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

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

Scaling-Up of Dental Pulp Stem Cells Isolated from Multiple Niches

Dental pulp (DP) can be extracted from child’s primary teeth (deciduous), whose loss occurs spontaneously by about 5 to 12 years. Thus, DP presents an easy accessible source of stem cells without ethical concerns. Substantial quantities of stem cells of an excellent quality and at early (2–5) passages are necessary for clinical use, which currently is a problem for use of adult stem cells. Herein, DPs were cultured generating stem cells at least during six months through multiple mechanical transfers into a new culture dish every 3–4 days. We compared stem cells isolated from the same DP before (early population, EP) and six months after several mechanical transfers (late population, LP). No changes, in both EP and LP, were observed in morphology, expression of stem cells markers (nestin, vimentin, fibronectin, SH2, SH3 and Oct3/4), chondrogenic and myogenic differentiation potential, even after cryopreservation. Six hours after DP extraction and in vitro plating, rare 5-bromo-2′-deoxyuridine (BrdU) positive cells were observed in pulp central part. After 72 hours, BrdU positive cells increased in number and were found in DP periphery, thus originating a multicellular population of stem cells of high purity. Multiple stem cell niches were identified in different zones of DP, because abundant expression of nestin, vimentin and Oct3/4 proteins was observed, while STRO-1 protein localization was restricted to perivascular niche. Our finding is of importance for the future of stem cell therapies, providing scaling-up of stem cells at early passages with minimum risk of losing their “stemness”

Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

<p>Abstract</p> <p>Background</p> <p>The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression.</p> <p>Methods</p> <p>Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes.</p> <p>Results and Discussion</p> <p>We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent <it>in situ </it>hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age.</p> <p>Conclusion</p> <p>Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.</p

A New Mouse Model for Marfan Syndrome Presents Phenotypic Variability Associated with the Genetic Background and Overall Levels of Fbn1 Expression

Marfan syndrome is an autosomal dominant disease of connective tissue caused by mutations in the fibrillin-1 encoding gene FBN1. Patients present cardiovascular, ocular and skeletal manifestations, and although being fully penetrant, MFS is characterized by a wide clinical variability both within and between families. Here we describe a new mouse model of MFS that recapitulates the clinical heterogeneity of the syndrome in humans. Heterozygotes for the mutant Fbn1 allele mgΔloxPneo, carrying the same internal deletion of exons 19–24 as the mgΔ mouse model, present defective microfibrillar deposition, emphysema, deterioration of aortic wall and kyphosis. However, the onset of a clinical phenotypes is earlier in the 129/Sv than in C57BL/6 background, indicating the existence of genetic modifiers of MFS between these two mouse strains. In addition, we characterized a wide clinical variability within the 129/Sv congenic heterozygotes, suggesting involvement of epigenetic factors in disease severity. Finally, we show a strong negative correlation between overall levels of Fbn1 expression and the severity of the phenotypes, corroborating the suggested protective role of normal fibrillin-1 in MFS pathogenesis, and supporting the development of therapies based on increasing Fbn1 expression