An approach to the production of soluble protein from a fungal gene encoding an aggregation-prone xylanase in Escherichia coli

The development of new procedures and protocols that allow researchers to obtain recombinant proteins is of fundamental importance in the biotechnology field. A strategy was explored to overcome inclusion-body formation observed when expressing an aggregation-prone fungal xylanase in Escherichia coli. pHsh is an expression plasmid that uses a synthetic heat-shock (Hsh) promoter, in which gene expression is regulated by an alternative sigma factor (σ(32)). A derivative of pHsh was constructed by fusing a signal peptide to xynA2 gene to facilitate export of the recombinant protein to the periplasm. The xylanase was produced in a soluble form. Three factors were essential to achieving such soluble expression of the xylanase: 1) the target gene was under the control of the Hsh promoter, 2) the gene product was exported into the periplasm, and 3) gene expression was induced by a temperature upshift. For the first time we report the expression of periplasmic proteins under the control of an Hsh promoter regulated by σ(32). One unique feature of this approach was that over 200 copies of the Hsh promoter in an E. coli cell significantly increased the concentration of σ(32). The growth inhibition of the recombinant cells corresponded to an increase in the levels of soluble periplasmic protein. Therefore, an alternative protocol was designed to induce gene expression from pHsh-ex to obtain high levels of active soluble enzymes.     

Clinical and molecular findings in osteoporosis-pseudoglioma syndrome

Mutations in the low-density lipoprotein receptor-related protein 5 gene (LRP5) cause autosomal recessive osteoporosis-pseudoglioma syndrome (OPPG). We sequenced the coding exons of LRP5 in 37 probands suspected of having OPPG on the basis of the co-occurrence of severe congenital or childhood-onset visual impairment with bone fragility or osteoporosis recognized by young adulthood. We found two putative mutant alleles in 26 probands, only one mutant allele in 4 probands, and no mutant alleles in 7 probands. Looking for digenic inheritance, we sequenced the genes encoding the functionally related receptor LRP6, an LRP5 coreceptor FZD4, and an LRP5 ligand, NDP, in the four probands with one mutant allele, and, looking for locus heterogeneity, we sequenced FZD4 and NDP in the seven probands with no mutations, but we found no additional mutations. When we compared clinical features between probands with and without LRP5 mutations, we found no difference in the severity of skeletal disease, prevalence of cognitive impairment, or family history of consanguinity. However, four of the seven probands without detectable mutations had eye pathology that differed from pathology previously described for OPPG. Since many LRP5 mutations are missense changes, to differentiate between a disease-causing mutation and a benign variant, we measured the ability of wild-type and mutant LRP5 to transduce Wnt and Norrin signal ex vivo. Each of the seven OPPG mutations tested, had reduced signal transduction compared with wild-type mutations. These results indicate that early bilateral vitreoretinal eye pathology coupled with skeletal fragility is a strong predictor of LRP5 mutation and that mutations in LRP5 cause OPPG by impairing Wnt and Norrin signal transduction.     

播娘蒿下胚轴高频率再生植株因素的研究

以播娘蒿（Descurainia sophia)下胚轴为外植体,研究影响其分化的若干因素。结果表明：用附加2,4－D的培养基,对下胚轴切段预培养5d可提高分化频率;1.7mg/L AgNO3对分化出芽有很大促进作用,最佳激素组合为2.0mg/L 6-BA 0.2mg/L NAA;黄化苗的下胚轴比绿苗的下胚轴对激素反应更敏感,最高分化频率可达86.05%。再生芽转至1／2MS 0.5mg/L NAA 0.5mg/L IBA的培养基中,生根率100％。     

角额壁蜂在苹果梨园生活力下降的原因分析

采用测定苹果梨和苹果的花粉量以及对其主要营养成分进行定量分析的方法,探椭角额壁蜂Osmia cornifrons(Radoszkowski)在苹果梨园生活力下降的主要原因。结果表明,苹果梨花粉的花粉量约为苹果花粉的1/3,是角额壁蜂在苹果梨园的繁殖比明显低于苹果园的主要原因;苹果梨花粉与红富士等苹果花粉相比,在主要营养物质方面具有高糖、高纤维,低蛋白、低水分的特点,在矿质营养方面,苹果梨花粉具有高Ca和Mg,低Fe和Zn的特点。这些差异是引起连续使用苹果梨园的角额壁蜂出茧率和活动能力逐年下降的主要原因。     

低氧诱导因子和白血病细胞分化

三氧化二砷（As2O3,ATO）是一种新发现的有效治疗急性早幼粒细胞白血病（acute promyelocytic leukemia,APL）的药物。研究发现,该药物在体外诱导细胞分化的能力不如体内明显。以此为基础,最近我们意外地发现模拟低氧化合物和中度低氧环境能够直接在体外诱导急性髓系白血病细胞分化,也选择性地加强三氧化二砷诱导的APL细胞分化。进一步地,间歇性低氧能够显著延长移植的白血病小鼠生存时间,并且抑制白血病细胞浸润并诱导其分化。以这些工作为基础,我们就低氧诱导白血病细胞分化的分子机制进行了深入研究。本文将就相关工作作一综述,并讨论有待进一步研究的问题。     

一种简易的家兔离体心脏灌流方法

生理实验中的经典实验,蛙心灌流给研究药物对心脏的影响提供了很好的实验平台,但是蛙心的结构毕竟区别于哺乳动物的心脏,研究结果不能说明药物对哺乳动物心脏的影响。然而哺乳动物是恒温动物,体外存活条件苛刻,设备复杂,非一般教学实验室能够实现。介绍了一种在常规生理教学实验室可以实现的简易的哺乳动物心脏离体灌流的方法。     

Expression of recombinant and mosaic Cry1Ac receptors from Helicoverpa armigera and their influences on the cytotoxicity of activated Cry1Ac to Spodoptera litura Sl-HP cells

Bacillus thuringiensis (Bt) toxin receptors play important roles in the killing of pests, and investigation on characterization of the receptors is essential for utilization of Bt and management of insect resistance. Here, recombinant and mosaic receptors of Bt Cry1Ac toxin from Helicoverpa armigera were expressed in Spodoptera litura Sl-HP cells and their influences on cytotoxicity of activated Cry1Ac toxin were investigated. When H. armigera aminopeptidase N1 (APN1), alkaline phosphatase 2 (ALP2) and cadherin fused with or without GFP tag were, respectively, expressed in Sl-HP cells, live cell-immunofluorescence staining detection revealed that the quantity of the toxin binding to cadherin or cadherin-GFP was much more than that binding to ALP2 and APN1 or their fusion proteins with GFP, and only the cadherin- or cadherin-GFP-expressing cells showed aberrant cell morphology after the treatment of the toxin at low concentrations. ALP2 and APN1 fused with or without GFP tag did not significantly enhance the cadherin-mediated cytotoxicity of the toxin. The mosaic ALP-TBR-GFP-GPI was located on cell membrane, but did not bind to the toxin. The mosaic truncated cadherin-GFP-GPI was not located on cell membrane even if the signal peptide was sustained. The concentrations of the toxin resulting in swelling of 50 % cells for noncadherin-expressing Sl-HP cells and cadherin-expressing Hi5 cells were 5.08 and 9.50 µg/ml within 1 h, respectively. Taken together, our data have indicated that the binding affinity of ALP2 and APN1 to activated Cry1Ac toxin is much weaker than that of cadherin and both ALP2 and APN1 do not enhance the cytotoxicity of the toxin even though cadherin is co-expressed, and the mosaic receptor of ALP2 inserted with cadherin toxin binding domain does not mediate cytotoxicity of the toxin. In addition, the noncadherin-expressing Sl-HP cells are more susceptible to activated Cry1Ac than the cadherin-expressing Hi5 cells.     

鹤山南亚热带草坡生态系统的热量平衡

本文以广东鹤山丘陵综合试验站草坡为研究对象,分析了鹤山南亚热带草坡的能量平衡特征,主要结果如下：1．辐射平衡的各分量中,蒸发散耗热占净辐射的74.7％,其年变化幅度较大;湍流热通量占净辐射的21．2％,其月变化没有明显的规律,波动较大,土壤热通量与其它分量相比,其比重要小得多。2．虽然鹤山年幅射量和降雨量较多,但水热条件有几个月配合欠佳,使得草坡的净辐射能有90％以上用于地面以上植被和空气湍流交换所消耗,小气候条件较差,影响了这一生态系统的生产潜力的发挥。3．太阳辐射透过大气层以后,仅有4775．2MJm-2a-1左右的辐射能到达草坡,草坡反射了822．5MJm-2a-1,其净幅射收人为2915．6MJm-2a-1,净辐射收人中又有2077．8MJm-2a-1用于蒸散作用,618.1MJm-2a-1用于湍流热交换,87．5MJm-2a-1用于土壤热交换,植被贮热约38.8MJm-2a-1;用于净光合作用耗热93．4MJm-2a-1。     

海南岛河港海莲红树林凋落物动态的研究

 本文应用凋落物收集网对海南岛海莲红树群落进行连续4年(1984.1—1987.12)的凋落物动态研究。结果表明： (1)在保护良好的河港海滩上,海莲群落的年凋落物量高达1255g／m2,是热带地区凋落物量最大的群落类型之一;与世界上红树群落凋落物量相比处于较高水平。(2)4年平均凋落物各组分占总量的比例为叶64.32%、花10.63%,果21.34%、枝3.71%。 (3)月凋落物量(Y,g／m2)与月均气温(X,℃)的回归公式为 Y=5.009X–13.18(r=0.44**,df=46),相关极显著。 (4)不同年份凋落物的变化率R=1.33。     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> prevents the development of colitis in IL-10-deficient mouse by reducing the intestinal permeability

It is reported that defects exist in the small intestinal epithelial barrier of inflammatory bowel disease, which might be associated with increased intestinal permeability at a very early stage. Our study aims to investigate the role of Lactobacillus plantarum on the decrease of epithelial permeability and the further protective effects on the intestinal epithelial barrier using the IL-10-deficient mouse model. Our study showed that tight junction associated proteins were increased after the pre-treatment of L. plantarum by fluorescence staining, western blot, real-time PCR and transmission electron microscope. Oral gavage of milk containing L. plantarum was effective in decreasing small intestinal permeability using methods of Ussing chamber assay and sugar probe. Assay of pro-inflammatory cytokines IFN-γ, TNF-α, MPO, and colonic histology by ELISA showed protective effects of L. plantarum on the intestinal epithelial barrier. Therefore, L. plantarum may prevent the development of colitis in IL-10-deficient mice by blocking changes in the expression of TJ proteins, TJ structure and intestinal permeability.