Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization,cDNA cloning,and expressed sequence tag analysis

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.     

Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties

AIMS: An investigation was carried out on an oxidative and SDS-stable alkaline protease secreted by Bacillus clausii of industrial significance. METHODS AND RESULTS: Maximum enzyme activity was produced when the bacterium was grown in the medium containing (g l-1): soyabean meal, 15; wheat flour, 10; liquid maltose, 25; K2HPO4, 4; Na2HPO4, 1; MgSO4.7H2O, 0.1; Na2CO3, 6. The enzyme has an optimum pH of around 11 and optimum temperature of 60 degrees C. The alkaline protease showed extreme stability towards SDS and oxidizing agents, which retained its activity above 75 and 110% on treatment for 72 h with 5% SDS and 10% H2O2, respectively. Inhibition profile exhibited by phenylmethylsulphonyl fluoride suggested that the protease from B. clausii belongs to the family of serine proteases. CONCLUSIONS: Bacillus clausii produced high levels of an extracellular protease having high stability towards SDS and H2O2. SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline protease from B. clausii I-52 is significant for an industrial perspective because of its ability to function in broad pH and temperature ranges in addition to its tolerance and stability in presence of an anionic surfactant, like SDS and oxidants like peroxides and perborates. The enzymatic properties of the protease also suggest its suitable application as additive in detergent formulations.     

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.     

Kinetics of Cerebral Uptake Processes In Vitro of L-Glutamine, Branched-Chain L-Amino Acids, and L-Phenylalanine: Effects of Ouabain

Abstract: Estimates have been made of the amounts and rates of uptake of radioactive branched-chain i-amino acids, L-phenylalanine, and L-glutamine into incubated rat brain cortex slices. Estimates have also been made of the binding of these amino acids to brain cell fragments. It is shown that such binding, as well as the process of passive diffusion, is not affected by the presence of ouabain (0.2 mM), which suppresses the energy-dependent concentrative uptakes of the amino acids investigated. The maximum specific binding of L-glutamine is about three times that of the other amino acids and amounts to about 11% of the total uptake of the amino acid by rat brain cortex slices in 12 min from a medium containing 0.25 mM-glutamine. The sodium-ion concentration of the medium appears not to play a significant role in determining the rate of L-glutamine uptake in brain slices except at relatively low concentrations (<20 mequiv./l). The presence of Na⁺, however, is essential for the attainment of a tissue-to-medium concentration ratio greater than 2.0 for L-glutamine. At relatively low concentrations (0.25 mM) the rapidity of uptake of L-glutamine into a suspension of nerve terminals exceeds that into brain cortex slices. The uptakes of L-glutamine (K_m's = 0.66 mM and 2.25 mM) and of the branched chain L-amino acids (K_m's approx. 0.3 mM and 2 mM) by rat brain cortex slices are characterized by a double affinity system, but that of L-phenylalanine has only one affinity system (K_m= 0.23 mM). The K_m's have been calculated after subtracting the ouabain-insensitive passive uptakes of the amino acids from the total observed uptakes.     

城郊景观动态模型研究──以沈阳市东陵区为例

基于渗透理论、马尔柯夫过程理论,采用中性模型方法,建立了3个不同的城郊景观动态模型,模型中分别介入不同自然因子和决策因子.利用模型对研究区景观进行了动态变化模拟.对模拟结果进行了评价,评价方法与指标包括:1)多分辨率拟合分析;2)最近邻概率;3)斑块大小和数目.结果发现,综合介入决策因素和自然因子的模型具有最好的效果.     

日本囊对虾Kunitz型蛋白酶抑制剂在毕赤酵母中的表达纯化及活性分析

SKPI(shrimp Kunitz-type protease inhibitor)是日本囊对虾(Marsupenaeus japonicus)体内的一个小分子多肽, 含有一个Kunitz型结构域, 属于丝氨酸蛋白酶抑制剂。目前已知丝氨酸蛋白酶抑制剂在节肢动物免疫系统中起着非常重要的作用, 为了了解SKPI在对虾天然免疫系统中的作用, 首先对其进行了重组表达。从日本囊对虾肝胰腺中扩增skpi的cDNA片段, 插入改造后的pPIC9K酵母表达载体, 获得的重组质粒转化至毕赤酵母GS115进行表达。由于改造的pPIC9K载体加入了6-His标签, 因此利用Ni?Sepharose?High?Performance对SKPI进行了高效纯化。初步的活性研究表明, 重组表达的SKPI能特异性地抑制胰蛋白酶的水解活性。