Hydrazines probe of interaction of horseradish peroxidase with cryo-hydrogel

The interaction between horseradish peroxidase (HRP) and the cryo-hydrogel was probed by using hydrazines which show high specificity of the reaction of the edge in the prosthetic heme of horseradish peroxidase. For comparison, the interaction of hydrazine with the horseradish peroxidase adsorbed on graphite electrode was also carried out by using steady-state response of the enzyme electrode and cyclic voltammetry. In order to obtain a proper explanation of the kinetic parameters for the enzymatic reaction, the theoretical expressions of I-max and K-M' in the Michaelis-Menten equation for the experimental system were provided. (C) 1997 Elsevier Science B.V.

Study on the structure of hemoglobin in organic solvents by in situ STM

In situ STM has been used to study the structure of hemoglobin(Hb) in two kinds of organic media. In hydrophobic organic solvent such as carbon tetrachloride, the structure of Hb is almost the same as in aqueous solution, similar to its native structure. However, when in hydrophilic organic solvent such as dimethylformamide, the two dimers of Hb molecule become separate and unfold to a certain extent.

COBALT PORPHYRIN NAFION FILM ON CARBON MICROARRAY ELECTRODE TO MONITOR OXYGEN FOR ENZYME ANALYSIS OF GLUCOSE

A microcarbon array electrode was modified by the placement of a Nafion film containing cobalt tetramethylpyridyl phorphyrin on its surface. This electrode was applied to the analysis of solution glucose when it was further modified by the immobilization of glucose oxidase on the outermost surface of the Nafion by the cross-linking of serum albumin with glutaraldehyde. The concomitant decrease in the concentration of oxygen, as it was consumed in the enzymatic reaction of glucose with glucose oxidase, was determined by either cyclic voltammetry or a double potential step method at the porphyrin-Nafion catalytic electrode. Glucose could be determined in the range of 0.01-4 mM rapidly, without interference from substances such as ascorbate or other saccharides.

Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.

Characterization of degQ(Vh), a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain

Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQ(Vh)), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQ(Vh) was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQVh in T4 was modulated by temperature, possibly through the sigma(E)-like factor. Enzymatic analyses demonstrated that the recombinant DegQVh protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQVh protein were 50 C and pH 8.0. A vaccination study indicated that the purified recombinant DegQVh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQVh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQVh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular beta-agarase. The E.coli strain harboring pAQ1 could express and secrete the chimeric DegQVh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQ(Vh) significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.

Characterization of degQ(Vh), a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain

Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQ(Vh)), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQ(Vh) was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQVh in T4 was modulated by temperature, possibly through the sigma(E)-like factor. Enzymatic analyses demonstrated that the recombinant DegQVh protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQVh protein were 50 C and pH 8.0. A vaccination study indicated that the purified recombinant DegQVh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQVh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQVh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular beta-agarase. The E.coli strain harboring pAQ1 could express and secrete the chimeric DegQVh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQ(Vh) significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.

A thioredoxin with antioxidant activity identified from Eriocheir sinensis

Thioredoxin, with a redox-active disulfide/dithiol in the active site, is the major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state. In the present study, the cDNA encoding thioredoxin-1 (designated EsTrx1) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsTrx1 was of 641 bp, containing a 51 untranslated region (UTR) of 17 bp, a 3' UTR of 306 bp with a poly (A) tail, and an open reading frame (ORF) of 318 bp encoding a polypeptide of 105 amino acids. The high similarity of EsTrx1 with Trx1s from other animals indicated that EsTrx1 should be a new member of the Trx1 sub-family. Quantitative real-time PCR analysis revealed the presence of EsTrx1 transcripts in gill, gonad, hepato-pancreas, muscle, heart and haemocytes. The expression of EsTrx1 mRNA in haemocytes was up-regulated after Listonella anguillarum challenge, reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. In order to elucidate its biological functions, EsTrx1 was recombined and expressed in E. coli BL21 (DE3). The rEsTrx1 was demonstrated to possess the expected redox activity in enzymatic analysis, and to be more potent than GSH in antioxidant capacity. These results together indicated that EsTrx1 could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to bacterial challenge. (C) 2009 Elsevier Ltd. All rights reserved.

Ontogenetic development of digestive enzymes and effect of starvation in miiuy croaker Miichthys miiuy larvae

The ontogenetic development of the digestive enzymes amylase, lipase, trypsin, and alkaline phosphatase and the effect of starvation in miiuy croaker Miichthys miiuy larvae were studied. The activities of these enzymes were detected prior to exogenous feeding, but their developmental patterns differed remarkably. Trypsin activity continuously increased from 2 days after hatching (dah), peaked on 20 dah, and decreased to 25 dah at weaning. Alkaline phosphatase activity oscillated at low levels within a small range after the first feeding on 3 dah. In contrast, amylase and lipase activities followed the general developmental pattern that has been characterized in fish larvae, with a succession of increases or decreases. Amylase, lipase, and trypsin activities generally started to increase or decrease at transitions from endogenous to exogenous feeding or diet changes, suggesting that these enzymatic activities can be modulated by feeding modes. The activities of all the enzymes remained stable from 25 dah onwards, coinciding with the formation of gastric glands and pyloric caecum. These results imply that specific activities of these enzymes underwent changes due to morphological and physiological modifications or diet shift during larval development but that they became stable after the development of the digestive organs and associated glands was fully completed and the organs/glands functioned. Trypsin and alkaline phosphatase were more sensitive to starvation than amylase and lipase because delayed feeding up to 2 days after mouth opening was able to adversely affect their activities. Enzyme activities did not significantly differ among feeding groups during endogenous feeding; however, all activities were remarkably reduced when delayed feeding was within 3 days after mouth opening. Initiation of larvae feeding should occur within 2 days after mouth opening so that good growth and survival can be obtained in the culture.

Purification and characterization of agarases from a marine bacterium Vibrio sp. F-6

Marine bacterium Vibrio sp. F-6, utilizing agarose as a carbon source to produce agarases, was isolated from seawater samples taken from Qingdao, China. Two agarases (AG-a and AG-b) were purified to a homogeneity from the cultural supernatant of Vibrio sp. F-6 through ammonium sulfate precipitation, Q-Sepharose FF chromatography, and Sephacryl S-100 gel filtration. Molecular weights of agarases were estimated to be 54.0 kDa (AG-a) and 34.5 kDa (AG-b) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH values for AG-a and AG-b were about 7.0 and 9.0, respectively. AG-a was stable in the pH range of 4.0-9.0 and AG-b was stable in the pH range of 4.0-10.0. The optimum temperatures of AG-a and AG-b were 40 and 55 degrees C, respectively. AG-a was stable at temperature below 50 degrees C. AG-b was stable at temperature below 60 degrees C. Zn2+, Mg2+ or Ca2+ increased AG-a activity, while Mn2+, Cu2+ or Ca2+ increased AG-b activity. However, Ag+, Hg2+, Fe3+, EDTA and SDS inhibited AG-a and AG-b activities. The main hydrolysates of agarose by AG-a were neoagarotetraose and neoagarohexaose. The main hydrolysates of agarose by AG-b were neoagarooctaose and neoagarohexaose. When the mixture of AG-a and AG-b were used, agarose was mainly degraded into neoagarobiose.

Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K-m value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.

海带根中α-glucosidase 和PTP-1B 靶向生物活性物质的分离纯化及其抗糖尿病作用研究

海带根是一种治疗糖尿病的民间中药，在沿海地区有很长的民间用药历史。食用海带根能够有效降低糖尿病患者的血糖，起到治疗作用。本文目的在于发现海带根中抗糖尿病的天然活性物质并分析它们在糖尿病治疗中的靶点；进一步开发一种低价且无毒副作用的化学类新药或中药新药。 α-glucosidase和 PTP-1B是II型糖尿病的两个重要靶点，海带根提取物能同时作用于这两个靶点。通过抑制这两种酶，降低血糖水平，85%乙醇粗提物对两种酶的IC50分别为1589ug/ml、IC50 1271ug/ml。乙酸乙酯相和石油醚相分别抑制α-glucosidase和 PTP-1B，IC50分别为380ug/ml和220ug/ml。因此以α-glucosidase和 PTP-1B的抑制活性为导向，用天然产物化学的方法对活性成分进行追踪分离，寻找单体活性物质进而鉴定其结构。由于乙酸乙酯相具有α-glucosidase抑制活性，用硅胶柱层析（石油醚：丙酮5：1、1：1），（二氯甲烷：甲醇60：1、20：1、5：1），凝胶柱层析Sephadex LH20（二氯甲烷：甲醇1：1），HPLC （80% 甲醇-水），对α-glucosidase抑制剂进行分离，得到组分IC50 为3.6ug/ml。用质谱仪和核磁共振确定结构。 生物活性测定结果表明α-glucosidase和 PTP-1B是两种不同的物质，分别位于乙酸乙酯相和石油醚相。光照实验和高温实验表明抑制α-glucosidase的活性成分对光照和温度敏感。光照48h或者50℃ 12h而且对α-glucosidase的抑制活性显著降低，TLC检测并用FeCl3显色初步表明抑制α-glucosidase的活性成分可能是多数酚类物质。动物实验显示在1450ug/kg剂量下，乙酸乙酯相能够显著降低糖尿病小鼠血糖，与阴性对照组差异极显著（P<0.01）。表明，海带根提取物在体内和体外均呈现出抗糖尿病活性，是一种潜在的抗糖尿病药物。

羟基自由基降解琼胶及其产物的活性研究

琼胶是一种从石花菜等红藻中提取的，目前生产工艺和结构等方面研究比较成熟的海藻多糖，广泛应用于医药、仪器等行业。但是，海藻多糖因为具有分子量大，粘度大，溶解度较小的等特点，而使其应用范围受到限制。利用降解的手段对其进行修饰，降低分子量和粘度，改善溶解性，可以拓展其应用范围。并且根据文献报道，琼 胶寡糖具有一些特殊的生物活性，如抗氧化性，抗炎症等。因此，对琼胶降解的研究具有生要意义。本研究中，为了选择一种合适的降解方法，进行了几种水解方法的尝试，其中包括在不同湿度和酸度下盐酸水解，过氧化氢和醋酸催化水解，Fenton体系羟基自由基降解。对于酸水解和Fenton体系氧化还原降解方法，通过粘度法对反应的速度进行了比较，表明氧化还原降解反应中琼胶的粘度降低比较快，并且具有代表性和新意，确定为本实验的降解琼胶的方法并对氧化还原降解所得的产物进行了活性实验。通过模仿自然界普遍存在的氧化还原降解反应，利用Vc诱导的Fenton体系产生的羟基自由基氧化还原降解琼胶得到低分子量的琼胶。降解产物经过高速离心、60％乙醇沉淀，除去分子量比较大的降解产物和磷酸盐，得到可溶于60％乙醇的分子量估计小于3000的降争产物，其产率为85％。利用经Sephadex-G25凝胶色谱分离所香的不同分子量的级分进行分子量和α－葡萄糖苷酶抑制活性关系的实验。降解产物对α－葡萄糖苷酶的抑制率和各级分的浓度呈线性正相关，并且各级分的IC_(50)则随着分子量的降低而降低。另外，对所得的降解产物混合物进行了红外吸收光谱、质子去偶核磁共震碳谱和负离子基质辅助激光诱导－飞行时间质谱结构分析。结果表明，氧化还原降解反应的专一性差，在得到寡糖的同时，在光谱图中出现一些比较复杂的副产物的结构信息。最后，根据MTT法的原理，以有体皮肤成纤维细胞为材料，通过紫外线辐射产生自由基造成氧化损伤，研究降解产物对成纤维细胞的保护作用。当无紫外线辐射时，降解产物对成纤维细胞具有显著的促进生长增殖作用：当经UVa、UBb辐射时则可以显著地表现出对损伤的保护作用，并且这种促进生长和保护作用呈显著的量效关系，表明降解产物具有清除基自由基的作用。但是，因为氧化还原降解以应的机理尚不十分明的以及琼羟胶的特殊结构，使得反应的副产物很难预测，也就使得分离工作难以进行，所以，根据目前所得的信息，尚不能确定是降解产物的什么级分产生的以上两种生物活性。

Modulatory effects of ammonia-N on the immune system of Penaeus japonicus to virulence of white spot syndrome virus

To study response to white spot syndrome virus (WSSV) under ammonia stress, Penaeus japonicus were exposed to 5 mg l(-1) ammonia-N and challenged orally with WSSV (NW). Controls consisted of an ammonia-N-exposed control group (N), a WSSV-challenged positive control group (W), and an untreated control group (control). Immune parameters measured were total haemocyte count (THC), haemocyte phagocytosis, plasma protein content and haemolymph enzymatic activities for prophenoloxidase (proPO), alkaline phosphatase (ALP), and nitric oxide synthase (NOS). THC and plasma protein had downward trends with time in all treatment groups (NW, N, and W) in contrast to the untreated control group (control). The percentage phagocytosis, NOS activity, and ALP and proPO activity of W and NW decreased initially then increased from 6 to 78 h (except for NOS and ALP, from 6 to 54 h) before declining thereafter until the end of the experiment. Compared with untreated controls (control), there was a downward trend for all measured parameters in the treatment groups (N, NW, and W), but the degree was W > NW > N. WSSV was detected at 78 h postchallenge in both W and NW. In conclusion, 5 mg l(-1) ammonia-N reduced the immunocompetence of P japonicus and may have decreased the virulence of WSSV (C) 2004 Elsevier B.V. All rights reserved.

Effects of photoperiod on growth, mortality and digestive enzymes in miiuy croaker larvae and juveniles

The growth, mortality and digestive enzymes (trypsin, amylase and lipase) in miiuy croaker Miichthys miiuy larvae and juveniles (2-53 dph) were investigated at four photoperiods: 24L:OD), 18L:6D, 12L:12D and OL:24D. Larvae could not feed at OL:24D and did not survive up to 7 dph. In the 24L:OD, 18L:6D, 12L:12D groups, photoperiod had not significant effects on the growth of the rniiuy croaker younger than 20 dph. However, their total length and specific growth rate (SGR) were significantly larger at 18L:6D and 24L:OD than 12L:12D after 20 dph. Photoperiod also affected the mortality of the first feeding larvae (5 dph). being apparently higher in 5 dph larvae at OL:24D (60%) than at other photopenods (20-27%), but no significant differences in mortality were found among other photoperiods. High mortality of the miiuy croaker in 12L:12D, 18L:6D and 24 L:OD groups mainly occurred from 5 (20-27%) to 11 dph (11-16%) and tended to decrease gradually from 15 dph onwards. Digestive enzymes activities in the rniiuy croaker larvae and juveniles had a similar change trend with age at all photoperiods. They underwent drastic changes with age. The specific activity of lipase was significantly higher at 18L:6D and 24L:0D than 12L:12D, but there were no significant differences in specific activities of either trypsin or amylase among photoperiods. With regard to the total length, SGR, survival and digestive enzyme activities, our findings suggested that the optimal light regime for the culture of miiuy croaker during the early life stage was 18L:6D. (C) 2008 Elsevier B.V. All rights reserved.

Oxalate decarboxylase from Agrobacterium tumefaciens C58 is translocated by a twin arginine translocation system

Oxalate decarboxylases (OXDCs) (E.C. 4.1.1.2) are enzymes catalyzing the conversion of oxalate to formate and CO2. The OXDCs found in fungi and bacteria belong to a functionally diverse protein superfamily known as the cupins. Fungi-originated OXDCs are secretory enzymes. However, most bacterial OXDCs are localized in the cytosol, and may be involved in energy metabolism. In Agrobacterium tumefaciens C58, a locus for a putative oxalate decarboxylase is present. In the study reported here, an enzyme was overexpressed in Escherichia coli and showed oxalate decarboxylase activity. Computational analysis revealed the A. tumefaciens C58 OXDC contains a signal peptide mediating translocation of the enzyme into the periplasm that was supported by expression of signal-peptideless and full-length versions of the enzyme in A. tumefaciens C58. Further site-directed mutagenesis experiment demonstrated that the A. tumefaciens C58 OXDC is most likely translocated by a twin-arginine translocation (TAT) system.