Antimycobacterial and cytotoxicity activity of synthetic and natural compounds

Antimycobacterial and cytotoxicity activity of synthetic and natural compounds. Secondary metabolites from Curvularia eragrostidis and Drechslera dematioidea, Clusia sp. floral resin, alkaloids from Pilocarpus alatus, salicylideneanilines, piperidine amides, the amine 1-cinnamylpiperazine and chiral pyridinium salts were assayed on Mycobacterium tuberculosis H37Rv. N-(salicylidene)-2-hydroxyaniline was the most effective compound with a minimal inhibitory concentration (MIC) of 8 µmol/L. Dihydrocurvularin was moderately effective with a MIC of 40 µmol/L. Clusia sp. floral resin and a gallocatechin-epigallocatechin mixture showed MIC of 0.02 g/L and 38 µmol/L, respectively. The cytotoxicity was evaluated for N-(salicylidene)-2-hydroxyaniline, curvularin, dihydrocurvularin and Clusia sp. floral resin, and the selectivity indexes were > 125, 0.47, 0.75 and 5, respectively.

Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when Nα-blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions.

Estudo clínico da aplicação de matriz inorgânica de osso associado a peptídeo sintético de adesão celular (MIO/P-15), PepGen P-15®, em lesões periodontais avançadas de cães

Os mecanismos biológicos desenvolvidos para aumentar a qualidade da regeneração óssea e da reparação tecidual de sítios periodontais específicos continuam a ser um desafio e têm sido complementado pela capacidade de adesão celular do colágeno do tipo I, promovida por um peptídeo sintético de adesão celular (P-15), associado a uma matriz inorgânica de osso (MIO) para formar MIO/P-15. O objetivo deste estudo foi avaliar a perda do nível clínico de inserção e a resposta da bolsa periodontal em dentes após 3 e 6 meses da aplicação de enxerto com MIO/P-15. Vinte e um cães do Hospital Veterinário da Universidade de São Paulo foram anestesiados para realização de tratamento periodontal e 132 faces dentais com perda de nível clínico de inserção foram tratadas, sendo que 36,4% (48 faces) receberam o peptídeo de adesão celular e 63,6% (84 faces) compuseram o grupo controle que recebeu tratamento convencional (retalho muco-gengival e aplainamento radicular). O procedimento foi documentado através de radiografia intra-oral e todas as sondagens de bolsas periodontais foram fotografadas. Depois de 3 e de 6 meses, os animais foram re-anestesiados a fim de se obter novas avaliações, radiografias, fotografias e sondagens periodontais. As 48 faces com perda de nível clínico de inserção que receberam material de enxertia apresentaram taxa de 40% de recuperação do nível clínico de inserção após 6 meses. O grupo controle de faces dentais não apresentou alteração do nível clínico de inserção. A face palatina foi a que apresentou melhor taxa de regeneração (40%) e os dentes caninos e molares mostraram as melhores respostas (57,14% e 65%, respectivamente). Não houve sinais de infecção pós-cirúrgica relacionadas à falta de higienização oral dos animais. Pode-se concluir que o MIO/P-15 auxilia na regeneração e re-aderência das estruturas periodontais, incluindo osso alveolar. Sua aplicação mostrou-se fácil e prática e a incidência de complicações pós-cirúrgicas foi baixa. Ainda assim, mais estudos e pesquisas são necessários para que se avalie a quantidade e a qualidade do osso e do ligamento periodontal formados.

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell-Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73 +/- 12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-gamma secretion, ratios of IFN-gamma/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNF alpha/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-gamma/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

Antileishmanial Activity of the Hydroalcoholic Extract of Miconia langsdorffii, Isolated Compounds, and Semi-Synthetic Derivatives

The in vitro activity of the crude hydroalcoholic extract of the aerial parts of Miconia langsdorffii Cogn. was evaluated against the promastigote forms of L. amazonensis, the causative agent of cutaneous leishmaniasis in humans. The bioassay-guided fractionation of this extract led to identification of the triterpenes ursolic acid and oleanolic acid as the major compounds in the fraction that displayed the highest activity. Several ursolic acid semi-synthetic derivatives were prepared, to find out whether more active compounds could be obtained. Among these ursolic acid-derived substances, the C-28 methyl ester derivative exhibited the best antileishmanial activity.

Study of the antimicrobial peptide indolicidin and a mutant in micelle medium by molecular dynamics simulation

The antimicrobial peptide indolicidin (IND) and the mutant CP10A in hydrated micelles were studied using molecular dynamics simulations in order to observe whether the molecular dynamics and experimental data could be sufficiently correlated and a detailed description of the interaction of the antimicrobial peptides with a model of the membrane provided by a hydrated micelle system could be obtained. In agreement with the experiments, the simulations showed that the peptides are located near the surface of the micelles. Peptide insertions agree with available experimental data, showing deeper insertion of the mutant compared with the peptide IND. Major insertion into the hydrophobic core of the micelle by all tryptophan and mutated residues of CP10A in relation to IND was observed. The charged residues of the terminus regions of both peptides present similar behavior, indicating that the major differences in the interactions with the micelles of the peptides IND and CP10A occur in the case of the hydrophobic residues.

Detection of Hepatitis B virus subgenotype A1 in a Quilombo community from Maranhao, Brazil

Background: The Brazilian population is mainly descendant from European colonizers, Africans and Native Americans. Some Afro-descendants lived in small isolated communities since the slavery period. The epidemiological status of HBV infection in Quilombos communities from northeast of Brazil remains unknown. The aim of this study was to characterize the HBV genotypes circulating inside a Quilombo isolated community from Maranhao State, Brazil. Methods: Seventy-two samples from Frechal Quilombo community at Maranhao were collected. All serum samples were screened by enzyme-linked immunosorbent assays for the presence of hepatitis B surface antigen ( HBsAg). HBsAg positive samples were submitted to DNA extraction and a fragment of 1306 bp partially comprising HBsAg and polymerase coding regions (S/POL) was amplified by nested PCR and its nucleotide sequence was determined. Viral isolates were genotyped by phylogenetic analysis using reference sequences from each genotype obtained from GenBank (n = 320). Sequences were aligned using Muscle software and edited in the SE-AL software. Bayesian phylogenetic analyses were conducted using Markov Chain Monte Carlo (MCMC) method to obtain the MCC tree using BEAST v.1.5.3. Results: Of the 72 individuals, 9 (12.5%) were HBsAg-positive and 4 of them were successfully sequenced for the 1306 bp fragment. All these samples were genotype A1 and grouped together with other sequences reported from Brazil. Conclusions: The present study represents the first report on the HBV genotypes characterization of this community in the Maranhao state in Brazil where a high HBsAg frequency was found. In this study, we reported a high frequency of HBV infection and the exclusive presence of subgenotype A1 in an Afro-descendent community in the Maranhao State, Brazil.

Sensitization Prevalence, Antibody Cross-Reactivity and Immunogenic Peptide Profile of Api g 2, the Non-Specific Lipid Transfer Protein 1 of Celery

Background: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. Methodology: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. Results: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. Conclusions: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.

Anti-Plasmodium Activity of Angiotensin II and Related Synthetic Peptides

Plasmodium species are the causative agents of malaria, the most devastating insect-borne parasite of human populations. Finding and developing new drugs for malaria treatment and prevention is the goal of much research. Angiotensins I and II (ang I and ang II) and six synthetic related peptides designated Vaniceres 1-6 (VC1-VC6) were assayed in vivo and in vitro for their effects on the development of the avian parasite, Plasmodium gallinaceum. Ang II and VC5 injected into the thoraces of the insects reduced mean intensities of infection in the mosquito salivary glands by 88% and 76%, respectively. Although the mechanism(s) of action is not completely understood, we have demonstrated that these peptides disrupt selectively the P. gallinaceum cell membrane. Additionally, incubation in vitro of sporozoites with VC5 reduced the infectivity of the parasites to their vertebrate host. VC5 has no observable agonist effects on vertebrates, and this makes it a promising drug for malaria prevention and chemotherapy.

Bothrops jararaca Peptide with Anti-Hypertensive Action Normalizes Endothelium Dysfunction Involved in Physiopathology of Preeclampsia

Preeclampsia, a pregnancy-specific syndrome characterized by hypertension, proteinuria and edema, is a major cause of fetal and maternal morbidity and mortality especially in developing countries. Bj-PRO-10c, a proline-rich peptide isolated from Bothrops jararaca venom, has been attributed with potent anti-hypertensive effects. Recently, we have shown that Bj-PRO-10c-induced anti-hypertensive actions involved NO production in spontaneous hypertensive rats. Using in vitro studies we now show that Bj-PRO-10c was able to increase NO production in human umbilical vein endothelial cells from hypertensive pregnant women (HUVEC-PE) to levels observed in HUVEC of normotensive women. Moreover, in the presence of the peptide, eNOS expression as well as argininosuccinate synthase activity, the key rate-limiting enzyme of the citrulline-NO cycle, were enhanced. In addition, excessive superoxide production due to NO deficiency, one of the major deleterious effects of the disease, was inhibited by Bj-PRO-10c. Bj-PRO-10c induced intracellular calcium fluxes in both, HUVEC-PE and HUVEC, which, however, led to activation of eNOS expression only in HUVEC-PE. Since Bj-PRO-10c promoted biological effects in HUVEC from patients suffering from the disorder and not in normotensive pregnant women, we hypothesize that Bj-PRO-10c induces its anti-hypertensive effect in mothers with preeclampsia. Such properties may initiate the development of novel therapeutics for treating preeclampsia.

Role of the gp85/Trans-Sialidases in Trypanosoma cruzi Tissue Tropism: Preferential Binding of a Conserved Peptide Motif to the Vasculature In Vivo

Background: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature. Methods: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied. Findings and Conclusions: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.

Purification and characterization of an antimicrobial peptide produced by Pseudomonas sp strain 4B

An antimicrobial peptide produced by a bacterium isolated from the effluent pond of a bovine abattoir was purified and characterized. The strain was characterized by biochemical profiling and 16S rDNA sequencing as Pseudomonas sp. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A major band on SDS-PAGE suggested that the antimicrobial peptide has a molecular mass of about 30 kDa. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, among other. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases tested. This substance showed different properties than other antimicrobials from Pseudomonas species, suggesting a novel antimicrobial peptide was characterized.

Participation of kinin receptors on memory impairment after chronic infusion of human amyloid-beta 1-40 peptide in mice

Chronic infusion of human amyloid-beta 1-40 (A beta) in the lateral ventricle (LV) of rats is associated with memory impairment and increase of kinin receptors in cortical and hippocampal areas. Deletion of kinin B1 or B2 receptors abolished memory impairment caused by an acute single injection of A beta in the LV. As brain tissue and kinin receptors could unlikely react to acute or chronic administration of a similar quantity of A beta, we evaluated the participation of B1 or B2 receptors in memory impairment after chronic infusion of A beta. Male C57BI/6 J (wt), knock-out B1 (koB1) or B2 (koB2) mice (12 weeks of age) previously trained in a two-way shuttle-box and achieving conditioned avoidance responses (CAR, % of 50 trials) were infused with AB (550 pmol, 0.12 mu L/h, 28 days) or vehicle in the LV using a mini-osmotic pump. They were tested before the surgery (TO), 7 and 35 days after the infusion started (T7; T35). In T0, no difference was observed between CAR of the control (Cwt = 59.7 +/- 6.7%; CkoB1 = 46.7 +/- 4.0%; CkoB2 = 64.4 +/- 5.8%) and A beta (A beta wt = 66.0 +/- 3.0%; A beta koB1 = 66.8 +/- 8.2%; A beta koB2 = 58.7 +/- 5.9%) groups. In T7, A beta koB2 showed a significant decrease in CAR (41.0 +/- 8.6%) compared to the control-koB2 (72.8 +/- 2.2%, P <0.05). In T35, a significant decrease (P <0.05) was observed in A beta wt (40.7 +/- 3.3%) and A beta koB2 (41.2 +/- 10.7%) but not in the A beta koB1 (64.0 +/- 14.0%) compared to their control groups. No changes were observed in the controls at T35. We suggest that in chronic infusion of BA, B1 receptors could playan important role in the neurodegenerative process. Conversely, the premature memory impairment of koB2 suggests that it may be a protective factor. (C) 2009 Elsevier Ltd. All rights reserved.

Acute and chronic effects of exercise on inflammatory markers and B-type natriuretic peptide in patients with coronary artery disease

Few studies have prospectively addressed the effects of exercise in the inflammatory activity of patients with coronary artery disease (CAD). We sought to evaluate the consequences of an acute bout of exercise on inflammatory markers and BNP in untrained CAD patients before and after randomization to a training program. 34 CAD patients underwent a 50-min acute exercise session on a cycle-ergometer at 65% peak oxygen uptake before and after blood sampling. They were then randomized to a 4-month chronic exercise program (15 patients) or general lifestyle recommendations (19 patients), undergoing a new acute session of exercise after that. In the overall population, acute exercise caused a significant increase in C-reactive protein [CRP; 1.79 (4.49) vs. 1.94 (4.89) mg/L, P < 0.001], monokine induced by interferon-gamma [Mig; 351 (324) vs. 373 (330) pg/mL, P = 0.027] and vascular adhesion molecule-1 [VCAM-1; 226 (82) vs. 252 (110) pg/mL, P = 0.02]. After 4-months, in exercise-trained patients, there was a significant decrease in the inflammatory response provoked by the acute exercise compared to patients in the control group reflected by a significant decrease in the differences between rest and post-exercise levels of CRP [-0.29 (0.84) mg/L vs. -0.11 (0.21) mg/L, P = 0.05]. Resting BNP was also significantly lower in exercise-trained patients when compared to untrained controls [15.6 (16.2) vs. 9.7 (11.4) pg/mL, P = 0.04 and 19.2 (27.8) vs. 23.2 (27.5) pg/mL, P = 0.76; respectively]. Chronic exercise training might partially reverse the inflammatory response caused by acute exercise in CAD patients. These results suggest that regular exercise is an important nonpharmacological strategy to the improvement in inflammation in CAD patients.

Equivalence between hot tensile and creep testing data for Kanthal A1 alloy

Hot tensile and creep tests were carried out on Kanthal A1 alloy in the temperature range from 600 to 800 degrees C. Each of these sets of data were analyzed separately according to their own methodologies, but an attempt was made to find a correlation between them. A new criterion proposed for converting hot tensile data to creep data, makes possible the analysis of the two kinds of results according to usual creep relations like: Norton, Monkman-Grant, Larson-Miller and others. The remarkable compatibility verified between both sets of data by this procedure strongly suggests that hot tensile data can be converted to creep data and vice-versa for Kanthal A1 alloy, as verified previously for other metallic materials.