Enterotoxigenicity and cytotoxicity of Bacillus thuringiensis strains and development of a process for Cry1Ac production

Bacillus thuringiensis is indistinguishable from Bacillus cereus except for the production of insecticidal crystal proteins (ICPs). B. thuringiensis strains may show enterotoxin profiles and toxin levels similar to those of B. cereus strains isolated from food-poisoning cases. It is important for the food industry and farmers to consider that with the application of B. thuringiensis strains to crops, their spores may be introduced into the human food chain. In this study, 59 B. thuringiensis strains were assayed for their hemolysin BL (HBL) using a BCET-RPLA kit and their cytotoxicity to Chinese hamster ovary (CHO) cells. The enterotoxin titer was as high as that of B. cereus diarrheal-type strain ATCC 49064. In an attempt to obtain a food safety strain for bioinsecticide use, in this study, a 3.5-kb cry1Ac DNA fragment was amplified with PCR from the total DNA of B. thuringiensis subsp. kurstaki CCRC 11502 and cloned into the Bacillus expression vector pHY300PLK. The alpha-amylase promoter, amyE, was then introduced into the promoter region and, afterward, the recombinant plasmid pHYe1Ac35 was introduced into a non-enterotoxigenic and non-cytotoxic B. thuringiensis subsp. kurstaki Tt14 strain. The transformant, without any detectable enterotoxigenicity or cytotoxicity, produced Cry1Ac toxin properly, and its insecticidal activity against Trichoplusia ni larvae was found to be satisfactory.     

Multiple forms of esterases in mouse,rat, and rabbit liver,and their role in hydrolysis of organophosphorus and pyrethroid insecticides

Six to seven esterases from mouse, rat, and rabbit liver microsomes were resolved by chromatofocusing in the pH range 7–4. Each esterase peak showed a different substrate specificity pattern with the substrates evaluated. Malathion and paraoxon hydrolysis always corresponded with p-nitrophenyl acetate and methylthiobutyrate hydrolysis, whereas the pattern of fenvalerate hydrolysis was more complicated. Phosphorotriester hydrolase activity was isolated, and was found to be more specific toward paraoxon than toward the other insecticides. Time-course studies of paraoxon hydrolysis indicated that the hydrolysis of paraoxon by carboxylesterase was an inhibitory reaction. This reaction and phosphorotriester hydrolase activity can serve as a detoxication reaction toward organophosphate insecticides.     

A characterization of the soils and sediments in contaminated sites and rivers using petroleum biomarker compounds

Journal of Soils and Sediments - The objective of this study is to reveal the presence of petroleum biomarkers in sediments of contaminated sites and to prove the existence of these compounds in...     

Purification and characterization of a novel extracellular tripeptidyl peptidase from Rhizopus oligosporus

A novel extracellular tripeptidyl peptidase (TPP) was homogenously purified from the culture supernatant of Rhizopus oligosporus by sequential fast protein liquid chromatography. The purified enzyme was a 136.5 kDa dimer composed of identical subunits. The effects of inhibitors and metal ions indicated that TPP is a metallo- and serine protease. TPP was activated by divalent cations, such as Co(2+) and Mn(2+), and completely inhibited by Cu(2+). Enzyme activity was optimal at pH 7.0 and 45 °C with a specific activity of 281.9 units/mg for the substrate Ala-Ala-Phe-pNA. The purified enzyme catalyzed cleavage of various synthetic tripeptides but not when proline occupied the P1 position. Purified TPP cleaved the pentapeptide Ala-Ala-Phe-Tyr-Tyr and tripeptide Ala-Ala-Phe, confirming the TPP activity of the enzyme.     

人CD14信号肽介导卵清蛋白在HEK293T细胞的高效表达

本试验将人CD14信号肽序列与卵清蛋白（ovalbumin,OVA）基因融合,旨在提高OVA在非输卵管上皮细胞中的表达。提取鸡输卵管上皮细胞总RNA,经反转录合成的cDNA,再利用修饰的特异性引物,通过PCR从cDNA中分别扩增出5’端与人CD14信号肽序列融合和非融合的OVA基因,并将它们分别插入到真核表达载体pcDNA3.1A中,构建成表达载体pcDNA-CD14sp-OVA和pcDNA-OVA。经磷酸钙介导,将质粒转染至HEK293T细胞,使其进行表达,用鼠抗OVA单克隆抗体通过免疫印迹（Western blot）法检测OVA表达水平。结果表明:本试验扩增的OVA基因序列与GenBank中的序列（登录号NM₂05152）一致,构建的人CD14信号肽序列融合OVA基因的表达载体结构正确。经过在HEK293T细胞的表达,融合人CD14信号肽序列的重组蛋白OVA的表达水平明显得到提高。结果提示,融合人CD14信号肽序列后的重组蛋白OVA在非输卵管上皮细胞中的表达水平明显提高,为OVA基因作为DNA疫苗模式抗原的应用提供了必要的条件。     

以電化學分析法探討堆肥對土壤三價砷活性的影響

台灣西南沿?貐^土壤中砷含量有偏高情形,砷含量過高會經由各種途徑對人體造成毒害。以X-射線繞射儀與傅立葉轉換紅外線吸收光譜儀證明普魯士藍對三價砷的孯恍晕綑C制,再以普魯士藍修飾電極搭配流動注入系統來偵測土壤三價砷,並探討堆肥對三價砷在不同土壤中吸附與脫附的影響。試驗結果得知電化學分析技術可快速、簡便且準確地偵測土壤三價砷,且結果中也指出堆肥處理會增加秀水與後里土壤對三價砷的吸附。此外,三種土壤不論有無施用堆肥,脫附百分率的大小順序爲興大>秀水>後里,其中秀水土壤雖在施用堆肥後吸附力大小明顯增加,但脫附百分率下降的情形較不明顯,顯示黏土礦物種類與含量控制三價砷的脫附行爲。     

Oceanographic influences on the distribution of Baltic cod, Gadus morhua, during spawning in the Bornholm Basin of the Baltic Sea

The Baltic Sea is a stratified, semienclosed sea typified by a low-salinity surface layer and a deep saline layer of varying volume, salinity, temperature and oxygen concentration. The relationships between these oceanographic factors and the distribution of Baltic cod are presented, utilizing results from a survey carried out during the 1995 spawning period in the Bornholm Basin, at present the main spawning area of this stock. Cod distribution, abundance and population structure were estimated from hydroacoustic and trawl data and related to hydrographic parameters as well as to bottom depth. In the central basin, cod were aggregated in an intermediate layer about 15 m thick. This area of peak abundance was defined at its upper limit by the halocline and at the lower limit by oxygen content. The majority of individuals caught in the basin centre were in spawning or pre-spawning condition with a high proportion of males to females. On the basin slopes, aggregations of cod were found near the bottom. These individuals were mainly immature and maturing stages with an increasing proportion of females to males with size. Salinity and oxygen conditions were found to be the major factors influencing the vertical and horizontal distribution of adult cod. Abundance of immature cod was also positively related to decreasing bottom depths. The effect of temperature was minor. The observed size- and sex-dependent spawning aggregation patterns, in association with habitat volume and stock size, may influence cod catchability and thereby the assessment and exploitation patterns of this stock.     

Self-organizing map network for automatically recognizing color texture fabric nature

The method of recognizing color texture brought forth in the present study is to employ unsupervised learning network to automatically recognize the fabric type and the main texture types. Firstly, the color scanner is adopted to extract fabric image which is afterwards saved as the digital image. Secondly,CIE-Lab color model is taken to obtain the feature value and wavelet transform is utilized to display the texture of the fabric image. Thirdly, co-occurrence matrix is employed to figure out the feature values of the texture structure such as angular second moment, entropy, homogeneity, contrast. Finally, self-organizing map (SOM) network is used as the classifier. The experiment result shows that the study can automatically and accurately classify the fabric types (including shuttle-woven fabric, jersey fabric and non-woven fabric) and main texture type of the fabric (such as plain weave, twill weave, satin weave, single jersey, double jersey and non-woven fabric).