A spider derived peptide, PnPP-19, induces central antinociception mediated by opioid and cannabinoid systems

Some peptides purified from the venom of the spider Phoneutria nigriventer have been identified as potential sources of drugs for pain treatment. In this study, we characterized the antinociceptive effect of the peptide PnPP-19 on the central nervous system and investigated the possible involvement of opioid and cannabinoid systems in its action mechanism. Methods Nociceptive threshold to thermal stimulation was measured according to the tail-flick test in Swiss mice. All drugs were administered by the intracerebroventricular route. Results PnPP-19 induced central antinociception in mice in the doses of 0.5 and 1 g. The non-selective opioid receptor antagonist naloxone (2.5 and 5 g), -opioid receptor antagonist clocinnamox (2 and 4 g), -opioid receptor antagonist naltrindole (6 and 12 g) and CB1 receptor antagonist AM251 (2 and 4 g) partially inhibited the antinociceptive effect of PnPP-19 (1 g). Additionally, the anandamide amidase inhibitor MAFP (0.2 g), the anandamide uptake inhibitor VDM11 (4 g) and the aminopeptidase inhibitor bestatin (20 g) significantly enhanced the antinociception induced by a low dose of PnPP-19 (0.5 g). In contrast, the -opioid receptor antagonist nor-binaltorphimine (10 g and 20 g) and the CB2 receptor antagonist AM630 (2 and 4 g) do not appear to be involved in this effect. Conclusions PnPP-19-induced central antinociception involves the activation of CB1 cannabinoid, - and -opioid receptors. Mobilization of endogenous opioids and cannabinoids might be required for the activation of those receptors, since inhibitors of endogenous substances potentiate the effect of PnPP-19. Our results contribute to elucidating the action of the peptide PnPP-19 in the antinociceptive pathway.

La Escuela de Salud Pública de México: innovación educativa y tecnológica en el nuevo milenio / School of Public Health of Mexico: Educational and technological innovation in the new millennium

Este artículo fue concebido para analizar la función de la Escuela de Salud Pública de México (ESPM) desde el año 2000 hasta el presente. Uno de sus puntos centrales es el análisis del proceso de reorientación de la labor educativa de la escuela con la finalidad de responder a los retos en materia de salud y educación surgidos a finales del siglo XX. Para exponer cómo ha evolucionado dicho proceso, retomamos tres ejes rectores que caracterizan la labor de la escuela en la actualidad: el cambio de modelo pedagógico, la incorporación de las tecnologías de la información y las comunicaciones, y la profesionalización de la docencia. Con la exposición de este tema, y a través del contraste entre el pasado y el presente, buscamos completar la historia de trabajo ininterrumpido de la Escuela durante sus 92 años de existencia, que ha trascendido los confines del país.

This article was conceived to analyze the work of the School of Public Health of Mexico (ESPM for is acronym in Spanish) from the year 2000 to the present day. One of the highlights that we will examine is the reorientation of the educational work of the school in order to meet the challenges in health and education that emerged during the end of the twentieth century. In order to explain the evolution of this process, we will describe the three main guiding principles that characterize the present work of the school: the pedagogical model's change, the incorporation of the information and communication technologies, and the professionalization in teaching. The purpose of this work is to define those guiding principles, and to expose, through the contrast between past and present, the complete history of uninterrupted work of the School of Public Health of Mexico during its ninety-two years of existence, that has gone beyond the boundaries of the country.

O sistema nervoso central como alvo das ações anti-hipertensivas de um peptídeo rico em resíduos de prolina do veneno da Bothrops jararaca / The central nervous system as target for anti-hypertensive actions of proline-rich peptide from Bothrops jararaca venom

Os peptídeos potenciadores da bradicinina (BPPs) presentes no veneno da serpente Bothrops jararaca são oligopeptídeos ricos em prolinas. Eles foram os primeiros inibidores naturais da enzima conversora de angiotensina (ECA) descritos. As propriedades bioquímicas e farmacológicas desses peptídeos foram essenciais para o desenvolvimento do captopril, o primeiro inibidor sítio-dirigido da ECA, usado para tratar a hipertensão humana. Recentes dados têm sugerido que a atividade farmacológica dos BPPs não pode ser explicada somente pela ação inibitória da atividade da ECA e que os efeitos dos BPPs devem envolver a participação do sistema nervoso central (SNC). Nesse trabalho foi caracterizada a sinalização de Ca2+ induzida pelo BPP-lOc [

Identificação e validação de um novo alvo funcional de um peptideo com atividade anti-hipertensiva do veneno da Bothrops jararaca / Identification and validation of a novel functional target of a peptide from Bothrops jararaca venom with anti-hypertensive activity

O BPP-10c é um decapeptídeo bioativo, rico em resíduos de prolina e é expresso em uma proteína precursora no cérebro e na glândula de veneno da Bothrops jararaca. Recentemente demonstramos que o BPP-10c tem um potente e sustentado efeito anti-hipertensivo em ratos espontaneamente hipertensos (SHR), sem, no entanto, causar qualquer efeito em ratos normotensos, por um mecanismo farmacológico independente da inibição da enzima conversora de angiotensina (ECA), levando à hipótese de que outro mecanismo poderia estar envolvido na atividade do peptídeo. Neste trabalho, usamos cromatografia de afinidade para isolar e identificar as proteínas renais com afinidade pelo BPP-10c e demonstramos que a argininosuccinato sintase (AsS) é a principal proteína a se ligar ao peptídeo. Além disso, mostramos que essa interação promove um aumento na atividade catalítica da enzima, de forma dose-dependente. A AsS é reconhecida como uma peça chave na regulação do ciclo da citrulina-óxido nítrico (NO), e sua ação é passo limitante na síntese de NO...

Production and Characterization of Anti-Staphylococcal Toxic Shock Syndrome Toxin-1 Monoclonal Antibody / 대한진단검사의학회지

BACKGROUND: Recently the association between the virulence factors of Staphylococcus aureus and the outcome of the patients infected with the organism appears to be the subject of active investigation. Toxic shock syndrome toxin-1 (TSST-1) is thought to be a clinically more significant virulence factor than other staphylococcal toxins. We attempted to produce and characterize monoclonal antibodies to staphylococcal TSST-1. METHODS: An important epitope of TSST-1, amino acids 1-15 region, was synthesized into a peptide antigen, and Balb/c mice were immunized by intraperitoneal injection of the synthetic antigen. Hybridomas were produced by fusing immunized murine splenocytes with immortal myeloma cells. Hybridomas were cloned through a limiting dilution method. Stable cultured hybridoma was injected into the peritoneal cavity of Balb/c mice, and peritoneal fluid containing the monoclonal antibody was produced. RESULTS: One IgG2b type monoclonal antibody and two IgM type monoclonal antibodies were obtained. The IgG2b type monoclonal antibody was able to detect 5 microgram of TSST-1 with Western blot analysis and showed a strong reactivity to TSST-1 with ELISA. CONCLUSIONS: Highly immunoreactive anti-TSST-1 monoclonal antibody was produced by the use of synthesized peptide antigen. Diagnostic and protective capacity of this monoclonal antibody should be evaluated in the future.

Evaluation of commercially available third-generation anti-hepatitis C virus enzyme-linked immunosorbent assay in patients on haemodialysis.

Restricted antibody reactivity to hepatitis C virus (HCV) synthetic peptides has been observed in HCV-infected patients on haemodialysis (HD). The aim of this study was to evaluate third-generation anti-HCV enzyme-linked immunosorbent assay (ELISA) test systems containing either synthetic peptide HCV antigens or recombinant HCV antigens or a combination of synthetic and recombinant antigens in screening of 69 chronic renal failure patients on HD for HCV infection. Seven patients were detected to have antibodies to HCV by the 'recombinant HCV antigens'-containing kits, of which the recombinant immunoblot assay for HCV confirmed four cases. The recombinant kits had a sensitivity of 100% and a specificity of 66%. However, the ELISA kits with only synthetic HCV antigens failed to detect antibodies in any of the cases (zero sensitivity). Hence a recombinant protein containing ELISA test system is ideal for screening of HCV infection in patients on hemodialysis.

Peptide synthesis: chemical or enzymatic

Peptides are molecules of paramount importance in the fields of health care and nutrition. Several technologies for their production are now available, among which chemical and enzymatic synthesis are especially relevant. The present review pretends to establish a non-biased appreciation of the advantages, potentials, drawbacks and limitations of both technologies. Chemical synthesis is thoroughly reviewed and their potentials and limitations assessed, focusing on the different strategies and challenges for large-scale synthesis. Then, the enzymatic synthesis of peptides with proteolytic enzymes is reviewed considering medium, biocatalyst and substrate engineering, and recent advances and challenges in the field are analyzed. Even though chemical synthesis is the most mature technology for peptide synthesis, lack of specificity and environmental burden are severe drawbacks that can in principle be successfully overcame by enzyme biocatalysis. However, productivity of enzymatic synthesis is lower, costs of biocatalysts are usually high and no protocols exist for its validation and scale-up, representing challenges that are being actively confronted by intense research and development in this area. The combination of chemical and enzymatic synthesis is probably the way to go, since the good properties of each technology can be synergistically used in the context of one process objective.

Identificação de epítopos de célula B na glicoproteína-E do envelope do vírus dengue sorotipo 3 / Identification of B-cell epitopes in the envelope glycoprotein of dengue virus type 3

As infecções pelo vírus dengue têm se tornado um problema crescente de Saúde Pública em regiões tropicais e subtropicais do mundo. O vírus pertence à família Flaviviridae com quatro sorotipos antigenicamente distintos (DENV-1 a DENV-4). Uma possível estratégia para evitar a patogenia associada com uma vacina para o dengue (que deve ser tetravalente), seria a construção de uma vacina quimérica composta de epítopos críticos selecionados dos quatro sorotipos. A maioria dos epítopos envolvidos na neutralização do vírus está presente na glicoproteína E do envelope, que é a maior proteína de superfície da partícula viral. O objetivo deste trabalho foi identificar epítopos de célula B na glicoproteína E do vírus dengue sorotipo 3. Para o mapeamento de epítopos imunodominantes, noventa e cinco peptídeos (15-mers cada, sobreposição de 10) foram sintetizados (Synpep, California-USA), a partir da sequência de 490 aminoácidos da glicoproteína E do envelope do DENV-3, de cepa circulante no Brasil. Estes peptídeos foram testados por ELISA contra um pool de soros de pacientes positivos e negativos para dengue, coletados durante a fase de convalescença da infecção por DENV-3. Os resultados mostraram que os soros de humanos reagiram com onze, dos noventa e cinco peptídeos testados, distribuídos em 5 regiões com aminoácidos na posições 51-65 (peptídeo 11), 71-90 (peptídeos 15 e 16), 131-170 (peptídeos 27, 28, 29, 30, 31 e 32), 196-210 (peptídeo 40) e 246-260 (peptídeo 50). A análise da curva ROC mostrou que, dentre os peptídeos identificados, nove seriam capazes de diferenciar entre pacientes com DENV-3 de pacientes não-dengue e três capazes de diferenciar a infecção por DENV-3 daquelas por outros sorotipos virais (DENV-1 e DENV-2). Os epítopos imunodominantes aqui descritos, junto com outros epítopos bem documentados, são potencialmente relevantes para o desenho de uma vacina para o vírus dengue e para o desenvolvimento de kits de diagnóstico específicos.

Structure-activity relationships of anti-HIV-1 peptides with disulfide linkage between D- and L-cysteine at positions i and i+3, respectively, derived from HIV-1 gp41 C-peptide

The constrained alpha-helical structure of a C-peptide is useful for enhancing anti-HIV-1 activity. The i and i+3 positions in an alpha-helical structure are located close together, therefore D-Cys (dC) and L-Cys (C) were introduced at the positions, respectively, to make a dC-C disulfide bond in 28mer C-peptides. Accordingly, this study tested whether a dC-C disulfide bond would increase the alpha-helicity and anti-HIV-1 activity of peptides. A C-peptide can be divided into three domains, the N-terminal hydrophobic domain (HPD), middle interface domain (IFD), and C-terminal hydrogen domain (HGD), based on the binding property with an N-peptide. In general, the dC-C modifications in HPD enhanced the anti-HIV-1 activity, while those in IFD and HGD resulted in no or much less activity. The modified peptides with no activity clearly showed much less alpha-helicity than the native peptides, while those with higher activity showed an almost similar or slightly increased alpha-helicity. Therefore, the present results suggest that the introduction of a dC-C bridge in the N-terminal hydrophobic domain of a C-peptide may be useful for enhancing the anti-HIV-1 activity.

Desenvolvimento de um método sorológico para o diagnóstico laboratorial da esquistossomose mansoni baseado em peptídeos sintéticos / Development of serologic method for laboratory diagnosis of the schistosomiasis mansoni based on synthetic peptides

Baseado nos algoritmos de hidrofilicidade e flexibilidade obtidos pelo uso do software ProtScale, acessível no endereço http://au.expasy.org/cgi-bin/protscale.pl, foram selecionados sete peptídeos, potencialmente antigênicos, das proteínas de Schistosoma mansoni, entre elas, catepsina B (Sm 31), heat shock protein de 70 kDa (HSPSm-70), cathodic circulating antigen (CCA), e uma seqüência da fase de leitura aberta do clone ET03. Estes peptídeos, produzidos por síntese química, foram denominados P1, P2, P3, P4, P5, P6 e P7. Em seguida, os peptídeos foram testados isoladamente contra pool de soros humanos positivos e negativos...

Synthetic peptides: a modern approach to vaccination.

For induction of humoral or cell-mediated immunity, development of synthetic peptide vaccines present a novel approach and a better alternative to conventional vaccines. Construction of synthetic peptides that mimic antigenic sites is the basis of this approach. In this article various methodologies involved in the peptide synthesis are discussed. This is followed by a discussion on the strategies involved in rendering poor or non-immunogenic peptides immunogenic. These include coupling to large carrier proteins, polymeric presentation and incorporation of an identified Th cell epitope into the peptide.

International clinical trials of HIV vaccines: II. phase I trial of an HIV-1 synthetic peptide vaccine evaluating an accelerated immunization schedule in Yunnan, China.

A Phase 1, double-blind, placebo controlled trial was conducted in Longchuan County, China, to evaluate the safety and immunogenicity of a prototype HIV-1 synthetic peptide vaccine in a target population at risk for HIV infection, and to establish the infrastructure for future large-scale HIV vaccine efficacy trials. Subjects were randomly assigned to receive 100 microg or 500 microg of vaccine or alum placebo, and were given three injections at an accelerated 0, 1, and 2 month schedule. The vaccine was well tolerated with no significant local or systemic reactions observed in any subjects. Fifty-five percent (100 microg dose) and 64% (500 microg dose) of subjects who received the vaccine produced binding antibody to the immunogen as determined by ELISA. However, HIV-1 (MN) neutralizing antibody was detected in only 23% (3/13) of subjects with detectable HIV-1 specific binding antibody. It was concluded that this prototype HIV-1 synthetic peptide vaccine was well tolerated, safe and immunogenic, and that a 0, 1, 2 month schedule was not as effective in stimulating HIV-1 specific neutralizing antibodies compared with previous trials utilizing a 0, 1, 6 month schedule. Finally, this trial demonstrated that well-designed HIV vaccine trials can be performed at this clinical trials site in Yunnan, China, and that this site should be considered for conducting larger safety, immunogenicity and efficacy trials of candidate HIV vaccines.

Synthesis and biological activities of some human gastrin analogs

The synthesis of analogs of the C-terminal tridecapeptide of gastrin in described. These pseudopeptide analogs were obtained either by replacing the C-terminal phenylalanine amide with 2-phenylethytalcohol or with 2-phenylethylamine, or by replacing the peptid bond between Trp and Leu, or between Leu and Asp with an aminomethylene (CH2NH). The ability of these compounds to stimulate gastric acid secretion in anesthetized rats and to inhibit binding of labeled CCK-8 to isolated cells from rabbit fundic mucosa was tested. [desPhe13, Leu11]-HG-12-I-beta-phenylethylester 33, [desPhe13, Leu11]-HG-12-II-beta-phenylethylester 38 [desPhe13, Leu11]-HG-12-I-beta-phenylethylamide 32, and [desPhe13, Leu11]-HG-12-II-beta-phenylethylamide 37 acted as gastrin receptor antagonists, while [Trp10-((CH2NH)-Leu11]-HG-13-I 31 and (Trp10-((CH2NH)-Leu11]-HG-13-II 36 acted as agonists. Unexpectedly, [Leu11-((CH2NH)-Asp12]-HG-13-I 30 and [Leu11-((CH2NH)-Asp12]-HG-13-II 35 were almost devoid of affinity for the gastrin receptor.

Structure and function of fibroblast growth factor

We describe some structural requirements od the fibroblast growth factor (FGF) signaling system for the stimulation of the mitogenic response in terms of the design, synthesis and mitogenic activity of linear peptides related to the human FGF-1 sequence and the structural requirements of heparin for the potentiation of the mitogenic activity of FGF-1. The best mitogenic peptide we have synthesized so far is Ac-WFVGLKKNGSSKRGPRT-NH2, that has been shown: 1)to bind to heparin-Sepharose columns with moderate affinity, requiring about 0.5 M NaCl to be eluted from the resin; 2) to be mitogenic upon BALB/c 3T3 fibroblasts in culture (ED50=10-20 muM) and 3)to compete with human FGF-1 for cellular binding (ID50=30-50 muM). The potentiating activity of heparin upon FGF-1 has shown to be dependent on the oligosaccharide size, degree of sulfation and carboxylation. Apparently, these same requirements hold for the heparan sulfate molecules. Based on the reported studies, ee propose some important requirements of an oligosaccharide to potentiate FGF-1 mitogenic activity: 1) to have a minimum of twelve units, organized as disaccharides where one of the units is a uronic acid and the second is glycosamine; 2) to have at least one iduronic acid sulfated at position 2 and 3) to have N-sulfated glycosamines, preferentially 6-O-sulfated. To have groups of negative charges is not enough: they need to be localized in a correct conformation.

Synthetic peptides related to the delta-receptor agonist, dermorphin gene associated peptide.

The delta-receptor selective dermorphin gen associated peptide (DGAP) and five of its analogues having structural modifications at positions 2, 4 and 5 were synthesized by the solid phase method using 9-fluorenylmethoxycarbonyl amino acid trichlorophenyl esters as coupling agents and rho-benzyloxybenzyl alcohol resin as the solid support. The delta-receptor selectivity of these peptides was determined by guinea pig ileum and mouse vas deferens assays. The latter assay was carried out using modified Kreb's solution aerated with pure oxygen instead of carbogen. All the synthetic peptides were found to be delta-receptor selective.

Inhibition of herpesvirus ribonucleotide reductase by new synthetic peptides

The prospect of total chemical synthesis of large biologically active peptides up to the protein size has always represented a powerful tool in bioorganic chemistry. The advances made in the chemical synthesis of peptides would not have been possible without the availability of solid phase peptide synthesis [SPPS] introduced by Merrifield[1]. Since the conception of [SPPS], the chemistry developed for its application has remained fairly standardized. Like all ingenious ideas, [SPPS] also has its short-comings, for example the repetitive acidolysis required for the deprotection of [Boc] group, which may affect the acid sensitive peptide bonds and also catalyze some undesirable side reactions result in failure sequences. This initiated the development of different N-alpha-aminoblocking groups. However, the most successful one was the base labile 9-fluorenyl methoxycarbonyl Fmoc group, which is considered one of the best choices for protecting the alpha-amino-group not only because of its cleavage in weak alkaline medium [e.g. piperidine in DMF] and can therefore be used in conjunction with acid labile linker, acid sensitive amino acids and side chain protecting groups, but also because it enables- as UV active group- continuous spectroscopic monitoring during all steps of synthesis cycle[2]

Enzyme-linked immunosorbent assay for riboflavin carrier protein and modified protein: application for epitope analysis.

Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards the conformational epitopes and antibodies to disulphide bond reduced carboxymethylated riboflavin carrier protein (RCM-RCP) to the sequential epitopes. Taking advantage of this premise and in order to map the epitopes of RCP recognized by the antibodies, enzyme-linked immunosorbent assays were validated for RCP and RCM-RCP using the Avidin-Biotin system. The usefulness of these assays were illustrated when antigenicity of peptides derived from RCM-RCP following trypsinization were examined. Two major (T1,T2) and one minor peptide (T3) fractions were obtained when the tryptic peptides were fractionated on DEAE-cellulose. RCP has a blocked N-terminal. Tryptic peptides (T1 and T2) on microsequencing revealed the absence of an N-terminal amino acid, indicating that these fragments emanate from the N-terminal region of RCP. In support of this observation is the finding that antipeptide antibody to cRCP (10-24) of cRCP interacted with T1 as well as T2 indicating the presence of the sequential epitope (10-24) of cRCP in these fragments. In RCP-ELISA, only T2 displaced RCP and peptides T1 and T2 displaced RCM-RCP in RCM-RCP ELISA. Differences in the ability of these fragments (T1 and T2) to displace RCP and RCM-RCP reflect the subtle changes in the spatial structures of these epitopes in RCP and RCM-RCP.

Síntesis química de un nonapéptido del antígeno de superficie del virus de hepatitis B / Synthesis of a nonapeptide of the surface antigen protein from hepatitis B virus

Una de las proteínas más estudiadas con el fin de realizar ensayos inmunológicos ha sido el antígeno de superficie del virus de la hepatitis B humana, tanto por su interés para la producción de una vacuna, como para disponer de nuevos métodos de diagnóstico de la enfermedad mediante la aplicación de la técnica de obtención de anticuerpos monoclonales. En este artículo se describe la síntesis por la vía química en solución, del péptido correspondiente a la región 130-138 de la región pre-S del antígeno de superficie del virus de la hepatitis B, así como el estudio de los resultados obtenidos por espectrometría de masas, análisis de aminoácidos y secuencia aminoacídica manual, siguiendo el método de degradación de Edman