Autodisplay of the protease inhibitor aprotinin in Escherichia coli

The Kunitz type protease inhibitor aprotinin, containing three intramolecular disulfide bonds, was expressed on the surface of Escherichia coli by Autodisplay. For this purpose, the aprotinin gene was fused in-frame to the transporter domain encoding DNA region of the AIDA-I autotransporter protein. Culture of cells supplied with the artificial gene at reducing conditions resulted in the translocation of aprotinin to the cell surface. Correct folding of aprotinin was shown by high affinity to its target enzyme HLE. No surface translocation was detectable under non-reducing conditions, indicating the degradation of aprotinin in the periplasm. By the use of periplasmic-protease defective E. coli strains PW147, PW151, and PW152, under non-reducing conditions, significant amounts of aprotinin appeared in the periplasm but not at the surface. Our results indicate that aprotinin molecules, reaching stable conformation before transport across the outer membrane, are degraded in the periplasm due to proteolysis. In case folding can be prevented, i.e., by blocking disulfide bond formation in the periplasm, aprotinin is translocated and can adopt its active conformation at the cell surface.     

A purification method for tag-free human cystatin C recombinant protein expressed in Escherichia coli

To obtain recombinant cystatin C (CysC) protein, which can be used in immunological diagnostic kits, we focused on the preparation of tag-free CysC. The 6?×?His–TF–CysC fusion protein was found to overexpress in soluble form in cells of BL21-Gold (DE3)/pCold TF–CysC, which had been induced with isopropyl-D-1-thiogalactopyranoside. Subsequently, we established a protein purification method for tag-free CysC using immobilized metal-affinity chromatography and size-exclusion chromatography. In this method, glutathione-S-transferase–human rhinovirus 3C proteases were used to remove the protein tags. High homogeneity of the purified CysC was determined by SDS-PAGE, while the purity of the tag-free CysC was ascertained to be above 95%. With a yield of 25?mg/L from bacterial culture, the biological activity of the tag-free CysC was evaluated as inhibitors like natural CysC. The performance of this purification method was successfully evaluated in the preparation of other low molecular weight heterologous proteins in Escherichia coli.     

Early biochemical effects of Microcystis aeruginosa extracts on juvenile rainbow trout (Oncorhynchus mykiss)

Microcystins (MC) are usually the predominant cyanotoxins associated with cyanobacterial blooms in natural surface waters. These toxins are well-known hepatotoxic agents that proceed by inhibiting protein phosphatase in aquatic biota; recent studies have also reported oxidative stress and disruption of ion regulation in aquatic organisms. In the present study, young trout (Oncorhynchus mykiss) were exposed to crude extracts of Microsystis aeruginosa for four days at 15 °C. The level of microcystins was calculated to confirm the presence of toxins in these crude extracts: 0, 0.75, 1.8 and 5 μg/L. Protein phosphatase measured in the liver increased by at least 3-fold and is significantly as a result of exposure to these sublethal concentrations of crude extract, his indicates an early defense response against protein phosphatase inhibition from cyanotoxins. This was corroborated by the decreased phosphate content in proteins found in the liver and brain. No increase in glutathione-S transferase (GST) activity was observed and lipid peroxidation was unaffected in both liver and brain tissue exposed to the cyanobacterial extracts. The data revealed that the proportion of the reduced (metal-binding) form of metallothionein (MT) decreased by two-fold relative to the control group (with a concomitant increase in the proportion of the oxidized form). The level of phosphate associated with MT increased by 1.5-fold at the highest concentration of crude extract. Acetylcholinesterase (AChE) activity in brain tissue was decreased after exposure to the highest concentration of crude extract, suggesting a slowdown in neural activity. However, no biotransformation processes or detoxification of GST was triggered. Our findings show early sign of biochemical effects of MC-LR in young trout.     

Functional expression of recombinant human stefin A in mammalian and bacterial cells

Recombinant human cysteine protease inhibitor, stefin A, was expressed in both Escherichia coli and BS-C-1 monkey kidney cells utilizing pET and recombinant vaccinia virus systems, respectively. The expressed protein was purified and analyzed by SDS-PAGE and Western blot analysis utilizing a polyclonal antibody against rat cystatin alpha. In both cases the purified protein appeared as a single band corresponding to the molecular weight of stefin A ( approximately 10kDa). Viability of the expressed stefin A was determined by the inhibition of the plant cysteine protease, papain. Recombinant human stefin A expressed in both E. coli and BS-C-1 cells, was shown to almost completely inhibit papain. The expression of a fully functional recombinant human stefin A in the bacterial system provides a highly efficient tool for the production of large quantities of the protein. This can be an important tool in kinetic studies as well as in production of antibodies for other analytical studies (immunoblot, immunohistochemical studies, etc.). Expression in the mammalian cells, on the other hand, can provide a significant research tool to study the functional roles of stefin A in mammalian systems such as regulation of cysteine proteases.     

Cloning of a cDNA Coding for Active Tyrosine Hydroxylase in the Rainbow Trout (Oncorhynchus mykiss): Comparison with Other Hydroxylases and Enzymatic Expression

Abstract: Although catecholamines are of critical importance for neuroendocrine function in teleost fishes, there has been no tool to give access to pretranslational regulation of their synthesis enzymes. In this study, we undertook cloning of the cDNA coding for the tyrosine hydroxylase (TH) of the rainbow trout ( Oncorhynchus mykiss ). First, we looked for a tissue sufficiently rich in TH to make an expression library. The cDNA coding for the rainbow trout TH (rtTH) was then cloned and sequenced. The rtTH sequence encodes a protein of 489 amino acids. Several domains and amino acids required for enzyme activity, like cysteines or phosphorylation sites, are highly conserved between species. Northern blot analysis showed a single rtTH messenger RNA of 4.2 kb expressed in the anteroventral brain. The ability of rtTH to hydroxylate l -tyrosine was analyzed by transient expression of the rtTH cDNA in COS-1 cells. In vitro TH activity, using COS-1 cell extracts, demonstrated that TH activity in transfected cells was 40-fold higher than in untransfected cells. Western blot analysis revealed a single protein of ∼65 kDa in both COS-1 cells and in trout brain. This rtTH cDNA provides us with a tool for further studies on pretranslational regulation of the enzyme in salmonids.     

肠道病毒71型3C蛋白酶在大肠杆菌中的表达及活性研究

本研究利用大肠杆菌表达系统构建肠道病毒71型3C蛋白酶,并进行纯化,对其酶活性进行研究。首先,将3C蛋白酶基因克隆至pET28a载体,在大肠杆菌BL21(DE3)中表达,Ni-NTA柱亲和层析纯化获得3C蛋白酶,经肠激酶酶切去除N端His标签后获得无His标签的3C蛋白酶,再以荧光多肽为底物进行酶活性研究。经过双酶切鉴定和测序证实,重组表达质粒pET28a-3C构建正确,表达的重组3C蛋白酶相对分子质量约22kD;纯化后有无His标签的3C蛋白酶均能催化荧光底物3B-3C,并且两者的酶动力学数据无显著差异,含有His标签的3C蛋白酶Km、Vmax、Kcat分别为22μM、434nM.Min-1、0.0669 Min-1;其最适反应pH为7.0,最佳反应温度为30℃～37℃。本实验成功表达并纯化了重组3C蛋白酶,该酶具有良好的活力,为抗病毒抑制剂、结构蛋白组装、疫苗开发及3C蛋白酶检测方法的研发奠定了基础。     

Compliance and smooth muscle reactivity of rainbow trout (Oncorhynchus mykiss) vessels in vitro

Systemic veins have a profound influence on cardiac output in mammals. Venoregulatory mechanisms have not been adequately studied in fish and their existence has been questioned. In the present study, two characteristics of vascular mechanics, compliance and agonist-induced tension development, were investigated in rainbow trout vessels in vitro. Rapid compliance in the anterior cardinal vein and efferent branchial artery was calculated from step-wise changes in the volume-pressure curve of isolated vessel segments. Agonist-induced tension development was examined in four veins; anterior and posterior cardinals, intestinal and duct of Cuvier. Venous compliance was not altered in response to epinephrine, norepinephrine or angiotensin II, while efferent branchial artery compliance was decreased by 10^-6 mol·l^-1 epinephrine and norepinephrine but not angiotensin II. The ratios of venous to arterial compliance in vessels from two rainbow trout strains were similar (21:1 and 32:1) and consistent with the ratio reported for mammalian viens (24:1). Trout veins contracted in response to agonists in both an, agonist- and vesselspecific manner. The greatest tension per vessel wet weight was produced in anterior cardinal vein. The response pattern of anterior cardinal vein and duct of Cuvier were similar; acetylcholine, arginine vasotocin, epinephrine and norepinephrine, and the thromboxane A₂ agonist, U-44,069, produced approximately identical contractions, whereas angiotensin II was virtually ineffective. Conversely, angiotensin II was more potent than epinephrine in posterior cardinal vein. In cumulative dose-response experiments, epinephrine was equipotent in anterior cardinal vein and duct of Cuvier, whereas the latter was less sensitive to acetylcholine. Both atrial natriuretic peptide and sodium nitroprusside relaxed precontracted veins. This is the first study to determine compliance in fish vessels and the contractile nature of different rainbow trout veins. These findings suggest that venous tone and therefore cardiac output in fish may be regulated by neural or humoral mechanisms.Abbreviations ACH acetylcholine - ACV anterior cardinal vein - ANG II salmon asn¹-val⁵ angiotensin II - ANP rat atrial natriuretic peptide - AVT arginine vasotocin - DNR Department of Natural Resources - DOC duct of Cuvier - EBA efferent branchial artery - EC₅ threshold dose producing 5% maximal contraction - EC₅₀ dose producing 50% maximal contraction - EPI epinephrine - HI K⁺ 80 mmol·l^-1 - KCl IV, intestinal vein - NEPI norepinephrine - PBS phosphate buffered saline - PCV posterior cardinal vein - SNP sodium nitroprusside - U-44,069 thromboxane A₂ agonist     

Evolution of Hox Clusters in Salmonidae: A Comparative Analysis Between Atlantic Salmon (Salmo salar) and Rainbow Trout (Oncorhynchus mykiss)

We studied the genomic organization of Hox genes in Atlantic salmon (Salmo salar) and made comparisons to that in rainbow trout (Oncorhynchus mykiss), another member of the family Salmonidae. We used these two species to test the hypothesis that the Hox genes would provide evidence for a fourth round of duplication (4R) of this gene family given the recent polyploid ancestry of the salmonid fish. Thirteen putative Hox clusters were identified and 10 of these complexes were localized to the current Atlantic salmon genetic map. Syntenic regions with the rainbow trout linkage map were detected and further homologies and homeologies are suggested. We propose that the common ancestor of Atlantic salmon and rainbow trout possessed at least 14 clusters of Hox genes, and additional clusters cannot be ruled out. Salmonid Hox cluster complements seem to be more similar to those of zebrafish (Danio rerio) than medaka (Oryzias latipes) or pufferfish (Sphoeroides nephelus and Takifugu rubripes), as both Atlantic salmon and rainbow trout have retained HoxCb ortholog, which has been lost in medaka and pufferfish but not in zebrafish. However, our data suggest that phylogenetically, the homologous genes within each cluster express mosaic relationships among the teleosts tested and, thus, leave unresolved the interfamilial relationships among these taxa. Sequence data from this article have been deposited within the EMBL/GenBank Data Libraries under the following accession numbers: AY677341, AY677342, AY677343, AY677344, AY677345, AY677346, AY677347, AY677348, AY677349, AY677350, AY677351, AY677352, AY677353, AY677354, AY677355, AY677356, AY677357, AY677358, AY677359, AY677360, AY677361, AY677362, AY677363, AY677364 and AY677365. [Reviewing Editor: Dr. Axel Meyer]     

Calcium signalling in isolated single chromaffin cells of the rainbow trout (Oncorhynchus mykiss)

Chromaffin cells were isolated from the posterior cardinal vein of rainbow trout (Oncorhynchus mykiss) to assess their suitability as a model system for studying mechanisms of catecholamine secretion in fish and to evaluate intracellular calcium changes associated with cholinoreceptor stimulation. Immunocytochemistry in concert with fluorescence microscopy was employed to identify characteristic chromaffin cell proteins and thus to confirm the presence of these specific cells in suspensions and cultures. Dopamine-β-hydroxylase, an enzyme of the catecholamine-synthesising Blaschko pathway, was identified in cytoplasmic vesicles of the isolated chromaffin cells. The actin filament-severing protein, scinderin, was co-localized with actin in the sub-plasmalemmal membrane of these chromaffin cells. Intracellular calcium [Ca²⁺]_i was measured in single chromaffin cells by microspectrofluorometry using the fluorescent dye Fura-2. Significant increases in [Ca²⁺]_i were observed in chromaffin cells in response to depolarisation of the cell membrane by high concentrations of K⁺ or by the stimulation of the cell by the cholinergic receptor agonists, nicotine, acetylcholine or carbachol. The response to the reversible agonist, nicotine, was attenuated following addition of the nicotinic receptor blocker hexamethonium. Such attenuation, however, did not occur when hexamethonium was added after stimulation with the non-specific irreversible cholinergic agonist, carbachol. These results demonstrate the presence of functional cholinoreceptors, linked to intracellular calcium signalling, on isolated trout chromaffin cells and reveal the potential of these cells as a model system for studying aspects of catecholamine secretion in fish.     

Major histocompatibility class II genes in rainbow trout (Oncorhynchus mykiss) exhibit temperature dependent downregulation

Major histocompatibility (MH) class II receptors are expressed on the surface of specialized antigen-presenting cells in vertebrate immune systems. Their function is to present peptides derived from exogenous pathogens to CD4+ T cells. Variation in the level of expression of these genes has been linked to pathogenesis in various diseases. Very little has been published on the function of MH class II receptors in teleost fish to date. In this study, we have produced polyclonal antibodies recognizing MH class II alpha and beta proteins of rainbow trout and employed them to characterize the expression pattern of these genes. Deglycosylation using N-glycosidase F and endoglycosidase H showed that MH class II alpha is glycosylated in rainbow trout. MH class II beta was also found to be glycosylated as reported previously. Results from Northern blotting revealed that the expression of these genes was not affected by exposure of rainbow trout to temperature of 5°C. However, at 2°C, downregulation of MH class II alpha and beta genes was evident at both the mRNA and protein levels as assessed by Northern and Western blotting, respectively. Because MH class II antigens play an important role in generating an immune response to bacterial and fungal pathogens, downregulation of these genes at low temperature could account for the susceptibility of fish to low temperature-related diseases such as bacterial cold-water disease and winter saprolegniosis.     

Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus)

Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of alpha-lactalbumin, beta-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young.     

Enhanced Aeromonas salmonicida bacterin uptake and side effects caused by low frequency sonophoresis in rainbow trout (Oncorhynchus mykiss)

Low frequency sonophoresis (LFS) has been recognized as one of the most advanced technologies in transdermal delivery of substances, due to the modification of the stratum corneum lipid bilayer, in focal skin applications in mammals. Based on these findings, LFS has been suggested as a potential technology to be used for enhancement in immersion fish vaccination. In contrast to mammals where LFS is applied to discrete regions of the skin, in fish the whole individual needs to be exposed for practical purposes. The current study evaluated the impact of LFS at 37 kHz on the uptake of an Aeromonas salmonicida bacterin and side effects of the treatment in rainbow trout. Quantitative real time PCR (qPCR) and immunohistochemistry were used to examine the bacterin uptake into skin and gill tissue. Side effects were assessed by behavioural examination, histology and blood serum analysis. The sonication intensity of 171 mW/cm² was enough for increasing skin permeability, but caused heavy erratic swimming and gill haemorrhages. Sonication intensities as low as 105 mW/cm² did not modify skin permeability and enhanced the bacterin uptake into the gill tissue by factor 15 compared to conventional immersion. Following sonication, the gill permeability for the bacterin decreased after 20 min and 120 min by factor 3 and 2, respectively. However, during sonication, erratic swimming of the fish raised some concerns. Further reduction of the sonication intensity to 57 mW/cm² did not induce erratic swimming, and the bacterin uptake into the gill tissue was still increased by factor 3. In addition, a decreasing albumin–globulin ratio in the serum of the rainbow trout within 40 min revealed that LFS leads to an inflammatory response. Consequently, based on both increased bacterin uptake and the inflammatory response, low intensity LFS has the potential to enhance vaccine immunity without significant side effects.     

Rainbow trout (Oncorhynchus mykiss) recombinant IL-1beta and derived peptides induce migration of head-kidney leucocytes in vitro.

The present work provides the first information concerning the chemoattractant activity of trout recombinant IL-1beta and its derived peptides, referred to as P1, P2 and P3. The predicted rainbow trout mature interleukin-1beta peptide was produced as a recombinant protein in Escherichia coli. The first peptide, P1, corresponded to fragment 146-157 (YVTPVPIETEAR) of the trout sequence and had an MW of 137 kDa. It was equivalent to a region known to be part of the receptor binding domain from the mammalian crystal structure of IL-1beta complexed to its receptor. P2 was used as control peptide, consisting of the same 12 amino acids as P1, but arranged in a random sequence (VVEEYIRAPPTT). P3 was synthesised to complex with an adjacent region of the IL-1 receptor, and corresponded to fragment 207-216 (YRRNTGVDIS) of the trout sequence, with an MW of 1.18 kDa. Migration was stimulated when leucocytes were exposed to concentrations of > or = 10 ng ml(-1) rIL-1beta. Peptide P3 also induced leucocyte migration, with an optimal dose of 0.25 mM being recorded. While P1 had no effect on cell migration when used alone, synergism was evident as a consequence of combining P1 with a suboptimal dose (0.01 mM) of P3. No synergism occurred when cells were exposed to a combination of P3 and the control peptide P2.     

叉角厉蝽丝氨酸蛋白酶及抑制剂基因的克隆与表达分析

为明确叉角厉蝽Eocanthecona furcellata (Wolff)丝氨酸蛋白酶基因EfSP1及抑制剂基因EfSPI20的基因序列特征和时空转录特征,为其生理功能研究奠定基础。利用PCR克隆技术获得叉角厉蝽唾液腺EfSPI20和EfSP1的完整开放阅读框(Open reading frame, ORF)序列,使用生物信息学软件进行序列分析以及系统进化分析,采用实时荧光定量PCR (Real time quantitativate PCR,RT-qPCR)分析两个基因分别在叉角厉蝽不同发育时期和组织中的表达特征。结果表明,EfSPI20与EfSP1基因完整开放阅读框长度分别为378 bp和921 bp,分别编码125个氨基酸和306个氨基酸,预测均为亲水蛋白质,理论分子量分别为13.48 kDa和33.82 kDa,等电点分别为6.68和5.80,分别有30个和23个氨基酸残基的信号肽序列,EfSPI20有跨膜结构域,EfSP1无跨膜结构域。序列比对显示叉角厉蝽EfSPI20与茶翅蝽Halyomorpha halys PPI同源性最高,氨基酸序列一致性达58%;EfSP1与稻绿蝽Nezara viridula SP同源性最高,氨基酸序列一致性达66%;系统发育树显示叉角厉蝽与同为蝽科的茶翅蝽和稻绿蝽物种亲缘关系近。EfSPI20基因在雌雄成虫和唾液腺中高表达,推测EfSPI20可能具有抑制胰蛋白酶活性的功能和与叉角厉蝽的捕食消化相关;EfSP1基因在卵期、卵巢和肠道中高表达,推测EfSP1可能与叉角厉蝽的生殖功能和蛋白消化相关。