Identificação de um novo motivo peptídico específico para a vasculatura cerebral e que diferencia as barreiras hematoenfálica e hematoretiniana / Identification of a new specific peptide motif to the brain vasculature that differentiates between the blood brain barrier and the bloodretinal barrier

O conceito de heterogeneidade vascular é bem aceito pela comunidade cientifica, desempenhando papel essencial em processos fisiológicos e patológicos. Uma vez que os vasos sanguíneos são importantes na organogênese, diferenciação e morfogênese de tecidos e órgãos, torna-se interessante desvendar a diversidade vascular cerebral, identificando novos marcadores moleculares para este órgão tão importante. Utilizando tecnologia combinatorial de phage display in vivo, identificamos um novo motivo peptídico, na qual os aminoácidos FenilalaninaArginina-Triptofano (Phe-Arg-Trp; FRW) predominam. Este motivo peptídico é um ligante seletivo para vasos sanguíneos cerebrais e não se acumula em outros órgãos, incluíndo tecidos como intestinos e gônadas, que também apresentam barreiras endoteliais especificas. No entanto, mais surpreendente foi a observação de que o motivo FRW não se liga aos vasos sanguíneos da retina, o que implica em uma diferença até então desconhecida entre duas barreiras: a barreira hematoencefálica e a barreira hematoretiniana. Combinando phage display in vivo e microscopia eletrônica de transmissão, observamos a presença de partículas de fago ligadas à vasculatura cerebral em um nível supramolecular: aglomerados de fagos filamentosos expressando o motivo FRW foram visualizados ligados às regiões de contato entre as células endoteliais. Por fim, a utilização do peptídeo CFFWKFRWMC permite imageamento in vivo, demonstrando que novas ferramentas para estudar e visualizar o cérebro podem surgir deste motivo

The concept of vascular heterogeneity is well accepted by the scientific community, playing an essential role in physiological and pathological processes. Since blood vessels are important in organogenesis, differentiation, and morphogenesis of tissues and organs, it becomes interesting to unveil the cerebral vascular diversity, identifying new molecular markers for such important organ. Using in vivo phage display, we show that a new peptide motif that emerged from our combinatorial screening of the vasculature binds selectively to blood vessels in the brain in vivo but not to vessels in other organs. Peptides containing a conserved motif in which amino acids Phenylalanine-Arginine-Tryptophan (Phe-Arg-Trp; FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial in all areas of the brain, including the optic nerve but not in other barrier containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif

Proteomic analysis of the acquired enamel pellicle formed on human and bovine tooth: a study using the Bauru in situ pellicle model (BISPM)

Abstract The acquired enamel pellicle (AEP) is an organic film, bacteria-free, formed in vivo as a result of the selective adsorption of salivary proteins and glycoproteins to the solid surfaces exposed to the oral environment. Objective: This study aimed to compare the proteomic profile of AEP formed in situ on human and bovine enamel using a new intraoral device (Bauru in situ pellicle model - BISPM). Material and Methods: One hundred and eight samples of human and bovine enamel were prepared (4×4 mm). Nine subjects with good oral conditions wore a removable jaw appliance (BISPM) with 6 slabs of each substrate randomly allocated. The AEP was formed during the morning, for 120 minutes, and collected with an electrode filter paper soaked in 3% citric acid. This procedure was conducted in triplicate and the pellicle collected was processed for analysis by LC-ESI-MS/MS. The obtained mass spectrometry MS/MS spectra were searched against human protein database (SWISS-PROT). Results: The use of BISPM allowed the collection of enough proteins amount for proper analysis. A total of 51 proteins were found in the AEP collected from the substrates. Among them, 15 were common to both groups, 14 were exclusive of the bovine enamel, and 22 were exclusive of the human enamel. Proteins typically found in the AEP were identified, such as Histatin-1, Ig alpha-1, Ig alpha 2, Lysozyme C, Statherin and Submaxillary gland androgen-regulated protein 3B. Proteins not previously described in the AEP, such as metabolism, cell signaling, cell adhesion, cell division, transport, protein synthesis and degradation were also identified. Conclusion: These results demonstrate that the proteins typically found in the AEP appeared in both groups, regardless the substrate. The BISPM revealed to be a good device to be used in studies involving proteomic analysis of the AEP.

Peptidomic investigation of Neoponera villosa venom by high-resolution mass spectrometry: seasonal and nesting habitat variations

Advancements in proteomics, including the technological improvement in instrumentation, have turned mass spectrometry into an indispensable tool in the study of venoms and toxins. In addition, the advance of nanoscale liquid chromatography coupled to nanoelectrospray mass spectrometry allows, due to its high sensitivity, the study of venoms from species previously left aside, such as ants. Ant venoms are a complex mixture of compounds used for defense, predation or communication purposes. The venom from Neoponera ants, a genus restricted to Neotropical regions, is known to have cytolytic, hemolytic, antimicrobial and insecticidal activities. Moreover, venoms from several Neoponera species have been compared and differences in their toxicity related to nesting habitat variation were reported. Therefore, the present study aimed to perform a deep peptidomic analysis of Neoponera villosa venom and a comparison of seasonal and nesting habitat variations using high-resolution mass spectrometry. Methods: Specimens of N. villosa ants were captured in Panga Natural Reserve (Uberlândia, MG, Brazil) from arboreal and ground-dwelling nests during summer and winter time. The venom glands were dissected, pooled and disrupted by ultra-sonic waves. The venom collected from different habitats (arboreal and ground-dwelling) and different seasons (summer and winter) was injected into a nanoACQUITY ULPC hyphened to a Q-Exactive Orbitrap mass spectrometer. The raw data were analyzed using PEAKS 7. Results: The results showed a molecular diversity of more than 500 peptides among these venoms, mostly in the mass range of 800-4000 Da. Mutations and post-translational modifications were described and differences among the venoms were observed. Part of the peptides matched with ponericins, a well-known antimicrobial peptide family. In addition, smaller fragments related to ponericins were also identified, suggesting that this class of antimicrobial peptide might undergo enzymatic cleavages. Conclusion: There are substantial differences among the venom of N. villosa ants collected in different seasons and from different nest habitats. The venom composition is affected by climate changes that influence prey availability and predator presence. Clearly, nano-LC-MS boosted the knowledge about ant venom, a rich source of unexplored and promising bioactive compounds.(AU)

Sequence analysis of the cDNA encoding for SpCTx: a lethal factor from scorpionfish venom ( Scorpaena plumieri )

Lethal factors are multifunctional oligomeric proteins found in the venomous apparatus of Scorpaeniformes fish. These toxins elicit not only an array of biological responses in vitro but also cardiovascular disorders and strong hemolytic, nociceptive and edematogenic activities in vivo. This work describes the cloning and molecular identification of two toxin subunits, denominated Sp-CTx-α and Sp-CTx-ß, from scorpionfish venom ( Scorpaena plumieri ). Methods: The primary structures were deduced after cDNA amplification by PCR with primers from conserved sequences described in Scorpaeniformes toxins. Following DNA sequencing and bioinformatic analysis, the tridimensional structures of both subunits were modeled. Results: The translated sequences (702 amino acids, each subunit) show homology with other lethal factors, while alignment between Sp-CTx-α and Sp-CTx-ß shows 54% identity. The subunits lack N-terminal signal sequences and display masses of approximately 80 kDa each. Both Sp-CTx subunits display a B30.2/SPRY domain at the C-terminal region with typically conserved motifs as described in these toxins. Secondary structure prediction identified six α-helices 18 residues long in both α and ß subunits, some of them amphiphilic with their N-terminal flanked by many basic residues, creating a cationic site associated with the cytolytic activity of these toxins. Antimicrobial potential sites were identified in Sp-CTx and share some features with other peptides presenting variable and broad-spectrum activity. A phylogenetic tree built to represent these toxins supports the proximity between scorpionfish, lionfish and stonefish. Conclusion: The study identified a putative toxin protein whose primary structure is similar to other fish toxins and with potential for production of antivenom against scorpionfish envenomation in Brazil. As a prelude to structure-function studies, we propose that the toxin is structurally related to pore-forming marine toxins.(AU)

Improvement of erectile dysfunction by the active pepide from urechis unicinctus by high temperature/pressure and ultra - wave assisted lysis in streptozotocin induced diabetic rats

ABSTRACT Introduction: We investigate the effect of active peptide from Urechis unicinctus (UU) by high temperature/pressure and ultra-wave assisted lysis on erectile dysfunction in streptozotocin-induced diabetic rats. Materials and Methods: Forty 12-week-old Sprague-Dawley rats were used in this study. Diabetes was induced by a one-time intraperitoneal injection of streptozotocin (50mg/kg). One week later, the diabetic rats were randomly divided into four groups: normal control, untreated diabetes control, and groups treated with 100 or 500mg/kg/d UU peptide. Rats were fed with UU peptide by intragastric administration for 8 weeks. After 8 weeks, penile hemodynamic function was evaluated in all groups by measuring the intracavernosal pressure after electrostimulating the cavernous nerve. Nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) activities were measured and endothelial nitric oxide synthase (eNOS) and neuronal NOS (nNOS) protein expression was determined by Western blot. Results: Maximum intracavernosal pressure in diabetic control rats decreased significantly compared to normal control rats, and was increased significantly compared to untreated diabetic rats after UU peptide supplementation. Treatment with the higher dose of UU peptide significantly increased the NO and cGMP levels compared with the diabetic control group. Decreased activity and expression eNOS and nNOS were found in the diabetic rats compared with the normal control group. Decreased eNOS and nNOS in diabetic rats were improved by UU peptide administration. Conclusions: Active peptide from UU ameliorates erectile function in a streptozotocin induced diabetic rat model of erectile dysfunction.

Initial peptidomic profiling of Brazilian sea urchins: Arbacia lixula, Lytechinus variegatus and Echinometra lucunter

Sea urchins can be found throughout the Brazilian coast and are reported to be one of the major causes of marine accidents on the shoreline. Although not lethal, these accidents are reported to be extremely painful. In order to understand the toxinology of the Brazilian urchins, a peptidomic approach was performed aiming to characterize the naturally occurring peptides in both the coelomic fluid and the spine. Methods Animals were collected without gender distinction and samples of the coelomic fluid and spines extracted were analyzed by RP-HPLC and mass spectrometry for peptide de novo sequencing. Results Several peptides were identified either in the coelomic fluid or the spine extract (except for E. lucunter). The peptide sequences were aligned with public deposited sequences and possible functions were inferred. Moreover, some peptides can be cryptides, since their sequences were identified within functional proteins, for example thymosin from Strongylocentrotus purpuratus. Conclusions Although preliminary, the peptidomic approach presented here reports, for the first time, the abundance of novel biological molecules derived from these animals. The discovery of such molecules may be of potential biotechnological application, as described for other organisms; nevertheless, further studies are required.

Péptidos inmunogénicos del gluten en deposiciones: nuevo método para evaluar la adherencia a la dieta en pacientes celíacos / Immunogenic gluten peptides in stools: new method to evaluate in the dietary adherence in celiac patients

Celiac disease (CD) is an autoimmune pathology caused by the ingestion of gluten in genetically susceptible people, currently considered multisystemic. The treatment of CD is a lifelong strict Gluten-Free Diet (GFD), which allows a symptomatic improvement in most patients and achieve intestinal mucosa healing confirmed with histological study. The adherence to the GFD is variable, arguing as possible factors related to failure the economic, cultural, social aspects and the consumption of gluten inadvertently. The management of celiac patients contemplates instructing in the proper follow-up of GFD and evaluating their adherence. So far, the only way to assess adherence to GFD is through surveys, self-reports of eating habits and serology, being the main disadvantage the subjectivity factor. Recently the immunogenic gluten peptides have acquired relevance for the objective evaluation of the adherence to the GFD and the measurement appears as an efficient and sensitive option to determine the gluten intake, providing relevant information for the clinical management.

Estudos biofísicos e de atividade de peptídeos correspondentes ao N-terminal das toxinas esticolisinais I e II - contribuição para a elucidação do mecanismo de ação / Biophysical and activity studies of peptides corresponding to the N-termini of sticholysins I and II - a contribution to the elucidation of the toxins' mechanism of action

Esticolisinas I e II, citolisinas purificadas da anêmona marinha Stichodactyla helianthus, agem lisando membranas biológicas e modelo. O mecanismo de ação proposto consiste na formação de um poro toroidal com o envolvimento do domínio N-terminal. Diferentes aspectos da interação entre peptídeos derivados do N-terminal das toxinas (StI1-31 and StI12-31 SELAGTIIDGASLTFEVLDKVLGELGKVSRK, e StII1-30 and StII11-30 ALAGTIIAGASLTFQVLDKVLEELGKVSRK) com membranas modelo - micelas e bicamadas - foram estudados com o objetivo de contribuir para a elucidação do mecanismo de ação das toxinas em nível molecular. O emprego dos peptídeos teve como base a hipótese de que fragmentos proteicos podem ser capazes de mimetizar a estrutura e atividade das proteínas inteiras. O análogo contendo o aminoácido paramagnético TOAC (N-TOAC-StII11-30) também foi estudado. Estudos conformacionais foram realizados empregando-se as técnicas espectroscópicas de dicroísmo circular (CD), ressonância paramagnética eletrônica (EPR) e fluorescência. Foram ainda realizados estudos de predição de estrutura e modelagem molecular. Espectros de CD mostraram que os peptídeos adquirem conformação em α-hélice ao interagir com membranas modelo, de acordo com a conformação observada nessa região para as toxinas. Variando a composição lipídica das membranas modelo estudadas, foi possível investigar a contribuição de forças eletrostáticas de de interações hidrofóbicas para a ligação do peptídeo. Ensaios de supressão de fluorescência de lípidos contendo grupamentos fluorescentes em diferentes posições pelo resíduo paramagnético TOAC e espectros de ressonância paramagnética eletrônica (EPR) permitiram localizar o resíduo TOAC na interface membrana-água, corroborando o modelo proposto do poro toroidal. A análise dos espectros de CD e EPR também permitiu obter as constantes de ligação dos peptídeos com micelas e bicamadas. Os peptídeos também foram capazes de mimetizar as toxinas do ponto de vista funcional, como mostrado por testes de vazamento de carboxifluoresceína e atividade hemolítica. Peptídeos curtos, contendo partes da sequência de StII1-30, sintetizados com o objetivo de examinar uma eventual atividade antimicrobiana, demonstraram baixa atividade, bem como ausência de atividade hemolítica e de toxicidade para células humanas

Sticholysins I and II, cytolysins purified from the sea anemone Stichodactyla helianthus, act by lysing biological and model membranes. The proposed mechanism of action consists in the formation of a toroidal pore with the involvement of the N-terminal domain [1]. Different aspects of the interaction between peptides from the toxins' N-termini (StI1-31 and StI12-31 SELAGTIIDGASLTFEVLDKVLGELGKVSRK, and StII1-30 and StII11-30 ALAGTIIAGASLTFQVLDKVLEELGKVSRK) and model membranes - micelles and bilayers - have been studied to contribute to the elucidation of the toxins mechanism of action at the molecular level. The use of peptides was based on the hypothesis that potein fragments can eventually mimic the structure and activity of the whole protein. An analogue containing the paramagnetic amino acid TOAC (N-TOAC-StII11-30) was also studied. Conformational studies were performed making use of the spectroscopic techniques circular dichroism (CD), electron paramagnetic resonance (EPR), and fluorescence. Studies of structure prediction and molecular modeling were also performed. CD spectra showed that the peptides acquired α-helical conformation upon interaction with model lipid membranes, in agreement with the conformation found for these segments in the whole proteins. Making use of membranes of variable lipid composition, it was possible to assess the contribution of electrostatic and hydrophobic interactions for peptide binding. Fluorescence quenching of labeled lipids by paramagnetic TOAC and EPR spectra allowed us to locate the TOAC residue at the membrane-water interface, corroborating the proposed model of the toroidal pore. The CD and EPR studies also allowed us to obtain the binding constants for the peptide-micelle and peptide-bilayer interaction. The peptides were also capable of mimicking the toxins function, as shown by assays of carboxyfluorescein leakage and hemolytic activity. Short peptides containing parts of StII1-30's sequence were synthesized with the aim of testing their antimicrobial activity. The peptides displayed low antimicrobial activity, as well as lack of hemolytic activity and toxicity against human cells

Estimation of N‑terminal telopeptides of type I collagen in periodontal health, disease and after nonsurgical periodontaltherapy in gingival crevicular fluid: A clinico ‑ biochemical study.

Aim: This study explored gingival crevicular fluid (GCF) N‑terminal telopeptides of type I collagen (NTx) levels in periodontal health, disease and after nonsurgical periodontal therapy along with its association with the clinical parameters. Materials and Methods: Study comprised of three groups of 10 subjects each: Healthy (Group I), gingivitis (Group II), and periodontitis (Group III), while Group III patients after scaling and root planning (SRP) constituted Group IV. Gingival index (GI), probing pocket depth (PPD), clinical attachment loss (CAL), and radiological parameters were recorded. GCF samples were analyzed by competitive‑enzyme‑linked immunosorbent assay. Results: Samples in Group III and Group IV tested positive for NTx whereas in Group I and Group II, NTx was not detected. Mean NTx levels were higher in Group III (6.79 ± 0.94 nanomole bone collagen equivalents per liter [nm BCE/L]) compared to Group IV (5.73 ± 0.95 nm BCE/L) which was statistically significant. Positive correlation was seen between the clinical parameters and the NTx levels in Group III and IV. Conclusion: As NTx is specific bone turnover marker, it is detected only in periodontitis Group and the values decline after SRP. Failure to detect NTx in Group I and II, relates to the minimum or no resorption at the sample sites.

Liver cancer stem cells are selectively enriched by low-dose cisplatin

Accumulating evidence has indicated the importance of cancer stem cells in carcinogenesis. The goal of the present study was to determine the effect of low-dose cisplatin on enriched liver cancer stem cells (LCSCs). Human hepatoblastoma HepG2 cells were treated with concentrations of cisplatin ranging from 1 to 5 μg/mL. Cell survival and proliferation were evaluated using a tetrazolium dye (MTT) assay. LCSCs were identified using specific markers, namely aldehyde dehydrogenase-1 (ALDH1) and CD133. The percentage of ALDH1+ or CD133+ cells was examined by flow cytometric analysis. The expression of ALDH1 and/or CD133 in HepG2 cells was determined by immunocytochemical analysis. Low-dose cisplatin treatment significantly decreased cell survival in HepG2 cells after 24 or 72 h. However, the percentage of LCSCs in the surviving cells was greatly increased. The percentage of ALDH1+ or CD133+ cells was increased in a time- and dose-dependent manner after treatment with 1-4 μg/mL cisplatin, whereas 5 μg/mL cisplatin exposure slightly reduced the number of positive cells. These findings indicate that low-dose cisplatin treatment may efficiently enrich the LCSC population in HepG2 cells.

Optimization of Bacillus subtilis cell growth effecting jiean-peptide production in fed batch fermentation using central composite design

Background Optimization of nutrient feeding was developed to improve the growth of Bacillus subtilis in fed batch fermentation to increase the production of jiean-peptide (JAA). A central composite design (CCD) was used to obtain a model describing the relationship between glucose, total nitrogen, and the maximum cell dry weight in the culture broth with fed batch fermentation in a 5 L fermentor. Results The results were analyzed using response surface methodology (RSM), and the optimized values of glucose and total nitrogen concentration were 30.70 g/L and 1.68 g/L in the culture, respectively. The highest cell dry weight was improved to 77.50 g/L in fed batch fermentation, which is 280% higher than the batch fermentation concentration (20.37 g/L). This led to a 44% increase of JAA production in fed batch fermentation as compared to the production of batch fermentation. Conclusion The results of this work improve the present production of JAA and may be adopted for other objective products' production.

Degree of hydrolysis and peptide profile of whey proteins using pancreatin / Grau de hidrólise e perfil peptídico das proteínas do soro de leite utilizando a pancreatina

In the present study, we used pancreatin for hydrolyzing whey proteins and evaluated the degree of hydrolysis (DH) and peptide size distribution. The following methods were used for DH determination: formol titration, soluble protein content, ortho-phthalaldehyde (OPA), and freezing point. Peptide size distribution was conducted by size-exclusion high-performance liquid chromatography (HPLC). The DH varied from 3.26% to 36.41%, and the highest yield was obtained by the soluble protein content method (mean of 35.85%). Formol titration was considered the most suitable method for assessing the DH, because it showed a marked increase with reaction time (from 1h-17.20% to 2h-24.86%). Significant positive correlation of strong intensity was observed between the following methods: formol titration and OPA (r=0.9616; p=0.0090), formol titration and freezing point (r=0.8784; p=0.0493), and OPA and freezing point (r=0.9515; p=0.0127). The highest contents of di- and tri-peptides and free amino acids were 9.07% and 8.22%, respectively. A significant positive correlation of strong intensity was also observed between the degree of hydrolysis and the fraction of medium-sized peptides by the formol titration (r =?0.9274; p=0.0232) and OPA (r=?0.8977; p=0.0386) methods.

En este trabajo, se utilizó una pancreatina para hidrolizar las proteínas del suero de leche, evaluándose el grado de hidrólisis (GH) y la distribución de tamaño de los péptidos. Los métodos de valoración con formol, determinación de proteínas solubles, ortoftalaldehído (OPA) y disminución del punto de congelación se emplearon para evaluar el GH. La distribución del tamaño de los péptidos se realizó por cromatografía líquida de exclusión molecular. El GH varió de 3,26% a 36,41% y los mejores resultados se obtuvieron con el método de determinación de proteínas solubles (media de 35,85%). La valoración con formol se consideró el método más adecuado para evaluar el GH, en función de los significativos incrementos con el tiempo de reacción (de 17,20% a 24,86%). Se observó una correlación positiva y de fuerte intensidad entre los siguientes métodos: valoración con formol y OPA (r=0,9616, p=0,0090); valoración con formol y disminución del punto de congelación (r=0,8784, p=0,0493); OPA con la disminución del punto de congelación (r=0,9515, p=0,0127). Los contenidos más altos de di-tripéptidos y de aminoácidos libres fueron de 9,07% y 8,22%, respectivamente. Una correlación positiva y de fuerte intensidad se verificó entre la fracción de péptidos medios y el GH evaluado con los métodos de la valoración con formol (r=?0,9274, p=0,0232) y OPA (r=?0,8977, p=0,0386).

Neste trabalho, uma pancreatina foi utilizada para hidrolisar as proteínas do soro de leite, avaliando-se o grau de hidrólise (GH) e a distribuição de tamanho dos peptídeos. Os métodos de titulação com formol, determinação de pro-teínas solúveis, ortoftalaldeído (OPA) e depressão do ponto de congelamento foram empregados para avaliar o GH. A distribuição do tamanho de peptídeos foi realizada por cromatografia líquida de exclusão molecular. O GH variou de 3,26% a 36,41% e os melhores resultados foram obtidos com o método de determinação de pro¬teínas solúveis (média de 35,85%). A titulação com formol foi considerada como o método mais adequado para avaliar o GH, em função dos significativos aumentos com o tempo de reação (de 17,20% a 24,86%). Observou-se uma correlação positiva e de forte intensidade entre os métodos de titulação com formol e OPA (r=0,9616, p=0,0090); titulação com formol e depressão do ponto de congelamento (r=0,8784, p=0,0493); OPA e depressão do ponto de congelamento (r=0,9515, p=0,0127). Os maiores teores de di-tripeptídeos e de aminoácidos livres foram de 9,07% e 8,22%, respectivamente. Correlação positiva e de forte intensidade foi verificada entre a fração de peptídeos médios e o GH avaliado pelos métodos da titulação com formol (r=?0,9274, p=0,0232) e OPA (r=?0,8977, p=0,0386).

The Cuban scorpion Rhopalurus junceus (Scorpiones, Buthidae): component variations in venom samples collected in different geographical areas

The venom of the Cuban scorpion Rhopalurus junceus is poorly study from the point of view of their components at molecular level and the functions associated. The purpose of this article was to conduct a proteomic analysis of venom components from scorpions collected in different geographical areas of the country. Results Venom from the blue scorpion, as it is called, was collected separately from specimens of five distinct Cuban towns (Moa, La Poa, Limonar, El Chote and Farallones) of the Nipe-Sagua-Baracoa mountain massif and fractionated by high performance liquid chromatography (HPLC); the molecular masses of each fraction were ascertained by mass spectrometry analysis. At least 153 different molecular mass components were identified among the five samples analyzed. Molecular masses varied from 466 to 19755 Da. Scorpion HPLC profiles differed among these different geographical locations and the predominant molecular masses of their components. The most evident differences are in the relative concentration of the venom components. The most abundant components presented molecular weights around 4 kDa, known to be K+-channel specific peptides, and 7 kDa, known to be Na+-channel specific peptides, but with small molecular weight differences. Approximately 30 peptides found in venom samples from the different geographical areas are identical, supporting the idea that they all probably belong to the same species, with some interpopulational variations. Differences were also found in the presence of phospholipase, found in venoms from the Poa area (molecular weights on the order of 14 to 19 kDa). The only ubiquitous enzyme identified in the venoms from all five localities studied (hyaluronidase) presented the same 45 kD molecular mass, identified by gel electrophoresis analysis. Conclusions The venom of these scorpions from different.

Periarticular Osteoporosis Is a Prominent Feature in Early Rheumatoid Arthritis: Estimation Using Shaft to Periarticular Bone Mineral Density Ratio

We aimed to quantify periarticular osteoporosis and investigate its significance in 45 patients with rheumatoid arthritis (RA) and 106 controls. Dual-energy X-ray absorptiometry (DXA) was used to determine the ratio of shaft to periarticular bone mineral density (BMD) as an index of periarticular demineralization. Periarticular osteoporosis was measured by conventional radiography. The BMDs of shaft and periarticular regions in eight designated areas on proximal phalanges were quantified. Clinical variables were examined to identify risk factors for periarticular osteoporosis. The assessment of periarticular osteoporosis on X-ray images reached a moderate degree of interobserver agreement among four physicians (k = 0.47). For BMD quantification, we designed three types of mathematical formulae: the ratio of shaft to periarticular BMD, the mean of the ratios, and the ratio of the sums. These ratios were significantly higher in the patients with early RA (disease duration < or = 3 yr) than in controls (P < 0.01). The findings were not as distinctive in patients with established RA. Body mass index, cumulative dose of corticosteroid, and C-terminal telopeptide were correlated with BMD ratios. Conclusively, DXA-assisted localized quantification and BMD ratio calculations are feasible for assessing periarticular demineralization. Periarticular osteoporosis is a relatively distinctive feature of early RA.

Different approaches, one target: understanding cellular mechanisms of Parkinson's and Alzheimer's diseases / Abordagens diferentes, um único objetivo: compreender os mecanismos celulares das doenças de Parkinson e de Alzheimer

Neurodegenerative disorders are undoubtedly an increasing problem in the health sciences, given the increase of life expectancy and occasional vicious life style. Despite the fact that the mechanisms of such diseases are far from being completely understood, a large number of studies that derive from both the basic science and clinical approaches have contributed substantial data in that direction. In this review, it is discussed several frontiers of basic research on Parkinson´s and Alzheimer´s diseases, in which research groups from three departments of the Institute of Biomedical Sciences of the University of São Paulo have been involved in a multidisciplinary effort. The main focus of the review involves the animal models that have been developed to study cellular and molecular aspects of those neurodegenerative diseases, including oxidative stress, insulin signaling and proteomic analyses, among others. We anticipate that this review will help the group determine future directions of joint research in the field and, more importantly, set the level of cooperation we plan to develop in collaboration with colleagues of the Nucleus for Applied Neuroscience Research that are mostly involved with clinical research in the same field.

Os transtornos neurodegenerativos são, sem dúvida, um problema crescente nas ciências da saúde, dado o aumento da expectativa de vida e de estilos de vida pouco saudáveis. Embora os mecanismos de tais doenças ainda estejam longe de ser esclarecidos, vários estudos que derivam tanto da ciência básica quanto de abordagens clínicas contribuíram nessa direção. Na presente revisão, são discutidas linhas de frente da pesquisa básica sobre as doenças de Parkinson e Alzheimer, em que grupos de pesquisas de três departamentos do Instituto de Ciências Biomédicas da Universidade de São Paulo estão envolvidos em um esforço multidisciplinar. O foco principal desta revisão envolve os modelos animais desenvolvidos para se estudar os aspectos celulares e moleculares daquelas doenças neurodegenerativas, incluindo o estresse oxidativo, a sinalização da insulina e as análises proteômicas, dentre outros. Antecipamos que esta revisão irá auxiliar o grupo a determinar as futuras direções da pesquisa conjunta nessa área e, o mais importante, estabelecer o nível de cooperação que planejamos desenvolver juntamente com colegas do Núcleo de Apoio à Pesquisa em Neurociência Aplicada que estão envolvidos com pesquisa clínica na mesma área.

Caracterización de factores asociados con comportamiento suicida en adolescentes estudiantes de octavo grado, en tres colegios bogotanos / Factor Characterization Associated with Suicidal Behavior in 8"' Grade Adolescent Students in Three Schools from Bogotá (Colombia)

Objetivos: Determinar la probabilidad de riesgo suicida y/o enfermedad mental y factores asociados en estudiantes de secundaria de tres colegios bogotanos. Métodos: Estudio de corte transversal con 309 adolescentes. Resultados: El promedio de edad fue de 13,83 ± 0,9 años, predominó el género femenino (58,6%) y el estrato socioeconómico 3 (68,3%). La probabilidad de riesgo para comportamiento suicida y/o síntomas mentales fue de 47,6%; 26,5% tuvo alguna manifestación suicida; 14,23% tuvo ideación suicida en los últimos tres meses; 3,55% tuvo intentos suicidas alguna vez en la vida, y 8,73% tuvo ideación suicida e intentos suicidas en los últimos tres meses. El riesgo de comportamiento suicida y/o enfermedad mental fue explicado conjuntamente por la depresión (OR = 27,9, IC95% = 3,5-223,1), la baja autoestima (OR = 11,8, IC95% = 2,5-56,5), la disfunción familiar severa (OR = 3,4, IC95% = 1,2-9,7), el sexo femenino (OR = 2,1, IC95% = 1,2-3,8) y la edad mayor o igual a 15 años (OR = 1,9, IC95% = 0,9-3,9). El maltrato psicológico seguido del abuso físico se asociaron con manifestación suicida y/o enfermedad mental, y la buena relación familiar, con menor probabilidad. Conclusión: La depresión, la baja autoestima, la disfuncionalidad familiar, el género femenino, la edad > 15 y la violencia intrafamiliar son factores asociados al riesgo suicida y/o enfermedad mental en adolescentes, y las buenas relaciones familiares se asocian con menor riesgo.

Objective: To establish the probability for suicide risk and/or mental disorders, together with related factors among high school students in 3 schools in Bogota. Methods: Cross sectional study of 309 adolescents. Results: The average age was 13.83 ± 0.9, female dominance (58.6%) and a 3rd socioeconomic stratum (68.3%). The suicidal risk behavioral probability and/or mental symptoms was 47.6%, 26.5% exhibited some suicide manifestations, 14.23% had experienced suicidal ideas in the last 3 months, 3.55% had had suicide attempts at least once in life, and 8.73% had suicidal ideas in the last 3 months with suicide attempts. The risk of suicidal behavior and /or mental disorders was explained jointly by depression (OR=27.9, 95% CI: 3.5-223. 1), low self-esteem (OR=11.8, 95% CI: 2.5-56.5), severe family dysfunction (OR=3.4, 95%CI 1.2-9.7), being female (OR=2.1, 95% CI: 1.2-3.8) and being 15 or older (OR=1.9, 95% CI: 0.967-3.9). Psychological abuse followed by physical mistreatment was associated with suicidal behavior and /or mental illness while good family relationships were associated to lower probability. Conclusion: Depression, low self-esteem, severe family dysfunction, female gender, older age (> 15) and domestic violence are risk factors associated with suicide and/or mental disorders in adolescents; good family relationships are associated with lower risk.

Peptidolytic microbial community of methanogenic reactors from two modified UASBs of brewery industries

We studied the peptide-degrading anaerobic communities of methanogenic reactors from two mesophilic full-scale modified upflow anaerobic sludge blanket (UASB) reactors treating brewery wastewater in Colombia. Most probable number (MPN) counts varied between 7.1 x 10(8) and 6.6 x 10(9) bacteria/g volatile suspended solids VSS (Methanogenic Reactor 1) and 7.2 x 10(6) and 6.4 x 10(7) bacteria/g (VSS) (Methanogenic Reactor 2). Metabolites detected in the highest positive MPN dilutions in both reactors were mostly acetate, propionate, isovalerate and, in some cases, negligible concentrations of butyrate. Using the highest positive dilutions of MPN counts, 50 dominant strains were isolated from both reactors, and 12 strains were selected for sequencing their 16S rRNA gene based on their phenotypic characteristics. The small-subunit rRNA gene sequences indicated that these strains were affiliated to the families Propionibacteriaceae, Clostridiaceae and Syntrophomonadaceae in the low G + C gram-positive group and Desulfovibrio spp. in the class d-Proteobacteria. The main metabolites detected in the highest positive dilutions of MPN and the presence of Syntrophomonadaceae indicate the effect of the syntrophic associations on the bioconversion of these substrates in methanogenic reactors. Additionally, the potential utilization of external electron acceptors for the complete degradation of amino acids by Clostridium strains confirms the relevance of these acceptors in the transformation of peptides and amino acids in these systems.

Evaluation of a lipopeptide biosurfactant from Bacillus natto TK-1 as a potential source of anti-adhesive, antimicrobial and antitumor activities / Avaliação de um lipopeptídio biosurfactante de Bacillus natto TK-1 com fonte potencial de atividade antiadesiva, antimicrobiana e antitumoral

A lipopeptide biosurfactant produced by Bacillus natto TK-1 has a strong surface activity. The biosurfactant was found to be an anti-adhesive agent against several bacterial strains, and also showed a broad spectrum of antimicrobial activity. The biosurfactant induced a significant reduction in tumor cells viability in a dose- dependent manner.

Um lipopeptídio biosurfactante produzido por Bacillus natto TK-1 apresenta intensa atividade de superfície. Verificou-se que o biosurfactante apresentou atividade antiadesiva contra várias cepas bacterianas, e também atividade antimicrobiana de amplo espectro. O biosurfactante causou uma redução significativa na viabilidade de células tumorais, de forma dose-dependente.

Doppler e marcadores séricos maternos na predição de complicações da gestação / Doppler and maternal serum screening in the prediction of pregnancy complications

OBJETIVO: Comparar a eficácia do Doppler das artérias uterinas e de marcadores séricos maternos na predição de complicações da gestação. MATERIAIS E MÉTODOS: Trata-se de um estudo prospectivo com 49 primigestas, incluídas no estudo na 18ª semana, sendo coletada a amostra sanguínea para a realização das dosagens séricas, realizadas pelo método de quimioluminescência (alfa-fetoproteína, gonadotrofina coriônica humana e óxido nítrico) e radioimunoensaio (peptídio atrial natriurético). O Doppler das artérias uterinas foi realizado entre 24-26 semanas, determinando a presença ou ausência de incisura na onda de velocidade de fluxo. Na análise estatística utilizou-se o teste de Mann-Whitney, para amostras não-paramétricas, e o teste exato de Fisher, para parâmetros qualitativos. RESULTADOS: Os valores de sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo foram, respectivamente, de 8,3 por cento, 97,0 por cento, 50,0 por cento e 74,4 por cento para a alfa-fetoproteína; 8,3 por cento, 87,9 por cento, 20,0 por cento e 72,5 por cento para a gonadotrofina coriônica humana; 16,7 por cento, 97,0 por cento, 33,3 por cento e 76,2 por cento para o peptídio atrial natriurético; e 16,7 por cento, 93,9 por cento, 50,0 por cento e 75,6 por cento para o óxido nítrico. A sensibilidade do Doppler foi de 75,0 por cento, especificidade de 63,6 por cento, valor preditivo positivo de 57,1 por cento e valor preditivo negativo de 87,5 por cento. CONCLUSÃO: O Doppler das artérias uterinas é melhor preditor de complicações da gestação quando comparado a alguns marcadores séricos em populações de baixo risco.

OBJECTIVE: To compare the effectiveness of uterine artery Doppler and maternal serum screening in the prediction of pregnancy complications. MATERIALS AND METHODS: Prospective study with 49 primigravidae at their 18th gestational week, when a blood sample was collected for serum dosage by chemiluminescence (alpha-fetoprotein, human chorionic gonadotropin and nitric oxide) and radioimmunoassay (atrial natriuretic peptide). Uterine artery Doppler was performed between the 24th and 26th gestational weeks, for determining the presence or absence of notch in the flow velocity waveform. The non-parametric Mann-Whitney test was utilized for statistical analysis, and the Fisher exact test for analysis of qualitative parameters. RESULTS: Sensitivity, specificity, positive and negative predictive values were, respectively, 8.3 percent, 97.0 percent, 50.0 percent and 74.4 percent for alpha-fetoprotein; 8.3 percent, 87.9 percent, 20.0 percent and 72.5 percent for human chorionic gonadotropin; 16.7 percent, 97.0 percent, 33.3 percent and 76.2 percent for atrial natriuretic peptide; and 16.7 percent, 93.9 percent, 50.0 percent and 75.6 percent for nitric oxide. The uterine artery Doppler sensitivity was 75.0 percent, specificity 63.6 percent, positive predictive value 57.1 percent, and negative predictive value 87.5 percent. CONCLUSION: Uterine artery Doppler is an effective method for prediction of pregnancy complications as compared with maternal serum screening in low risk populations.

Decrease of mosquito salivary gland proteins after a blood meal: an implication for pathogenesis of mosquito bite allergy.

Salivary gland protein profiles ofAedes aegypti (L.) and Culex quinquefasciatus (Say) pre- and post-blood feeding were analyzed. SDS-PAGE studies before blood feeding of Ae. aegypti demonstrated 8 major polypeptide bands of 20, 35, 37, 42, 45, 47, 70 kDa and a high molecular weight band >118 kDa, whereas those of Cx. quinquefasciatus demonstrated 9 major polypeptide bands of 20, 26, 36, 38, 45, 47, 49 kDa and 2 high molecular weight bands >118 kDa. After a blood feeding, salivary gland polypeptides of Ae. aegypti at 35, 37, 45, 47, 70 kDa and high molecular weight band >118 kDa were depleted, while the polypeptide bands of 20, 26, 36, 38 kDa were depleted in Cx. quinquefasciatus. The presented study suggests that these major polypeptides were introduced into vertebrate hosts when a mosquito took a blood meal. Further investigation in molecular, biochemical and immunological aspects of these salivary gland polypeptides may provide information for better understanding in the role of these proteins in mosquito bite allergy.