Effects of class-specific,synthetic, and natural proteinase inhibitors on life-history traits of the cotton bollworm Helicoverpa armigera

Herbivorous insects have more difficulty obtaining proteins from their food than do predators and parasites. The scarcity of proteins in their diet requires herbivores to feed voraciously, thus heavily damaging their host plants. Plants respond to herbivory by producing defense compounds, which reduce insect growth, retard development, and increase mortality. Herbivores use both pre- and postdigestive response mechanisms to detect and avoid plant defense compounds. Proteinase inhibitors (PIs) are one example of plant compounds produced as a direct defense against herbivory. Many insects can adapt to PIs when these are incorporated into artificial diets. However, little is known about the effect of PIs on diet choice and feeding behavior. We monitored the diet choice, life-history traits, and gut proteinase activity of Helicoverpa armigera larvae using diets supplemented with synthetic and natural PIs. In choice experiments, both neonates and fourth-instar larvae preferred the control diet over PI-supplemented diets, to varying degrees. Larvae that fed on PI-supplemented diets weighed less than those that fed on the control diet and produced smaller pupae. Trypsin-specific PIs had a stronger effect on mean larval weight than did other PIs. A reduction of trypsin activity but not of chymotrypsin activity was observed in larvae fed on PI-supplemented diets. Therefore, behavioral avoidance of feeding on plant parts high in PIs could be an adaptation to minimize the impact of this plant's defensive strategy.     

猪流行性腹泻病毒(PEDV)与抗病毒天然免疫

猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)是引起猪流行性腹泻病等肠道疾病的一种动物冠状病毒.PEDV与宿主系统相互作用,特别是其对宿主抗病毒天然免疫调节作用和机制是目前动物冠状病毒研究的基础科学问题之一.基于作者近几年来对人类重要冠状病毒对宿主抗病毒天然免疫系统调节作用的研究,本文对PEDV基因组与编码蛋白主要功能以及PEDV调节宿主抗病毒天然免疫反应及其可能机制的进展和现状进行了分析.与人类冠状病毒相似,PEDV编码的木瓜样蛋白酶(papain like protease,PLP)是一个多功能蛋白酶,除了蛋白酶活性外,还具有去泛素化酶(DUB)活性和宿主干扰素拮抗活性,是PEDV编码的一种新型病毒来源DUB和宿主干扰素拮抗蛋白.这些研究为阐明PEDV对宿主抗病毒天然免疫反应调节作用和其致病机制提供了重要的理论依据,为研制新型PEDV免疫防治措施提供了重要理论基础.     

Active site specificity of plasmepsin II.

Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues.     

五种新化合物对亚洲玉米螟幼虫生长发育和消化生理的影响(英文)

研究了五个新化合物对亚洲玉米螟 (Ostriniafurnacalis)五龄幼虫的拒食活性以及对其生长发育和中肠蛋白酶活性的影响 ,并与川楝素的作用效果进行了比较。结果表明 ,川楝素以及C19、C2 3 、C2 4、C2 6、C2 8在 5 0 0mg kg浓度下拒食活性分别为 5 1 16 %、5 7 6 1%、4 2 8%、5 1 0 8%、36 73%、5 1 6 7% ,其中C19、C2 4、C2 8拒食活性与川楝素相当。在 2 0mg kg浓度 ,生长发育实验表明C19、C2 6、C2 8具有较好的抑制幼虫生长作用 ,其中C2 8的作用与川楝素相当 ;药剂对中肠蛋白酶的影响结果表明 ,川楝素和C2 8对弱碱性类胰蛋白酶、类胰凝乳蛋白酶表现为激活作用 ,C19、C2 4、C2 6对两种蛋白酶在 2 4h激活 ,4 8h抑制。对相关作用机理进行了探讨。     

蛋白酶分泌菌对重金属胁迫的反应

本文研究了Pb、zn、cu和Cd对3种蛋白酶产生菌:碱性地衣芽孢杆菌2709(Bacilluslicheniformis 2709)、中性枯草杆菌1.398(Bacillus subtilis1.398)和酸性宇佐美曲霉537(Aspergillususamii 537)生长及其酶活性的影响,并采用生长抑制皿分析(GIPA)方法针对3种菌株在Pb、zn、Cu、Cd不同胁迫浓度下的生长量进行打分,分析其耐性与抗性指标(MTC与MIC).结果表明4种重金属在超过一定临界浓度后,对3种菌株的生长和蛋白酶活性均产生了较大的抑制作用.碱性蛋白酶对4种重金属均具有较大的适应性,其次是中性蛋白酶对Zn和Cd具有一定的适应性.而酸性蛋白酶对4种重金属均表现出被抑制状态.3种菌株对Pb和Zn耐性与抗性最高(2.0 mmol/L～6.0 mmol/L),其次是对Cd也具有一定的抗性(0.5 mmol/L～0.75 mmol/L).     

Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli

Summary Two multicopy plasmids carrying either the umuDC or the mucAB operon were used to compare the efficiency of UmuDC and MucAB proteins in UV mutagenesis of Escherichia coli K12. It was found that in recA ⁺ uvr ⁺bacteria, plasmid pIC80, mucAB ⁺mediated UV mutagenesis more efficiently than did plasmid pSE117, umuDC ⁺. A similar result was obtained in lexA51(Def) cells, excluding the possibility that this was due to a differential regulation by LexA of the umuDC and mucAB operons. We conclude that some structural characteristic of the UmuDC and MucAB proteins determines their different efficiency in UV mutagenesis. This characteristic could be also responsible for the observation that in the recA430 mutant, pIC80 but no pSE117 can mediate UV mutagenesis. In the recA142 mutant, pIC80 also promoted UV mutagenesis more efficiently than pSE117. In this mutant, the recombination proficiency, the protease activity toward LexA and the mutation frequency were increased by the presence of adenine in the medium. In recA ⁺ uvrB5 bacteria, plasmid pSE117,umuDC caused both an increase in UV sensitivity as well as a reduction in the mutation frequency. These nagative effects resulting from the overproduction of UmuDC proteins were higher in recA142 uvrB5 than in recA ⁺ uvrB5 cells. In contrast, overproduction of MucAB proteins in excision-deficient bacteria containing pIC80 led to a large increase in the mutation frequency. We suggest that the functional differences between UmuDC and MucAB proteins might be due to their different dependence on the direct role of RecA protease in UV mutagenesis.     

碱性蛋白酶基因在Bacillus licheniformis 2709中的整合和扩增

将含有去掉启动子的枯草杆菌碱性蛋白酶基因和Ｃｍ抗性基因的整合质粒ｐＭＰ以原生质体ＰＥＧ法转化Ｂ．ｌｉｃｈｅｎｉｆｏｒｍｉｓ２７０９在１０ｕ／ｍｌＣｍ平板上筛选整合子,并通过不断提高整合子的抗氯霉素能力来扩增染色体碱性蛋白酶基因,最终选到一株产酶比生产菌２７０９提高６０％的高表达整合菌ＢＬ－２０３,该菌在无选择压力下遗传性状相当稳定。     

Purification and Properties of a Protease Produced by Bacillus Subtilis CN2 Isolated from a Vietnamese Fish Sauce

Bacillus subtilis CN2 isolated from a Vietnamese fish sauce produced a large quantity of an alkaline protease, when grown on a soy peptone medium. The protease was purified to an electrophoretically homogeneous state and crystallized in its pure condensed solution. The molecular weight was determined to be 27,636 Da, and the N-terminal amino acid sequence was AQSVPYGISQIKAPAL. The optimum pH and temperature were pH 10.0 and 50 °C, respectively. The protease was active over a wide pH range of pH 7.0–11.0, and also active over a broad temperature range of 30–60 °C. The enzyme was potently inhibited by 1 mM phenylmethanesulphonyl fluoride, but resistant to 1 mM sodium dodecyl sulphate (SDS). This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation and characterization of a protease inhibitor from spinach leaves

An acid-stable and heat-labile proteinous protease inhibitor which was found in spinach leaves but not in seeds was isolated by sequential chromatography and preparative isoelectric focusing. The isoelectric point of this inhibitor was 4.5. The inhibitor had a M_r of ca 18 000 and was rich in aspartic acid and glycine; it had 4 half-cystine, 2 tryptophan and no methionine residues. Its extinction coefficient (E|_cm^%) was 13.7 at 280 nm. The inhibition was competitive and the dissociation constant was 3.32 × 10^?13 M. The inhibitor was specific to serine proteases and strongly inhibited trypsin and weakly inhibited α-chymotrypsin and kallikrein.     

Cadmium elevates level of protein, amino acids and alters activity of proteolytic enzymes in germinating rice seeds

When seeds of two rice cvs. Ratna and Jaya were germinated under increasing levels of cadmium nitrate (0, 100 and 500 μM) in the medium, a marked decrease in germination percentage was observed with Cd treatments, as compared to controls. There was more absorbed Cd in embryo axes than in endosperms. More uptake resulted with increasing Cd levels in the growth medium in embryo axes. In both rice cultivars, during a germination period of 0 – 120 h, an increased level of protein as well as free amino acids was noted in Cd treatments. Protease activity in general decreased in both embryo axes as well as endosperms due to Cd treatment. In vitro studies showed an enhancement in protease activity in Cd treatments at low Cd levels (50–100 μM), whereas concentrations above this caused inhibition in enzyme activity. Under 500 μM Cd treatments in vivo there was about 30 to 50 percent decline in leucine aminopeptidase (LAP) activity in endosperms, however, carboxypeptidase activity showed a marked increase in endosperms beyond 24 h under Cd treatments. In embryo axes of germinating seeds there was always a decline in peptidase activities, under the influence of cadmium. The leucine amino peptidase and protease activity were always greater in embryo axes in cv. Ratna than cv. Jaya. However, the carboxypeptidase activity was higher in Jaya when compared to Ratna in endosperms under Cd treatments. The results suggest possible suppression of protease and peptidase activities due to Cd treatments in germinating rice seeds leading to altered levels of protein and amino acids.