Interaction of grammistins with lipids and their antibacterial activity

ABSTRACT: Grammistins are hemolytic and ichthyotoxic peptides in the skin secretion of soapfishes and are structurally characterized by their abundance in amphiphilic α-helicity. In the present study, their interaction with lipids and lipid vesicles as well as antibacterial activity were examined using four grammistins (Gs 1 and Gs 2 from Grammistes sexlineatus and Pp 1 and Pp 3 from Pogonoperca punctata ). The hemolytic activity of grammistins was inhibited by phospholipids but not by cholesterol. Moreover, grammistins released carboxyfluorescein entrapped within liposomes made of phosphatidylcholine. In contrast, grammistins were found to have antibacterial activity with a broad spectrum against nine species of bacteria, including both Gram-negative and Gram-positive groups. The potency of their antibacterial activity was not related to that of hemolytic activity, suggesting that grammistins bind to membrane phospholipids but lyse erythrocyte and bacterial membranes via different mechanisms. Conclusively, grammistins are new members of the family of cell non-selective membrane-lytic peptides with amphiphilic α-helices, being similar to pardaxins, which are secreted from the skin of soles, and to melittin, which is derived from bee venom.     

Primary and secondary structures of grammistins, peptide toxins isolated from the skin secretion of the soapfish Pogonoperca punctata

SUMMARY: Six peptides (grammistins Pp 1, 2a, 2b, 3, 4a and 4b) with both hemolytic and ichthyotoxic activities were isolated from the skin secretion of the soapfish Pogonoperca punctata by gel filtration and reverse-phase high performance liquid chromatography. They were completely sequenced and found to be simple peptides differing from the previous suggestion that they are peculiar peptides with tertiary amine or quaternary ammonium base moieties. Grammistins Pp 1, 2a and 2b were composed of 13 residues, Pp 3 of 25 residues and Pp 4a and 4b of 24 residues. While grammistins Pp 4a and 4b were identical and highly homologous with grammistin Gs 2 previously isolated from another soapfish Grammistes sexlineatus , the other grammistins did not have homologies with any proteinic or peptidic toxins known to date. Judging from circular dichroism data, helical wheel projections and hydrophobic moment plots, all the isolated grammistins are surface-seeking peptides with an abundance in amphiphilic α-helicity, similar to pardaxins from the skin secretion of two species of soles and melittin from bee venom as well as the G. sexlineatus grammistins.     

Recovery of Proteins from Squid By-Products with Enzymatic Hydrolysis and Increasing the Hydrolysate’s Bioactivity by Maillard Reaction

ABSTRACT

The Maillard reaction is an effective method for enhancing the activity of bioactive peptides. In this study, squid by-product hydrolytic peptides (SPHPs) were obtained. The effects of the Maillard reaction on the antioxidant and antibacterial activities of the SPHPs were explored. The results showed most of the peptides in the < 500 Da fraction of SPHPs reacted with D-arabinose to form the 500–1000 Da fraction (89.60%). The Maillard reaction products (MRPs) possessed more potent antioxidant activities, including free radical scavenging activity, ferrous chelating capacity, and reducing power, than the SPHPs. Although both the SPHPs and MRPs had broad-spectrum antibiotic activities and could inhibit the growth of both gram-negative and gram-positive bacteria, the antibacterial activities of the MRPs were higher than those of the SPHPs. Furthermore, the efficacy of the MRPs lasted significantly longer than that of ampicillin and could last for more than 30 days. In addition, both the SPHPs and MRPs had good functional properties and many potential applications. The results suggested that the Maillard reaction has potential to be used to improve the activities and physicochemical properties of bioactive peptides.     

Antioxidant and ACE Inhibitory Activities of Kawakawa (Euthynnus affinis) Protein Hydrolysate Produced by Skipjack Tuna Pepsin

ABSTRACT

Pepsin enzyme from skipjack tuna was extracted for the production of kawakawa (Euthynnus affinis) fish protein hydrolysate. Using ultra-fractionation, Kawakawa protein hydrolysates were separated into four different fractions, including fractioned protein hydrolysate I (FPH I) (< 1 kDa), FPH-II (1–3 kDa), FPH-III (3–10 kDa), and FPH-IV (> 10 kDa). The antioxidant activity was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, inhibition of linoleic acid oxidation, reducing power tests, and chelating activity of metal ions. Results indicated that FPH II fraction peptides had higher antioxidant activity in comparison with the other fractions, followed by FPH I. Further, the fractions were evaluated for angiotensin converting enzyme (ACE) inhibition, and IC₅₀ value ranged from 0.45 to 1.86 mg/ml with higher activity in FPH I (IC₅₀ 0.45). Finally, the amino acid profile of different fractions was analyzed. The fractions exhibited significant amounts of hydrophobic amino acids, which could perform as hydrogen donors, frustrate the free radicals, and inhibit the ACE. The recovered pepsin from the viscera was used to produce hydrolysates with good biological activities. Peptides lower than 3 KDa had antioxidant activity as positive controls and significant ACE activity. These are very important findings that could be used to conduct further research in a preclinical study of these peptides.     

鱼类抗菌肽的研究进展

抗菌肽是一类广泛存在于生物界的小分子短肽,具有广谱的抗菌、抗病毒、抗寄生虫及抗癌等生物活性。它是鱼类自身先天免疫系统的重要组成部分,结构复杂,种类多样。本文综述了目前发现的鱼类抗菌肽的结构特点、种类、生物学功能、抗菌机制以及基因工程上的研究进展。     

Thermal stability of the synthetic peptides with the sequence of fish fast skeletal muscle tropomyosin

Tropomyosins from fish skeletal muscle show high amino acid sequence homology, although their thermal stability is clearly different among species. In order to determine the regions that are responsible for the stability of this protein, five synthetic peptides of 30mer were synthesized by Fmoc method, based on the sequence of walleye pollack Theragra chalcogramma fast skeletal muscle tropomyosin, namely, N terminal Met¹-Lys³⁰, the variable region Asp⁸⁴-Leu¹¹³, the middle region Val¹²⁸-Ala¹⁵⁷, the region containing the conservative Cys (Leu¹⁷⁶-Lys²⁰⁵), and C terminal Asp²⁵⁵-Ile²⁸⁴. The thermal stability of these peptides was measured by circular dichroism and differential scanning calorimetry. The helical contents of these peptides were decreased in a temperature-dependent manner, although they showed no clear melting temperature, suggesting that the enthalpy necessary for the complete denaturation of these peptides was low. Peptides Asp²⁵⁵-Ile²⁸⁴ and Asp⁸⁴-Leu¹¹³ showed the highest and second highest α-helical contents, respectively, and the other peptides gave rise to lower α-helical contents.     

Production, release and olfactory detection of sex steroids by the tench (Tinca tinca L.)

The present study is concerned with pheromone communication in tench (Tinca tinca L.), establishing firstly whether males have a high olfactory sensitivity to some typical teleost sex steroids and prostaglandins; and secondly whether males and females might be able to synthesise and release some of these steroids into the water. The C₂₁ steroid, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was found to give large electro-olfactogram responses with an estimated threshold of detection of 10⁻¹² M. The male tench were equally sensitive to glucuronidated 17,20β-P (10^−11.6 M) but 100 times less sensitive to sulphated 17,20β-P (11^−9.7 M). Preliminary data from cross-adaptation studies suggest that both the free and conjugated forms are detected by the same olfactory receptor(s). Male tench also had high olfactory sensitivity to prostaglandin F_2α (PGF_2α) and 15-keto PGF_2α (11^−11.5 and 10^−11.4 M). They were relatively insensitive, however, to testosterone (T), androstenedione (AD), 11-ketotestosterone (11-KT), 17β-oestradiol (E₂), 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) and 17,20α-dihydroxy-4-pregnen-3-one (17,20α-P). Radioimmunoassays were used to measure the steroids in plasma and water and all samples were processed for the measurement of free, sulphated and glucuronidated fractions. In females, free 17,20β-P, 17,20α-P, free and glucuronidated T, and AD in plasma showed the largest increases in response to injection with mammalian gonadotropin-releasing hormone analogue (GnRHa) or Ovaprim (a mixture of GnRHa and a dopamine inhibitor). Free 17,20β-P was released into the water at the greatest rate. Plasma concentrations of the two conjugated forms of 17,20β-P were also elevated 18 h after the administration of GnRHa, but not by as much as the free steroid. In males, AD and 11-KT showed the greatest increase in response to GnRHa and were moreover released into the water at a higher rate in the treated group than in the control. The data support a possible pheromonal role for free and glucuronidated 17,20β-P. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sex steroids in intersexual fishes

Studies of thein vitro gonadal steroidogenesis in intersexual fishes, using labelled testosterone as precursor, showed large species variation. The protogynousMonopterus albus produced predominantly 5α-reduced metabolites while the protandrousRhabdosargus sarba produced mainly 5β-reduced products. Both fishes synthesized 11-oxotestosterone; the synthesis of which appeared to mediate mainly through adrenosterone inM. albus butvia 11β-hydroxytestosterone inR. sarba. When the plasma levels of androstenedione, testosterone, 11-oxotestosterone, 11β-hydroxytestosterone, estrone and 17β-estradiol among the male, intersexual and female phase of the same species were compared, available data showed that either there was no obvious difference among the different sexual phases or the differences could be accounted for by the seasonal reproductive activities of the animal. Except for androstenedione, there are no marked changes in plasma testosterone, 11-oxotestosterone, 11β-hydroxytestosterone, estrone and 17β-estradiol levels in the intersexual phase compared with the female and male, it is unlikely that these classical sex steroids act as a primary trigger of natural sex reversal in these fishes; the role of androstenedione awaits further elucidation. Department of Zoology, University of Hong Kong     

Acute effects of benzo[a]pyrene on liver phase I and II enzymes,and DNA damage on sea bream <Emphasis Type="Italic">Sparus aurata</Emphasis>

In the present study biotransformation and detoxification responses to acute exposure to the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) were investigated in the liver of Sparus aurata (sea bream). Sexually immature gilthead sea bream were treated by intraperitoneal injection of B[a]P (20 mg kg⁻¹) for 6, 12, 24, and 48 h. B[a]P accumulation was quantified in sea bream liver by mean of gas phase chromatography (GPC-MS) after the various exposure periods. The following biological responses were measured: (1) ethoxyresorufin-O-deethylase (EROD) activity, as a phase I biotransformation parameter; (2) liver glutathione S-transferase (GST) activity as a phase II conjugation enzyme. DNA damage was assessed over time using the single-cell gel electrophoresis comet assay. B[a]P bioaccumulation in the liver resulted in a biphasic curve with an increasing uptake up to 5.55 ± 0.67 μg g⁻¹ dry weight after only 6 h exposure and 4.67 ± 0.68 μg g⁻¹ dry weight after 48 h exposure. EROD activity showed a nonsymmetrical bell-shaped kinetic with a maximum at 24 h and lower but significant activities at 12 and 48 h with respect to control animals. Hepatic GST activities were only significant after 48 h exposure. Comet assay showed an increase in liver cells DNA damage with a maximum after 48 h exposure reaching up to 12.17 %DNA in the tail.     

大眼金枪鱼头蛋白酶解物1 ku超滤组分的抗氧化活性及其理化性质

研究大眼金枪鱼头蛋白酶解物1 ku超滤组分体外的还原力、自由基清除能力及对衰老小鼠体内抗氧化能力的影响,分析1 ku超滤组分的一般成分、氨基酸组成及分子量分布,为进一步分离纯化金枪鱼头抗氧化肽提供基础。体外结果显示,1 ku超滤组分对羟基自由基、超氧阴离子和DPPH自由基的清除活性随浓度的增加而增强,IC50分别为1.38、0.73与0.93mg/mL,还原力也随浓度的增加而增大,在浓度为12.5 mg/mL时为0.763;体内结果显示,灌胃30 mg/kg的1 ku超滤组分连续42 d,D-半乳糖致衰老小鼠肝组织的超氧化物歧化酶(SOD)活性、肝组织和血清的谷胱甘肽过氧化物酶(GSH-PX)活性显著提高(P<0.05),血清丙二醛(MDA)含量显著降低(P<0.01);理化分析结果显示,1 ku超滤组分(干基计)蛋白质含量为96.40%,脂肪0.11%,灰分4.86%,疏水性氨基酸占氨基酸总量的35.8%,活性组分分子量在1 802~2 519 u和422~922 u。     

Epidermal response of the Japanese eel to environmental stress

Nonspecific responses of Japanese eels to environmental stress were monitored by assaying various lytic activities in eel epidermal extract. In fish maintained at 10 and 30 °C for up to 10 days, epidermal proteolytic activities due to serine protease and aminopeptidase and hemolytic activity varied within a 2-fold value range. Other proteolytic activities, due to cathepsins B and L, in the fish at 30 °C increased for up to 8 days and were 3.4 and 2.9-fold over those in fish maintained at 10 °C, respectively. This was accompanied by a 3.0-fold increase in bacteriolytic activity. Other forms of stress were exerted on the fishes by immersing them in a suspension of Flavobacterium columnare or giving them intraperitoneal injections of Edwardsiella tarda over 72 h. Although serine protease and aminopeptidase activities and hemolytic activity in the fishes exposed to F. columnare changed marginally, and were similar to those in the control fish, cathepsins B and L activities in the infected fishes increased more than 1.5-fold over their initial values over a 48 h period, along with a 4.5-fold increase in bacteriolytic activity. No marked change was detected in any of the lytic activities of the fishes exposed to E. tarda. These findings indicate that epidermal cathepsins B and L probably participate in bacteriolysis associated with Japanese eel skin and that their activities are elicited by environmental stimuli and may be an important nonspecific response of eels. Abbreviations: Cbz – carbobenzoxy; MCA – 4-methylcoumaryl-7-amide.     

淇河鲫不同组织器官溶菌酶与抑菌活性比较

选择天然三倍体淇河鲫(Carassius auratus in Qihe River),通过试验研究其不同组织器官溶菌酶和抑菌活性,以了解淇河鲫不同组织器官的抗菌能力。结果表明,淇河鲫不同组织器官溶菌酶和抑菌活性存在差异,溶菌酶活性测定值依次为:肝胰脏肾脏鳃脑肠脾脏;抑菌活性测定值依次为:肝胰脏肾脏脑鳃肠脾脏(P0.01);淇河鲫抗菌活性较高的组织为肝胰脏和肾脏,且肝胰脏又极显著高于肾脏(P0.01),肾脏和鳃的抗菌能力极显著高于脾脏(P0.01);表明鱼类不同组织器官免疫能力和抗菌活性存在差异,这种差异与组织器官的生理功能和抗病响应机制不同有关;同时说明鱼类不同组织器官在致病菌干预过程中将发挥不同的作用。     

Effect of extracts from heshiko , a fermented mackerel product, on cholesterol metabolism in Wistar rats

Heshiko extract was separated into peptide and amino acid fractions by ion-exchange column chromatography. Heshiko extract and these fractions were administered to rats in a diet enriched with lipid and cholesterol for 30 days. In the heshiko extract group, increase of triglyceride, total cholesterol, and low-density lipoprotein (LDL)-cholesterol levels in plasma and accumulation of total lipids in the liver were suppressed, while amounts of both lipid and cholesterol excreted to feces were increased. Administration of heshiko extract tended to increase fecal bile acids and promoted the activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthesis of bile acid from cholesterol in the liver. However, activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol synthesis system in the liver, decreased due to regulation by the feedback of lipid transportation from diet to the liver. The same effect was observed in the peptide and free amino acid fraction groups. These data suggest that both excretion of cholesterol and bile acid to feces and promotion of cholesterol 7α-hydroxylase activity are related to the hypocholesterolemic effects of heshiko extract and that both peptides and amino acids contribute to that effect.     

家蝇抗菌肽对凡纳滨对虾生长性能及免疫相关指标的影响

通过8周的生长实验研究了饲料中添加家蝇幼虫抗菌肽提取物对初始体质量为(0.86±0.01)g的凡纳滨对虾(Litopenaeus vannamei)的生长性能、体成分及免疫相关指标的影响。家蝇抗菌肽提取物添加量分别为0、1000mg/kg、2000mg/kg、3000mg/kg、4000mg/kg、5000mg/kg,每组饲料设4个重复,每个重复饲养40尾虾。结果显示,在一定添加水平范围内,家蝇抗菌肽能显著提高凡纳滨对虾成活率、增重率、特定生长率、饲料效率(P0.05),当抗菌肽提取物添加量为2000～3000mg/kg时,以上4个指标均达最高,且显著高于对照组(P0.05)。以增重率为评价指标,凡纳滨对虾饲料中家蝇抗菌肽提取物的最适添加量为2900mg/kg。当抗菌肽提取物添加量为2000～3000mg/kg时能显著提高虾体的粗蛋白和灰分含量,虾体粗脂肪不受添加抗菌肽水平的显著影响。添加2000～5000mg/kg抗菌肽提取物组与对照组相比能显著提高对虾血细胞数量;添加3000mg/kg抗菌肽提取物时对虾血细胞吞噬率最高。当抗菌肽提取物添加量为2000～3000mg/kg时,对虾血清中酚氧化酶PO、过氧化物酶POD、碱性磷酸酶AKP、溶菌酶LZM的活性和总抗氧化能力T-AOC均维持在一个较高的水平,且显著高于对照组;超氧化物歧化酶SOD的活性在各组组间差异不显著(P0.05)。肝胰腺中AKP、SOD、T-AOC活性各组间差异均不显著(P0.05);LZM活性以3000mg/kg组最高,显著高于其他组。结果表明,饲料中添加适量的家蝇抗菌肽对凡纳滨对虾有一定的促生长作用,并能提高对虾的免疫相关指标。     

Effect of extracts from <Emphasis Type="Italic">narezushi</Emphasis>, a fermented mackerel product,on cholesterol metabolism in Wistar rats

Narezushi extract was separated into peptide and nonpeptide fractions by ion-exchange column chromatography. The narezushi extract and fractions were administered to rats in a diet enriched with lipid and cholesterol for 30 days. In the narezushi extract and nonpeptide fraction groups, increases in triglyceride, total cholesterol, and low-density lipoprotein-cholesterol levels in the plasma and accumulation of total lipids and triglyceride in the liver were suppressed, while both lipid and cholesterol fecal excretion were increased. In the peptide fraction group, these effects were also observed, except for the suppressing effect on liver lipid accumulation. Narezushi extract administration tended to increase fecal bile acids and promoted the activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthesis of bile acid from cholesterol in the liver. However, the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol synthesis system in the liver, decreased due to regulation by the feedback of lipid transportation from diet to the liver. These results suggest that both the increase in cholesterol and bile acid fecal excretion and the promotion of cholesterol 7α-hydroxylase activities are related to the hypocholesterolemic effects of narezushi extract. Amino acids and organic acids, which are abundantly contained in the nonpeptide fraction, seemed to have more intensive hypocholesterolemic effects than peptides existing in the peptide fraction.     

Biochemical and molecular differences in diploid and triploid ocean-type chinook salmon (<Emphasis Type="Italic">Oncorhynchus tshawytscha</Emphasis>) smolts

There is increasing evidence for complex dosage effects on gene expression, enzyme activity and phenotype resulting from induced ploidy change. In this study, ocean-type chinook salmon were bred using a 2 × 2 factorial mating design to create four families and test whether triploidization resulted in changes in growth performance and smolting. Eggs were pressure shocked after fertilization to create triploid fish from a subset of each family. In June, fish were sampled for size, plasma insulin-like growth factor 1 (IGF-1), gill Na⁺–K⁺-ATPase activity, and expression of two Na⁺–K⁺-ATPase α subunits in the gill. Diploids were significantly heavier than triploids, and there were significant differences due to family. Despite a significant positive correlation between plasma IGF-1 and fish size, plasma IGF-1 did not differ between diploid and triploid smolts. Diploids also had significantly greater gill Na⁺–K⁺-ATPase enzyme activities than triploids and there was a strong family effect. Gill Na⁺–K⁺-ATPase α1b isoform expression differed significantly by family, but not ploidy, and generally families with lower Na⁺–K⁺-ATPase enzyme activity had higher α1b isoform gene expression. Na⁺–K⁺-ATPase α1a isoform expression did not differ among any of the groups. Although diploids were larger and had higher specific activities of Na⁺–K⁺-ATPase in the gills, there was no difference in gene expression or circulating hormone levels. The strong family effect, however, suggests that strain selection may be useful in improving performance of triploids for aquaculture.     

Identification of a protein kinase which phosphorylate α4 subunit of the 26S proteasome in goldfish oocytes

To investigate the regulatory mechanism for the proteasome in the meiotic cell cycle, we purified the 26S proteasome from immature (in G2-phase) and mature (in M-phase) oocytes, and compared its subunits by immunoblotting. A monoclonal antibody, GC3β (anti-goldfish 20S proteasome component 3β) cross-reacted with two bands in the 26S proteasome from immature oocytes, however the upper band was absent in the 26S proteasome from mature oocytes. cDNAs which encode the α4 subunit of goldfish 20S proteasome (α4 _ca ) were isolated by an immuno-screening method using GC3β. Phosphatase treatment of the 26S proteasome revealed that a part of α4 _ca phosphorylated in G2-phase and dephosphorylated in M-phase. By the assay using recombinant α4 _ca as a substrate, a kinase was purified by column chromatographs. Amino acid sequence analysis was performed for resulting partial purified fraction. A protein band, which well corresponded to the kinase activity, was identified as Casein kinase-1α (CK-1α). The result suggests that CK-1α phosphorylate α4 subunit of the 26S proteasome in immature oocyte of goldfish. This revised version was published online in July 2006 with corrections to the Cover Date.     

Studies on digestibility and digestive enzyme activities in Labeo rohita (Hamilton) juveniles: effect of microbial α-amylase supplementation in non-gelatinized or gelatinized corn-based diet at two protein levels

A 60-days feeding trial was conducted to delineate the effect of both gelatinized and non-gelatinized corn with or without supplementation with exogenous α-amylase at two dietary protein levels (35% and 28%) on dry matter digestibility, digestive enzymes and tissue glycogen content of Labeo rohita juveniles. Three hundred and sixty juveniles (average weight 10±0.15 g] were randomly distributed into 12 treatment groups with each of two replicates. Twelve semi-purified diets containing either 35% or 28% crude protein were prepared by including gelatinized (G) or non-gelatinized (NG) corn as carbohydrate source with different level of microbial α-amylase (0, 50, 100 and 150 mg kg⁻¹). The dry matter digestibility of G corn fed groups was significantly higher (P < 0.05) than that of the NG corn fed groups. Hepatosomatic index (HSI), liver glycogen and intestinal amylase activity of G starch fed groups were significantly higher (P < 0.05) than those of the NG corn fed groups. However, the reverse trend was found for gastrosomatic index (GSI), muscle glycogen and intestinal protease activity. Addition of 50 mg α-amylase kg⁻¹ feed improved the dry matter digestibility of NG starch fed groups, which was similar to that of the G corn fed groups or NG corn supplemented with 100/150 mg α-amylase kg⁻¹ feed. HSI, liver glycogen and intestinal amylase activity were significantly increased (P < 0.05) at minimum level of α-amylase in the feed (50 mg kg⁻¹) and did not increase due to further inclusion of amylase in the diet. Supplementation with α-amylase at 50 mg kg⁻¹ increased the intestinal amylase activity beyond which no significant changes were observed. Protease activity of liver and intestine was highest (P < 0.05) in higher crude protein (CP) fed groups, but protease activity of the intestine was significantly higher in the α-amylase supplemented groups. Hence, it was concluded that feed with 28% CP containing either G corn without α-amylase or NG corn with 50 mg α-amylase kg⁻¹ may be used as the alternative carbohydrate source for L. rohita juveniles.     

Application of α-glucosidase activity and drug susceptibility tests to epidemiological studies on the fish pathogen <Emphasis Type="Italic">Nocardia seriolae</Emphasis>

This study determined the minimum inhibitory concentrations (MICs) for oxytetracycline hydrochloride (OTC) and erythromycin (Em), along with the α-glucosidase (α-glu) activities in 110 Nocardia seriolae strains isolated in Miyazaki and Kagoshima prefectures in 2008–2009. The strains were examined for the presence of the tet(K), tet(L), tet(M), tet(O), tet(S), erm(A), erm(B), mph(A), mef(A), and msr(D) genes. All the α-glu-positive strains (n = 15) were OTC resistant and Em sensitive, with MICs of 32–64 and <0.125 μg/ml, respectively. All the α-glu-negative strains (n = 95) were OTC sensitive, with MICs of 2–4 μg/ml, and most of them (93 of 95) were Em resistant, with MICs of >128 μg/ml. The MICs for Em in the remaining 2 α-glu-negative strains were <0.125 μg/ml. The 15 OTC-resistant strains possessed the tet(K) and/or tet(L) gene(s), and all of the 93 Em-resistant strains possessed both the mef(A) and msr(D) genes. The relationship between α-glu activity and drug sensitivity of the N. seriolae strains may explain the difference in prevalence of each phenotype. Nevertheless, the relationship should be further explored using N. seriolae isolates collected from more prefectures and farms.     

Tissue-specific distribution of amylase in the mosquitofish (Gambusia affinis holbrooki)

Tissue-specific expression of alpha-amylase (E.C.3.2.1; α 1,4-glucan-4-glucano-hydrolase) was studied in the mosquitofish (Gambusia affinis holbrooki). Gut activity patterns, immunohistochemical distribution in the hepatopancreas and tissue-specific activities and amount of amylase protein were characterized. In the gut, activity was observed all along the gut and no variation in this pattern was observed in individuals from two natural populations. In the hepatopancreas, amylase is found in pancreatic tissue. Highest amylase activity was observed in gut tissue but highest amount of amylase protein was found in the hepatopancreas. Paper III in the series on the amylase gene-enzyme system in fishes.