Membrane-translocating peptides and toxins: from nature to bedside

Today, different functional classes of bioactive peptides and toxins isolated from diverse sources of living organisms are known. In medicine, these polypeptides present the potential to be used structurally unmodified or to serve as templates for molecular design of improved derivatives. Here, we refer to members of three classes of remarkable peptides and toxins that act at the cell membranes level and membrane trafficking systems: (i) the binary toxins (ii) the antimicrobial peptides and (iii) the cell penetrating peptides. Binary toxins have been genetically manipulated to generate specific immunotoxins, while antimicrobial peptides are in use as alternative agents against resistant microbes and tumor cells. Cell penetrating peptides have applications as diverse as cell transfection and transport of nanomaterials. Our group is dissecting the capacity of crotamine, a peptide from rattlesnake venom, to translocate cell membranes and use it as a delivery system in the transducing technology and molecular imaging.Atualmente, diferentes classes funcionais de peptídeos e toxinas biologicamente ativas isolados de diversos organismos são conhecidas. Em medicina, esses polipeptídios podem ser diretamente utilizados ou podem servir como modelos para a geração de moléculas derivadas. Aqui, nós fazemos referência a três classes de peptídeos e toxinas que agem sobre membranas celulares ou sobre sistemas de transporte por membranas: (i) toxinas binárias; (ii) peptídeos antimicrobianos; (iii) peptídeos penetradores de células. As toxinas binárias têm sido geneticamente manipuladas para gerar imunotoxinas específicas, enquanto os peptídeos antimicrobianos são usados como agentes alternativos contra células tumorais e microbianas resistentes. Os peptídeos penetradores de células têm aplicações que vão desde a transfecção celular quanto ao transporte intracelular de nanopartículas. Nosso grupo vem investigando a capacidade da crotamina, um peptídeo do veneno de cascavel, em translocar membranas celulares, bem como de utilizar a crotamina como sistema de transporte molecular e de análise de imagens.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de Pernambuco Departamento de BioquímicaClínica e Centro de Pesquisa em Reprodução Humana Roger AbdelmassihInstituto Butantan Laboratório de HerpetologiaUniversidade Federal de São Paulo (UNIFESP) Departamento de FarmacologiaInstituto Butantan Centro de Toxinologia AplicadaInstituto Butantan Laboratório de GenéticaCentro de Biotecnologia da AmazôniaInstituto de Pesquisas Energéticas e NuclearesUNIFESP, Depto. de FarmacologiaSciEL

Unraveling the antifungal activity of a South American rattlesnake toxin crotamine

Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the beta-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50->200 mu g/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 mu g/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 mu g/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5-50.0 mu g/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. the peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications. (C) 2012 Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)University of Maryland Baltimore County Designated Research Initiative FundUniversidade Federal de São Paulo UNIFESP, Dept Farmacol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Med, BR-04044020 São Paulo, BrazilUniv São Paulo, Dept Bioquim & Imunol, BR-14049 Ribeirao Preto, BrazilUniv Maryland, Sch Med, Inst Human Virol, Baltimore, MD 21201 USAUniv Maryland, Dept Biochem & Mol Biol, Baltimore, MD 21201 USAUniversidade Federal de São Paulo UNIFESP, Dept Bioquim, BR-04044020 São Paulo, BrazilInst Butantan, Lab Bioquim & Biofis, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Ginecol, BR-04044020 São Paulo, BrazilCBA, Lab Bioquim & Biol Mol, Manaus, Amazonas, BrazilUniv Maryland Baltimore Cty, Dept Chem & Biochem, Baltimore, MD 21250 USAInst Butantan, Ctr Toxinol Aplicada CAT CEPID, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Farmacol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Ginecol, BR-04044020 São Paulo, BrazilWeb of Scienc

State of the Art in the Studies on Crotamine, a Cell Penetrating Peptide from South American Rattlesnake

Animal venoms comprise a naturally selected cocktail of bioactive peptides/proteins and other molecules, each of which playing a defined role thanks to the highly specific interactions with diverse molecular targets found in the prey. Research focused on isolation, structural, and functional characterizations of novel natural biologics (bioactive peptides/proteins from natural sources) has a long way to go through from the basic science to clinical applications. Herein, we overview the structural and functional characteristics of the myoneurotoxin crotamine, firstly isolated from the South American rattlesnake venom. Crotamine is the first venom peptide classified as a natural cell penetrating and antimicrobial peptide (CPP and AMP) with a more pronounced antifungal activity. in contrast to other known natural CPPs and AMPs, crotamine demonstrates a wide spectrum of biological activities with potential biotechnological and therapeutic values. More recent studies have demonstrated the selective in vitro anticancer activity of crotamine. in vivo, using a murine melanoma model, it was shown that crotamine delays tumor implantation, inhibits tumor cells proliferation, and also increases the survival of mice engrafted with subcutaneous melanoma. the structural and functional properties and also the possible biotechnological applications of minimized molecules derived from crotamine are also discussed.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Inst Butantan, Genet Lab, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Farmacol, São Paulo, BrazilUniv Fed Ceara, Labomar Inst Ciencias Mar, Fortaleza, CE, BrazilUniv Estado Amazonas, Manaus, AM, BrazilCBA, Lab Bioquim & Biol Mol, Manaus, AM, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Farmacol, São Paulo, BrazilWeb of Scienc

Kinetic characterization of gyroxin, a serine protease from Crotalus durissus terrificus venom

This work describes for the first time the characterization of the enzymatic features of gyroxin, a serine protease from Crotalus durissus terrificus venom, capable to induce barrel rotation syndrome in rodents. Measuring the hydrolysis of the substrate ZFR-MCA, the optimal pH for proteolytic cleavage of gyroxin was found to be at pH 8.4. Increases in the hydrolytic activity were observed at temperatures from 25 degrees C to 45 degrees C, and increases of NaCl concentration up to 1 M led to activity decreases. the preference of gyroxin for Arg residues at the substrate P1 position was also demonstrated. Taken together, this work describes the characterization of substrate specificity of gyroxin, as well as the effects of salt and pH on its enzymatic activity. (C) 2012 Elsevier Masson SAS. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Dept Farmacol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilInst Ciencias Mar UFC, BR-60165081 Fortaleza, Ceara, BrazilUniv Estado Amazonas, Escola Super Ciencias Saude INCT, BR-69065001 Manaus, Amazonas, BrazilUniversidade Federal de São Paulo, Dept Farmacol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

The density and biomass of mesozooplankton and ichthyoplankton in the Negro and the Amazon Rivers during the rainy season: The ecological importance of the confluence boundary

The boundary zone between two different hydrological regimes is often a biologically enriched environment with distinct planktonic communities. In the center of the Amazon River basin, muddy white water of the Amazon River meets with black water of the Negro River, creating a conspicuous visible boundary spanning over 10kmalong the Amazon River. Here, we tested the hypothesis that the confluence boundary between the white and black water rivers concentrates prey and is used as a feeding habitat for consumers by investigating the density, biomass and distribution of mesozooplankton and ichthyoplankton communities across the two rivers during the rainy season. Our results show that mean mesozooplankton density (2,730 inds. m-3) and biomass (4.8 mg m-33) were higher in the black-water river compared to the white-water river (959 inds. m-33; 2.4 mg m-33); however an exceptionally high mesozooplankton density was not observed in the confluence boundary. Nonetheless we found the highest density of ichthyoplankton in the confluence boundary (9.7 inds. m-3), being up to 9-fold higher than in adjacent rivers. The confluence between white and black waters is sandwiched by both environments with low (white water) and high (black water) zooplankton concentrations and by both environments with low (white water) and high (black water) predation pressures for fish larvae, and may function as a boundary layer that offers benefits of both high prey concentrations and low predation risk. This forms a plausible explanation for the high density of ichthyoplankton in the confluence zone of black and white water rivers. © 2017 Nakajima et al

マイワシ SARDCNOPS MELANOSTICA カク ブイ カラ チョウセイ シタ リン シ シツ コンゴウ ブツ ノ シ シツ ソセイ

前報で生の魚体から分離,回収したリン脂質混合物に雛の成長促進効果があること,および生の魚体からタンパク質分解酵素を用いてリン脂質を回収するための前処理としての酵素処理条件について報告した。本報告では,工業的にリン脂質を回収する場合を想定し,魚加工場で発生する加工副産物(頭,骨,内臓)中のリン脂質含量を知ることを目的とし,生の魚体を肉部,頭・中骨混合部および内臓部の3部位にわけ,それぞれの総脂質含有量,総脂質中リン脂質含有量と脂肪酸組成および各部位から回収したリン脂質混合物,リン脂質混合物中のリン脂質含量などにつき検討した。その結果は次のようにまとめられた。1.マイワシ全魚体の総脂質量は約13%で,総脂質中リン脂質は約11%であった。2.マイワシ全魚体の脂肪酸組成は食品成分表に示されたものとほぼ同じで,全脂肪酸中n-3の合計は32%であった。3.マイワシの3部位中,総脂質含量がもっとも多い部位は内臓部20%で,肉部と頭・中骨混合部は9～12%であった。しかし,各部位の総脂質中リン脂質含量は10～13%と部位による差は少なかった。4.各部位から回収したリン脂質混合物の回収率は肉部と頭・中骨混合部が19～20%で,内臓部は8%であった。5.リン脂質混合物中の総脂質量は乾物換算で肉部29%,内臓部23%,頭・中骨混合部19%であった。また総脂質中のリン脂質含量は内臓部が57%ともっとも高く,肉部,頭・中骨混合部は19～20%と低かった。6.以上より,マイワシ各部から回収できるリン脂質は生原料に対し肉部が1.2%,内臓部が1.1%で,頭・中骨混合部は0.7%であった。In the previous paper, it was reported that the phospholipid mixture prepared from fish had a growth promoting effect on chick, and the conditions necessary for enzymatic preparation of phospholipids from fish using protease was determined. The aim of this report is to estimate the content of phospholipids in the by-product in the fish-manufacturing factories for effective use of the industrial recovery of phopholipids. The total lipid content was examined together with, the content of phospholipids in total lipids and the fatty-acid composition in the three parts of raw sardine and phospholipid mixture in three parts of raw sardine, meat, head-and-bone mixture and viscera were determined. The results are summarized as follows. 1. The content of total lipids, phospholipids in total lipids in raw sardine was 13% and 11%. 2. Fatty-acid composition of raw sardines was similar to that shown in the table of food composition, and the sum of n-3 fatty acids in the total fatty acids was 32%. 3. The content of total lipids in raw sardines was highest in the viscera (20%) and lowest in the meat and head-and-bone mixture (9-12%). However, the content of phospholipids in the total lipids from each part was 10 to 12%, and there was only a small difference among the three parts. 4. The recovery percentage of phospholipids from the meat and head-and-bone mixture was 19 to 20% and that from the viscera was 8%. 5. The content of total lipids in the phospholipid mixture was 29% on a dry weight basis in the meat, 23% in the viscera and 19% in the head-and-bone mixture. The content of phospholipids in the total lipids was highest in the viscera (56%) and low in the meat and head-and-bone mixture (19 to 20%). 6. The amount of recoverable phospholipids from raw meat, viscera and head-and-bone mixture was estimated to be 1.2, 1.1 and 0.7%, respectively

マイワシ SARDCNOPS MELANOSTICA カラ リン シ シツ コンゴウ ブツ ノ ブンリ

生の魚体から分離,濃縮したリン脂質混合物には雛に対する成長促進効果がある。本研究では生の魚体から高濃度,安定的にリン脂質混合物を分離,回収するためのタンパク質分解酵素の添加率,反応時間および反応温度について検討した。試験では,タンパク質分解酵素(プロテアーゼP)の添加率を,生の原料に対し0.004,0.008,0.016および0.032%の4段階,反応時間を25,50および75分の3段階,反応温度を42～43,47～48および52～53℃の3段階に設定し,それぞれを組み合わせた10条件について,リン脂質混合物の収率について検討し,混合物中の総脂質量および総脂質中のリン脂質,トリアシルグリセロール等の含有量を検討した。その結果,生の魚体からリン脂質混合物とリン脂質を最大限に分離,回収するためのタンパク質分解酵素の添加率,反応温度および反応時間は,それぞれ0.008%,42℃,50分であり,回収できたリン脂質混合物は生の原料に対し約16%,リン脂質混合物中総脂質量は約21%で,総脂質中リン脂質量は約18%であった。Following the previous report, the concentrated mixture of phospholipids obtained from raw fish is considered to have a growth promoting effect in chicks. In the present study, the conditions for effective isolation of phospholipids from raw sardine, and the optimal amount of protease, reaction time and reaction temperature were examined. The yield of phospholipids, total lipid content, and percentage of the components in the total lipids such as phospholipids and triacylglycerol obtained by protease treatment of sardine using 10 treatment conditions consisted of a combination of the following : Protease added at the rates of 0.004, 0.008, 0.016 and 0.032% ; reaction time of 25, 50 and 75 minutes ; and reaction temperatures of 42-43, 47-48, and 52-53℃. The highest yield of lipids and phospholipids was obtained by the reaction using 0.008% protease, at a temperature of 42℃ and for 50min. Under these conditions, the yield of phospholipids in the raw sardine was about 16%, amount of total lipids was 21% and total amount of phospholipids was 18%