Calcium-permeable acid-sensing ion channel is a molecular target of the neurotoxic metal ion lead

Acid-sensing ion channels (ASICs) are emerging as fundamental players in the regulation of neural plasticity and in pathological conditions. Here we showed that lead (Pb2+), a well known neurotoxic metal ion, reversibly and concentration-dependently inhib

Insecticide resistance in Chinese populations of the Culex pipiens complex through esterase overproduction

In most parts of China, mosquitoes have been subjected to organophosphate (OP) insecticide treatments since the mid-1960s, and resistance gene monitoring in the Culex pipiens complex (Diptera: Culicidae) started in only a few locations from the end of the 1980s. Many resistant alleles at the Ester locus have been found in field populations, including those commonly found around the world (Ester(B1) and Ester(2)), and those endemic to China (Ester(B6), Ester(B7), Ester(8), and Ester(9)). This situation is atypical, and may represent a complex situation for the evolution of insecticide resistance genes in China. To increase our understanding of the Chinese situation and our ability to manage resistance in the C. pipiens complex, a large study was performed. Twenty field populations were sampled from Beijing to Guangzhou. Bioassays with five insecticides (dichlorvos, parathion, chlorpyrifos, 2-sec-butylphenyl methyl carbamate, and propoxur) disclosed resistance levels variable according to the geographic origin, and up to 85-fold for dichlorvos. Six overproduced esterases were identified, including two that have not been previously described. Most of them were found in all samples, although at variable frequencies, suggesting variable selection or a transient situation, e.g., each one was recently restricted to a particular geographic area. The results are discussed in the context of recent alterations to insecticide campaigns, and of the evolution of resistance genes in Chinese C. pipiens populations.

Application of matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in determining arginine esterase from snake venom

Using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The homogeneities and molecular weights of three arginine esterases from snake venom, which possessing therapeutic use in myocardial infarction, were determined and compared, MALDI-TOF-MS is possessed of high accuracy, high sensitivity and rapidity. MALDI-TOF-MS and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can provide complementary and confirmatory results information. MALDI-TOF-MS can be directly used as an important method for the purification of snake venom complexes successfully.

Biological activities of skin secretions of the salamander Tylototriton verrucosus

Water-soluble skin secretions of salamander Tylototriton venucosus, first described by Anderson in 1871, were studied for their biological and enzymatic activities. They were found to be toxic to mice with an intraperitoneal LD50 of 11.5 mg/kg. Using Sephadex G-75 gel filtration, it was proven that the toxic components of the secretions are proteins with molecular weights ranging from 30,000 to 50,000 Da. The secretions of T. venucosus display a wide spectrum of antimicrobial activities and also contain both proteolytic activity and trypsin inhibitory activity. In contrast, neither hemolytic nor hemorrhagic activities were found. The secretions were determined to have phospholipase A(2) activity; however, no acetylcholine esterase activity was detectable under the assay conditions.

ISOLATION AND PROPERTIES OF A BLOOD-COAGULATION FACTOR-X ACTIVATOR FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

Biochemical and biological properties of Trimeresurus jerdonii venom and characterization of a platelet aggregation-inhibiting acidic phospholipase A(2)

Several biochemical and biological activities such as phospholipase A(2), arginine esterase, proteolytic, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase, thrombin-like, anticoagulant, and hemorrhagic activities were determined for whole desiccated venom of Trimeresurus jerdonii. An acidic phospholipase (named TJ-PLA(2)) was purified by anionic exchange chromatography, gel filtration, and reverse phase HPLC. TJ-PLA(2) had a molecular weight of 16,000 and a pI of 4.8. TJ-PLA(2) was non-lethal to mice up to an i.p. dose of 15 mg/kg body weight and lacked neurotoxicity and myotoxicity. It induced edema in the footpads of mice. The purified enzyme inhibited ADP- and collagen-induced human platelet aggregation in a manner which was both dose- and time-dependent.

Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms

Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5' nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes. (C) 2003 Elsevier Ltd. All rights reserved.

A novel serine protease inhibitor from Bungarus fasciatus venom

By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta -bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor. (c) 2007 Elsevier Inc. All rights reserved.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Artificial interspecific hybridization between Macrobrachium species

Viable F-1 hybrids were obtained from crosses of female Macrobrachium nipponense and male Macrobrachium hainanense involving spermatophore transfer and artificial insemination. This represents the first successful known case of hybridization of two Macrobrachium species by means of artificial insemination. The hatching rate was over 90%. About 20-60% of newly hatched larvae metamorphosed to postlarvae. The morphological characteristics of the hybrids resembled a combination of features of both parents. Malate dehydrogenase (MDH) and esterase (EST) isozyme electrophoresis indicated parents and F-1 hybrids showed co-dominant expression of the paternal and maternal alleles controlling the isozymes and confirmed the hybridization. (C) 2004 Elsevier B.V. All rights reserved.

Genetic diversity among different clones of the gynogenetic silver crucian carp, Carassius auratus gibelio, revealed by transferrin and isozyme markers

Genetic diversity among four clones (A, D, E, F) of gynogenetic silver crucian carp was studied using transferrin and isozymes in the blood as markers. Of the five proteins investigated, three (transferrin, esterase and superoxide dismutase) indicated polymorphism and eight polymorphic loci were detected. These loci were probably encoded by codominant alleles and their inheritance patterns were analyzed. Intraclonal homogeneity and interclonal heterogeneity were observed in these clones, which allowed us to infer the clonal nature and evolutionary relationship between them. Clonal diversity in this population of silver crucian carp in China was also compared with data reported from gynogenetic crucian carp in Germany.

己酸乙酯酯化真菌筛选及发酵条件研究

本文主要研究了具有己酸乙酯酯化活性的真菌菌株的筛选和发酵条件优化。从大曲和糟醅样品中分离纯化获得79株产生透明圈的丝状真菌，菌落形态初步识别结果显示分离菌株包括红曲霉属、根霉属及曲霉属等菌株。其中菌株EM-56酯化酶活力最强，发酵获得的粗酶制剂酶活为172.36 u。根据显微形态、菌落形态及生理生化特征，初步鉴定该菌株为曲霉科红曲霉属紫色红曲霉（Monascus purpureus）。 在此基础上重点研究了菌株EM-56在不同培养基成分及不同培养条件下的产酶情况，确定了最佳培养基和培养条件。通过单因素实验确定在基础培养基中添加最佳碳源为葡萄糖，最佳氮源为蛋白胨。正交优化实验结果确定了最佳培养基组成：以麸皮为基础培养基，添加葡萄糖 2%，蛋白胨 0.3 %，KH2PO4.3H2O 0.05 %，MgSO4.7H2O 0.06 %。菌株EM-56在上述培养基中的最佳发酵条件为：初始pH 5.5，发酵温度为35°C，发酵时间7d，种龄48h，接种量8%，装瓶量50g / 瓶（500mL）。在最佳培养基和发酵条件下，菌株EM-56发酵获得的粗酶制剂酶活达到241.56 u，比优化前提高了40.15%。 In this paper, the research focuses on the selection of fungus with esterifying activity and optimization of fermentation conditions. We isolated 79 strains which had transparent zones from Daqu and fermented grains. The isolated strains contained Monascus、Rhizopus and Aspergillus through primary morphology analysis. The strain of EM-56 which produces strongest esterase was selected. The enzyme activity reached 172.36u. According to related literature, EM-56 was identified as Monascus purpureus through morphology analysis and biochemical determination. We also studied the effects of different medium and fermentation conditions on the esterase production of strain EM-56. The optimal medium and fermentation conditions were determined. Single factor experiment result shows that the optimal carbon source added is glucose and the optimal nitrogen source added is peptone. The optimal fermentation medium determined by orthogonal optimization test is as follows: wheat bran as substrate, glucose 2%, peptone 0.3%, KH2PO4.3H2O 0.05%，MgSO4.7H2O 0.06%. The optimal fermentation conditions are: initial pH 5.5, cultural temperature 35°C, cultural time 7d, seed age 48h, inoculation 8%, medium mass 50g / flask（500mL）. The esterse activity of EM-56 cultivated in the optimal medium and fermentation conditions reached 241.56u and increased by 40.15% compared with the original activity.

石油污染土壤生物修复过程中微生物生态研究

The analysis on microbiological ecology for four types of oil contaminates soils showed that the bacteria utilizing the oil as carbon sources increase,wheras the fugi become less .Zooloea and Bacillu are the dominant bacteria ; Mocor and Cunninghamella ,and Fursarium are the dominant fungi streptomyces take the superiority among the actinomyces.The anaiysis on esterase activity showed that the microbes above mentioned have abilies of degrading esters. The biodeg radationrates are 55.45%,56.74%,38.37% and 45.19%respectively,after 53 days,the biodegradation rate can be increased by 12.6% when the dominant microbes are added.

Mass spectrometry study on snake venoms and enzymes

In this paper, three kinds of snake venoms and lour kinds of enzymes (phospholipase A(2), fibrinolytic enzyme, arginine esterase and L-amino acid oxidase) isolated from the snake venom were analyzed. As the snake venom was different, the MALDI/TOF/MS showed difference, The MALDI/TOF/MS determination results could be affected Ly the concentrations of snake venom enzymes, And the mechanisms of desorption and ionization was also given in this study, By using MALDI/TOF/MS we obtained the accurate molecular weights and homogeneities of the enzymes. The apparent characteristics of the positive MALDI/TOF/MS of enzymes composed by two subunits were also given out, The results showed that MALDI/TOF/MS is an effective analytic method for discovering new components from snake venom complexes. And it is reliable to use this method to determine the molecular weights and purifies of protein molecules.

同步荧光法研究白眉蝮蛇蛇毒酶发色团的微环境

利用同步荧光技术对长白山白眉蝮蛇蛇毒中纯化得到的4种酶：磷脂酶A2(phospholipase A2,PLA2)、精氨酸酯酶(arginine esterase,AEase)、纤溶酶(fibrinolytic enzyme,FEase)和L-氨基酸氧化酶(L-amino acid oxidase,L-a a oxidase)进行了研究． 结果表明：当Δλ＝20nm时， 酶的荧光主要由酪氨酸(Tyr)残基所贡献； 当Δλ≥75nm时，酶的荧光主要由色氨酸(Try)所贡献．而且，长白山白眉蝮蛇蛇毒4种酶的Tyr和Trp残基所处的微环境并不相同.