缺陷型逆转录病毒上清及只具空白载体质粒pLXSN的病毒上清这四种病毒颗粒用于导入人肝癌、胃癌细胞,可获得单克隆化的细胞因子基因修饰株PCR,Southern,RT－PCR及Northern杂交证明,有相应细胞因子及筛选标记基因的转入和表达生物活性分析也证实各基因修饰株细胞培养上清液中有一定活性的相应细胞因子．结果表明逆转录病毒介导的细胞因子基因转移是成功的     

反义前C／C基因转移表达抗乙型肝炎病毒作用的研究

为了观察逆转录病毒载体包装细胞系统介导反义基因转移表达的抗乙型肝炎病毒（HBV）作用,将HBVayw前C／C基因（preC／C）片段反向插入逆转录病毒载体质粒。将重组体转染逆转录病毒包装细胞PA317后,获得重组逆转录病毒。用重组逆转录病毒感染2．2．15细胞后发现,感染后第3天,细胞培养上清HBV表面抗原（HBsAg）和e抗原（HBeAg）表达量即明显减少,抑制作用于感染后第5天达到高峰,其中对HBsAg表达的抑制率为27．0％,对HBeAg表达的抑制率为59.5％。反义基因重组逆转录病毒感染2.2．15细胞对HBV抗原表达的抑制作用至少可以持续到转导后的第11天。空载及正义基因重组逆转录病毒感染对2．215细胞HBV抗原表达无明显抑制作用。此外,反义基因重组逆转录病毒感染对2.2.15细胞HBVDNA复制也有抑制作用．无细胞毒性。     

Dosage differential effects of permethrin impregnated into bednets on pyrethroid resistant and susceptible genotypes of the mosquito Anopheles stephensi

Abstract Effects of bednets impregnated with permethrin 200 mg and 500mg/m² on pyrethroid resistant and susceptible strains of Anopheles stephensi and their F hybrid progeny were studied, using free-flying female mosquitoes of these three genotypes, in a room with a human subject under a polyester net, having one of his arms in contact with the treated netting. Unexpectedly an apparently higher feeding rate, but lower knockdown and mortality rates, of mosquitoes were obtained for each of the three genotypes with the higher concentration of 500mg/m² compared with the lower dose of 200mg/m². At the lower dose there was 100% mortality 24 h after exposure of all three genotypes, suggesting that there would not be selection for resistance at this dose. However, at the higher dose there was significantly higher mortality of the susceptible strain than of the F hybrids, suggesting incomplete recessiveness of this resistance and that there would therefore be effective selection for resistance by this dose.
When female mosquitoes were confined in bioassay cones on treated netting, the resistant strain of An.stephensi showed significantly less irritability (scored as the time until first flight take-off) in response to each dose, as compared with the susceptible strain and F, hybrids. The higher dose provoked more irritation of each genotype; this could explain the greater knockdown and mortality rates of mosquitoes exposed to the lower dose which was less irritating and hence more effectively insecticidal. Thus a dose of 200mg/m² is preferable to 500mg/m² for malaria vector control.     

Construction of expression vectors and study on single-chain antibody and reshaping single-domain antibody against CD3

Two vectors, pWAI80 and pROH80, for expression of single-chain Fv fragments (ScFv) were constructed. The anti-CD3 VH and VL genes were amplified from UCHT1 cells by RT-PCR and sequenced. Both genes were cloned in pWAI80 to express native ScFv and pROH80 for GST-ScFv fusion protein expression. The expression products were analysed by ELISA and Western blot. The combining site of OKT3 was modeled. Human Ig LS1 and Nd were selected as acceptors of CDRs of OKT3 VL and VH to construct a reshaping antibody against CD3. By comparing OKT3, LS1 and Nd with their own family sequences, some residues were changed and the reshaping VL and VH genes were designed. The full VH gene was assembled in three steps with eight chemically synthesized oligonucleotide fragments using overlapping PCR and sequenced. The VH gene was expressed as active protein in pCOMB3 and as inclusion bodies in pGEX-4T-1 by ELISA and Western blot analysis. Project supported by the National Natural Science Foundation of China and “863” Plan.     

Selective reversal of drug resistance in drug-resistant lung adenocarcinoma cells by tumor-specific expression of MDR1 ribozyme gene mediated by retrovirus

According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDRl ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzedin vitro andin vivo; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDRl ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effective treatment method for patients with CEA-producing lung cancers which was usually refractory to conventional chemotherapy