Biological activities of synthetic grammistins and analogous peptides

Grammistins, peptide toxins isolated from the skin secretions of two species of soapfishes Grammistes sexlineatus and Pogonoperca punctata, are new members of the family of linear membrane-lytic antibacterial peptides characterized by the abundance of amphiphilic α-helices. As the first step toward future use of grammistins to examine the mode of membrane lysis and design new antibiotics, C-terminally free and amidated forms were chemically synthesized for each of five grammistins (Gs 1, Gs 2, Pp 1, Pp 2b and Pp 3), and evaluated for hemolytic and antibacterial activities. No difference in biological activities was observed between C-terminally free and amidated forms of Gs 1, while the C-terminally amidated forms of the other grammistins exhibited much higher activities than the C-terminally free forms. Comparison with the biological activities of natural grammistins, together with our preliminary cloning experiments, showed that natural grammistins, except for Gs 1, are C-terminally amidated. As for Pp 1, six analogous peptides were further synthesized. Determination of their biological activities revealed that the amphiphilic α-helical structure and positive charge are important for the hemolytic and antibacterial activities, respectively, of Pp 1.     

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.     

Purification and characterization of a 630 kDa bacterial killing metalloprotease (KilC) isolated from plaice Pleuronectes platessa (L.), epidermal mucus

Antibacterial chemicals in the mucus of fish such as lysozyme, lectins, peptides and proteases provide an efficient first line of defence against pathogens. This study shows that there are at least three antibacterial proteins in plaice skin mucus in addition to lysozyme. One of these proteins is responsible for approximately 74% of the antibacterial activity and is a 630 kDa protease complex designated KilC (bacterial killing metalloprotease C). Purified KilC kills the bacteria Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa efficiently. The protease activity of KilC is dependent upon the divalent cation Mg(2+) and shows pH dual optima of 5.0 and 8.0. The enzyme has a temperature optimum of 25 degrees C and is made up of at least five different sized peptides. Studies with protease inhibitors show that the catalytic site of KilC may be cysteine- or serine protease-like. KilC may kill bacterial cells by acting directly upon the bacteria or by producing low molecular weight bioactive compounds such as peptides.     

Distribution, excretion and biotransformation of 14C-piromidic acid in goldfish Carassius aumtus (L.)

Abstract. The pharmacokinetic and metabolic disposition of piromidic acid (PA) was studied in goldfish orally administered a single dose of 40 mg/kg of PA or ¹⁴C-PA with the following results. PA was rapidly absorbed from the alimentary tract and became widely distributed to give maximal organ and tissue levels mostly after 1 h post dosing. Organ and tissue levels of PA as measured by the radioactivity were 1.4–2.2 times as high as those obtained by the bioassay, though their distribution patterns were similar. PA was partly biotransformed to unconjugated antibacterial metabolites, i.e., α -OHPA and β -OHPA and partly to more hydrophilic glucuronides which were excreted into bile. Bioactive metabolites along with PA disappeared almost entirely from the fish within 48 h post dosing. The antibacterial activity of α -OHPA and β -OHPA against major pathogenic bacteria isolated from fish showed two to four-fold greater activity as compared with PA. This pharmacokinetic and metabolic disposition, together with the antibacterial activity of PA and its active metabolites, suggest a possible practical application of PA against bacterial fish diseases.     

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.     

Antibacterial and hemolytic effects of aqueous and organic extracts from different tissues of sea urchin <Emphasis Type="Italic">Echinometra mathaei</Emphasis> on pathogenic streptococci

In this study, the antibacterial and hemolytic effects of the gonadal tissue, test, spines and Aristotle’s lantern of the sea urchin (Echinometra mathaei) were investigated. The aerobic Gram-positive pathogenic bacteria Streptococcus mutans and Streptococcus sobrinus were selected because of their importance in oral diseases. The samples of sea urchin were collected from an intertidal zone in the Persian Gulf and then dissected. Finally, the crude extracts of the gonadal tissue, test, spines and Aristotle’s lantern of the samples were separately prepared by the solvents, including phosphate-buffered saline (PBS), 96 % ethanol (Et) and 80 % acetonitrile (ACN). The antibacterial activities of the extracts were evaluated using the well diffusion method in two concentrations of 1500 and 600 µg well^?1. The antibacterial effects against streptococcal species were observed in the organic extracts (Et and ACN) of the gonads and test while the extracts of spines and Aristotle’s lantern showed no antibacterial activity. The hemolytic activity of the PBS, 96 % Et, and 80 % ACN extracts from the sea urchin were evaluated using horse red blood cells. Hemolytic activity was observed only in the 80 % Et and ACN extracts of the gonad and test. In conclusion, the organic extracts of gonadal tissue showed the most inhibitory activity on the growth of S. mutans and S. sobrinus.     

鲢鱼鱼皮蛋白肽的制备与抗氧化活性评价

为制备抗氧化活性良好的鲢鱼鱼皮蛋白肽,采用胰蛋白酶、碱性蛋白酶、菠萝蛋白酶和木瓜蛋白酶等4种常见的商业酶对鲢鱼鱼皮进行酶解,测定酶解物的ABTS自由基清除力和Fe2+螯合力来评价其抗氧化活性,并用超滤及凝胶层析对酶解物进行分离,以期得到活性更好的酶解物分离组分。酶解后产物的抗氧化活性均有所提高,其中碱性蛋白酶酶解2 h产物活性较强。对此酶解物用截留分子量为10 k Da、5 k Da和3 k Da的中空纤维超滤膜进行超滤,得到的4个组分中,分子量越小的组分抗氧化活性越强。分子量小于3 k Da的组分经Sephadex G-15凝胶层析得到3个组分,其中分子量最大的组分活性较好,在0.51 mg/m L质量浓度下测定其ABTS自由基清除率和Fe~(2+)螯合力分别为(79.65±0.87)%和(93.40±0.20)%。该研究成果对鲢鱼鱼皮抗氧化肽的开发具有较好的指导作用。     

一种新型抗菌肽APNT-6的溶血性和急性毒性评价

研究了一种以抗菌脂肽类物质surfactin、iturin和fengycin为主要成分的新型抗菌肽APNT-6的体外溶血和小鼠口服急性毒性,为食品应用安全性提供初步评估。将抗菌肽作100倍稀释置于兔血琼脂平板上的牛津杯中观察溶血情况;结果显示,经100倍稀释的抗菌肽在体外对血细胞有较强的溶血毒性。将60只小鼠按雌雄各半随机分成6组,分别以8、40、200、1000、5000mg每千克体重剂量的抗菌肽对小鼠进行一次性灌胃,7d内观察小鼠毒性体征并测定相关指标。结果表明,当小鼠被给予该抗菌肽5000mg/kg剂量时,观察7d无任何毒性反应,说明高剂量的抗菌肽经口服在小鼠体内不能达到有效溶血浓度,其口服半数致死剂量(LD50)大于5000mg/kg小鼠体质量,急性毒性评级属实际无毒级。根据以上结果可知,新型抗菌肽APNT-6在体外有溶血毒性但经口服对小鼠没有急性毒性作用。     

健康养殖南美白对虾肠道细菌的抗菌活性

用双层琼脂扩散法检测从健康养殖南美白对虾肠道内分离出的111株菌对8株病原菌的抗菌活性。结果表明:111株菌对白假丝酵母菌,非01群霍乱弧菌,嗜水气单胞菌指示菌无抗菌活性,48.6%的菌株对余下的5株指示菌有抗菌活性,其中气单胞菌属和发光杆菌属对荧光假单胞菌与金黄色葡萄球菌的抑制性最强。各菌属中以气单胞菌属,发光杆菌属的抗菌谱最广,弧菌属、肠杆菌科、黄单胞菌属、芽孢杆菌属、小球菌属次之。土壤杆菌属、棒状杆菌属、黄杆菌属、产碱杆菌属、假单胞菌属和色杆菌属则没有抗菌活性。     

仿刺参补体B因子溶血活性初步研究

本文应用高等哺乳动物血清补体B因子溶血检测技术检测仿刺参个体体内补体B因子。通过溶血法检测了健康仿刺参以及人工诱导化皮仿刺参体腔液补体B因子活性。结果显示仿刺参体腔液有补体B因子存在,且含补体B因子仿刺参体腔液溶血活性明显高于去补体B因子体腔液的溶血活性。实验对仿刺参体腔液进行加热处理结果显示其补体B因子是对热不稳定的分子,此特性与高等哺乳动物B因子特征相同。仿刺参体内存在补体B因子,它与脊椎动物补体B因子性质相似,在免疫旁路途径中起重要作用。     

乳酸杆菌L1对致病弧菌的抑制作用

以致病弧菌T1、T2为检测菌,采用平板抑菌圈检测法,研究乳酸杆菌L1及其代谢产物的抑菌能力。结果表明:L1发酵上清液对两种弧菌的抑制效果和发酵液对弧菌的抑菌作用相似,即对弧菌T1的抑制作用强于T2;L1 发酵液对弧菌T1、T2的抑制能力强于上清液,表明乳酸杆菌及其代谢物质对弧菌具有协同抑制作用;L1的发酵液经3倍稀释仍对弧菌T1有抑制作用;乳酸杆菌L1生长不同时期的代谢产物对弧菌的抑菌活性不同,代谢产物的抑菌能力在衰退期>稳定期>生长期;L1的发酵液经沸水浴处理15 min其抑菌活性不变,表明抑菌代谢物质具有较好的耐热性。     

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.     

黄鳝溶血毒素性质的初步研究

通过磷酸缓冲液提取、Sephadex G-50凝胶层析和DEAE Fast Flow离子交换等分离纯化技术,从黄鳝(Monopterus albus)肌肉中分离到具有溶血活性的MAM-Ⅰ-2毒素,并对其性质进行了初步研究。结果表明:MAM-Ⅰ-2能使鸡血发生溶血现象,具有丝氨酸蛋白酶抑制剂和抗凝血活性,对嗜水气单胞菌也有较强的抑制作用。经SDS-PAGE凝胶电泳测定,该毒素蛋白相对分子质量约为43 000。对MAM-Ⅰ-2的前体物质MAM-Ⅰ的研究表明:MAM-Ⅰ除具有MAM-Ⅰ-2的特性外,还对大肠杆菌、枯草芽孢杆菌等细菌以及白色念珠菌有抑制作用;不同来源的红细胞对MAM-Ⅰ毒素的敏感性不同,其中牛红细胞反应最为敏感,其次是兔、鼠和鸡的红细胞。     

大弹涂鱼皮肤黏液的抑菌活性和蛋白质组学

皮肤黏液是鱼类免疫防御的第一道防线。大弹涂鱼皮肤黏液对其免疫防御、渗透压维持以及适应水陆生活等方面具有重要意义。为深入了解大弹涂鱼皮肤黏液的蛋白质分子组成,对其皮肤黏液开展了抑菌活性分析和蛋白质组学分析。通过电刺激法收集大弹涂鱼皮肤黏液,采用打孔法比较了皮肤黏液和血清的抑菌活性差异;进一步利用鳗弧菌对大弹涂鱼进行诱导,采用生长曲线抑制法分析和比较了诱导前后皮肤黏液的抑菌活性差异。利用Shotgun质谱技术,结合大弹涂鱼皮肤转录组数据库,对大弹涂鱼皮肤黏液开展了蛋白质组学分析;进一步利用String软件对所鉴定的蛋白质开展了蛋白质相互作用预测。大弹涂鱼皮肤黏液具有广谱抑菌活性,鳗弧菌诱导后的大弹涂鱼皮肤黏液与诱导前相比,对部分革兰氏阴性菌的抑菌活性明显上升,但对其他菌株的抑菌活性差异不明显。从大弹涂鱼皮肤黏液中共鉴定各类蛋白质分子97种,基本分子组成与其他硬骨鱼类皮肤黏液蛋白相似,但也有少数蛋白如泛素蛋白、胸腺素蛋白等未在其他鱼类皮肤黏液中报道。此外,其他鱼类中所鉴定到的部分蛋白如热休克蛋白、抗菌肽等在大弹涂鱼皮肤黏液中未能鉴定到。大弹涂鱼皮肤黏液中已鉴定的蛋白多数具有与免疫功能相关的结构域,且存在一个以肌动蛋白为核心的相互作用网络。大弹涂鱼皮肤黏液具有明显的广谱抑菌活性,其蛋白质组成与其他鱼类皮肤黏液具有一定的相似性。本研究为深入了解大弹涂鱼皮肤黏液的分子多样性及功能机制奠定了基础。     

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。     

Comparative activity and stability under salinity conditions of different antimicrobial peptides isolated from aquatic animals

This study reports the in vitro activity of six antimicrobial peptides (AMPs) produced by aquatic animals (most marine invertebrates): tachyplesin (Tach), magainin (Mag), clavanin (Clav), penaeidin (Pen), mytilin (Myt) and antilipopolysaccharide factor (ALF) against marine vibrios, filamentous fungi and yeast. Their stability under salinity conditions and seawater was also examined. The results showed that Mag, Myt and especially Tach and ALF (minimum inhibitory concentration<1.5 μM) had a potent activity against all tested vibrio species, whereas Clav and Pen were ineffective (up to 50 μM). With respect to the antifungal activity, each AMP had a different potency according to the fungal species. In general terms, Tach was the most potent peptide, followed by Mag. Interestingly, Tach, Myt and ALF had a significant effect on the filamentous fungus Fusarium solani that could be pathogenic to marine organisms. All AMPs had a tendency to decrease or lose their activity at high salinity (>225 mM NaCl). Tach and Myt were the most stable peptides, maintaining significant activity under seawater salinity (450 mM). Curiously, all peptides lost their effect under seawater conditions. The results suggest that Tach, ALF and Myt are the most promising candidates for potential therapeutic use in farmed-marine species, because all have a significant and broad antimicrobial activity maintained at high salinity.     

厚壳贻贝抗菌肽Mytilin的初步鉴定

厚壳贻贝是我国具有重大经济价值的水产养殖贝类,对其抗菌肽的研究有助于人们了解厚壳贻贝的免疫机制。为了解厚壳贻贝血清中抗菌肽Mytilin的分子组成和特性,采用多维高效液相色谱对厚壳贻贝血清进行分离纯化,从中获得3种对革兰氏阳性菌以及阴性菌均有抑制作用的抗菌肽分子,序列分析表明3种抗菌肽具有较高的序列相似性,均属于贻贝抗菌肽Mytilin家族,分别命名为Mytilin-1,Mytilin-2和Mytilin-3。其中,Mytilin-1为34个氨基酸残基构成的多肽,其分子量为3885.17u,含8个半胱氨酸,形成4对二硫键。根据所测Mytilin-1的氨基酸序列设计特异性引物,通过菌落PCR方法筛选厚壳贻贝cDNA文库,获得Mytilin-1的cDNA基因并进行了序列分析。以上研究结果表明,作为贻贝的主要抗菌肽家族,Mytilin在厚壳贻贝血清中具有较高丰度,对其蛋白质序列以及基因序列的研究为深入了解厚壳贻贝Mytilin抗菌肽的分子多样性奠定了基础。     

Effects of cecropin peptides on bacteria pathogenic to fish

The antibacterial effects of synthetic cecropin B and cecropin P1 were tested against the fish-pathogenic bacteria Vibrio anguillarum, Vibrio salmonicida, Aeromonas salmonicida , Edwardsiella ictaluri and Yersinia ruckeri. Both cecropins were active against all bacteria tested, but the effect was strongly influenced by the growth media used. In brain heart infusion medium, the minimum inhibitory concentrations of cecropin B ranged from 0.3 to 1.3 μ m and from 0.3 to 1.0 μ m for cecropin P1, except for E. ictaluri , which was noticeably less sensitive to cecropin P1 (61 μ m ). The present authors have compared the bactericidal activity of these two peptides, showing that the killing rate for the selected bacteria was higher for cecropin B than for cecropin P1 . V. anguillarum was the most sensitive to the cecropins, and in the present study, no colony forming units were detected after 4 and 8 min of treatment with cecropin B and P1, respectively. Electron microscopy was performed to document the effect of cecropin on the bacterial surface.     

Self-feeding activity patterns in gilthead sea bream (Sparus aurata), red porgy (Pagrus pagrus) and their reciprocal hybrids

This study was conducted to monitor feeding activity of gilthead sea bream (Sa) and red porgy (Pp) and to investigate the hereditary effects on feeding in their hybrids (female Sa×male Pp, female Pp×male Sa), obtained after artificial crosses. All fish groups were held in 500-l tanks under ambient temperature (15–25°C) and light conditions (daylength from 9 h 30 min to 14 h 48 min). Fish (initial body weight 100–165 g) were fed by means of self-feeders, which were connected to a computer, recording fish feeding activity for a year. Results showed that there was a 24-h periodicity in all fish types over the experiment. In their daily feeding patterns, both parental species were diurnal: red porgies had a stable daily pattern over the experiment, while gilthead sea bream shifted their activity from a general daylight fluctuation in warm periods to an afternoon peak in cold periods. The Sa×Pp hybrids showed intermediate feeding patterns compared to their parents in warm periods and a pattern that was similar to the red porgy in cold periods, while the Pp×Sa hybrids had rather unchangeable daily feeding patterns, which were not related to the parental ones. The proportion of night feed demands increased in cold periods independent of fish group. The relationship between temperature and feed intake was species-dependent. Finally, this study indicated that feeding expression in hybrids was related to the combination of the parental genome, supporting our hypothesis for a hereditary influence on feeding activity in fish.     

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。