Analysis of Bothrops jararacussu venomous gland transcriptome focusing on structural and functional aspects: I - gene expression profile of highly expressed phospholipases A(2)

Snake venom glands are a rich source of bioactive molecules such as peptides, proteins and enzymes that show important pharmacological activity leading to in local and systemic effects as pain, edema, bleeding and muscle necrosis. Most studies on pharmacologically active peptides and proteins from snake venoms have been concerned with isolation and structure elucidation through methods of classical biochemistry. As an attempt to examine the transcripts expressed in the venom gland of Bothrops jararacussu and to unveil the toxicological and pharmacological potential of its products at the molecular level, we generated 549 expressed sequence tags (ESTs) from a directional cDNA library. Sequences obtained from single-pass sequencing of randomly selected cDNA clones could be identified by similarities searches on existing databases, resulting in 197 sequences with significant similarity to phospholipase A(2) (PLA(2)), of which 83.2% were Lys49-PLA(2) homologs (BOJU-1), 0.1% were basic Asp49-PLA(2)s (BOJU-II) and 0.6% were acidic Asp49-PLA(2)s (BOJU-III). Adjoining this very abundant class of proteins we found 88 transcripts codifying for putative sequences of metalloproteases, which after clustering and assembling resulted in three full-length sequences: BOJUMET-I, BOJUMET-II and BOJUMET-III; as well as 25 transcripts related to C-type lectin like protein including a full-length cDNA of a putative galactose binding C-type lectin and a cluster of eight serine-proteases transcripts including a full-length cDNA of a putative serine protease. Among the full-length sequenced clones we identified a nerve growth factor (Bj-NGF) with 92% identity with a human NGF (NGHUBM) and an acidic phospholipase A2 (BthA-I-PLA(2)) displaying 85-93% identity with other snake venom toxins. Genetic distance among PLA(2)s from Bothrops species were evaluated by phylogenetic analysis. Furthermore, analysis of full-length putative Lys49-PLA(2) through molecular modeling showed conserved structural domains, allowing the characterization of those proteins as group II PLA(2)s. The constructed cDNA library provides molecular clones harboring sequences that can be used to probe directly the genetic material from gland venom of other snake species. Expression of complete cDNAs or their modified derivatives will be useful for elucidation of the structure-function relationships of these toxins and peptides of biotechnological interest. (C) 2004 Elsevier SAS. All rights reserved.

Cloning and Identification of a Complete cDNA Coding for a Bactericidal and Antitumoral Acidic Phospholipase A(2) from Bothrops jararacussu Venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A microsatellite-based, gene-rich linkage map for the AA genome of Arachis (Fabaceae)

Cultivated peanut (Arachis hypogaea) is an important crop, widely grown in tropical and subtropical regions of the world. It is highly susceptible to several biotic and abiotic stresses to which wild species are resistant. As a first step towards the introgression of these resistance genes into cultivated peanut, a linkage map based on microsatellite markers was constructed, using an F-2 population obtained from a cross between two diploid wild species with AA genome (A. duranensis and A. stenosperma). A total of 271 new microsatellite markers were developed in the present study from SSR-enriched genomic libraries, expressed sequence tags (ESTs), and by data-mining sequences available in GenBank. of these, 66 were polymorphic for cultivated peanut. The 271 new markers plus another 162 published for peanut were screened against both progenitors and 204 of these (47.1%) were polymorphic, with 170 codominant and 34 dominant markers. The 80 codominant markers segregating 1:2:1 (P < 0.05) were initially used to establish the linkage groups. Distorted and dominant markers were subsequently included in the map. The resulting linkage map consists of 11 linkage groups covering 1,230.89 cM of total map distance, with an average distance of 7.24 cM between markers. This is the first microsatellite-based map published for Arachis, and the first map based on sequences that are all currently publicly available. Because most markers used were derived from ESTs and genomic libraries made using methylation-sensitive restriction enzymes, about one-third of the mapped markers are genic. Linkage group ordering is being validated in other mapping populations, with the aim of constructing a transferable reference map for Arachis.

Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A review of complete albinism in bats with five new cases from Brazil

Complete albinism is a rare phenomenon that occurs in all vertebrate groups. In bats, albinism has been recorded in several species but this information has not previously been reviewed in detail nor has there been an analysis of its importance. In this study, cases of albinism from the literature are checked and new cases are presented. Complete albinism in bats is documented in eight families, 38 species, and at least 64 individuals (47.4% female female and 52.6% males; n = 38). of these, 39 individuals were observed and/or captured in sheltered roosts, such as caves (51.3%), mines and galleries (20.5%), buildings (17.9%), and hollow-trees and bird boxes (7.7%). Only one albino bat (2.6%) was captured in an external roost (foliage). This individual is the fruit bat, Artibeus planirostris, which is recorded here for the first time. Information on four additional cases of albino individuals of the common vampire bat, Desmodus rotundus, is also presented. It is suggested that sheltered roosts favour survival of albino bars, offering protection against sunlight, water loss, and visually hunting predators.

Quasispecies of hepatitis C virus genotype 1 and treatment outcome with Peginterferon and Ribavirin

The majority of patients with chronic hepatitis C fail to respond to antiviral therapy. The genetic basis of this resistance is unknown. The quasispecies nature of HCV may have an important implication concerning viral persistence and response to therapy. The HCV nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy. To evaluate whether the NS5A quasispecies pre-treatment composition of HCV 1a/1b is related to responsiveness to combined pegylated interferon (PEG-IFN) and Ribavirin therapy, detailed analyses of the complete NS5A were performed. Fifteen full-length NS5A clones were sequenced from 11 pretreatment samples of patients infected with genotype 1 HCV (3 virological sustained responders, 4 non-responders, and 4 end-of-treatment responders). Our study could not show a significant correlation between the mean number of mutations in HCV NS5A before treatment and treatment outcome, and the phylogenetic construction of complete NS5A sequences obtained from all patients failed to show any clustering associated with a specific response pattern. No single amino acid position was associated with different responses to therapy in any of the NS5A regions analyzed, and mutations were clustered downstream the ISDR, primarily in the V3 region. We observed that the CRS and NLS regions of the NS5A protein were conflicting for some variables analyzed, although no significant differences were found. If these two regions can have antagonistic functions, it seems viable that they present different mutation profiles when compared with treatment response. The patient sample that presented the lowest genetic distance values also presented the smallest number of variants, and the most heterogeneous pattern was seen in the end-of-treatment patients. These results suggest that a detailed molecular analysis of the NS5A region on a larger sample size may be necessary for understanding its role in the therapy outcome of HCV 1a/1b infection. (C) 2008 Elsevier B.V. All rights reserved.

New Insight into the Mechanism of Action of Wasp Mastoparan Peptides: Lytic Activity and Clustering Observed with Giant Vesicles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Oral manifestations of diabetes mellitus in complete denture wearers

Statement of problem. The oral mucosa has been reported to show a variety of changes in subjects with diabetes mellitus.Purpose. The purpose of this study was to compare diabetic and nondiabetic subjects wearing complete dentures with regard to salivary flow, salivary buffering capacity, denture retention, and oral mucosal lesions.Material and methods. Sixty subjects, 30 with and 30 without a diagnosis of diabetes, were matched for gender, race, and age. Salivary flow, salivary buffering capacity, glycemia, blood pressure, presence of mucosal lesions, denture retention, use of medications, and behavioral factors (controlled or uncontrolled diet, alcohol consumption, and smoking) reported by the subjects, were evaluated. For the salivary buffering capacity test, 1 mL of saliva was pipetted into a test tube containing 3 mL 0.005 N of hydrochloric acid, and the pH was measured with indicator strips. Group differences were statistically analyzed using the Student t test and the Mann-Whitney test for quantitative variables and the chi-square test for qualitative variables (alpha = .05).Results. Mean (SD) salivary flow was 1.14 (0.87) mL/min in the nondiabetic subjects and 0.95 (0.61) mL/min in the diabetic subjects. Evaluation of self-reported denture retention revealed no significant difference between groups. Denture retention was observed in 66.7% (20/30) of the control group and in 50% (15/30) of the diabetic group. The prevalence of mucosal lesions was 90% (27/30) in the control group and 83.3% (25/30) in the diabetic group. Salivary buffering capacity was 5.80 (0.85) in the control group and 5.26 (0.83) in the diabetic group (P = .017).Conclusions. Within the limitations of this study, no significant differences were observed in salivary flow, denture retention, or oral lesions in diabetic and nondiabetic subjects.

Vinegar as an antimicrobial agent for control of Candida spp. in complete denture wearers

The use of denture is known to increase the carriage of Candida in healthy patients, and the proliferation of Candida albicans strains can be associated with denture-induced stomatitis. The aim of this study was to evaluate the use of vinegar as an antimicrobial agent for control of Candida spp. in complete upper denture wearers. Fifty-five patients were submitted to a detailed clinical interview and oral clinical examination, and were instructed to keep their dentures immersed in a 10% vinegar solution ( pH less than 3) overnight for 45 days. Before and after the experimental period, saliva samples were collected for detection of Candida, counting of cfu/mL and identification of species by phenotypical tests ( germ tube formation, chlamidoconidia production, and carbohydrate fermentation and assimilation). The results were analyzed using Spearman's correlation and Student's t-test (p=0.05). Candida yeasts were present in 87.3% of saliva samples before the treatment. A significant reduction was verified in CFU/mL counts of Candida after treatment. A positive correlation between Candida and denture stomatitis was verified, since the decrease of cfu/mL counts was correlated with a reduction in cases of denture stomatitis. Although it was not able to eliminate C. albicans, the immersion of the complete denture in 10% vinegar solution, during the night, reduced the amounts (cfu/mL) of Candida spp. in the saliva and the presence of denture stomatitis in the studied patients.

Ultrasonic versus high-speed cavity preparation: Analysis of increases in pulpal temperature and time to complete preparation

Statement of problem. The use of ultrasonic tips has become an alternative for cavity preparation. However, there are concerns about this type of device, particularly with respect to intrapulpal temperatures and cavity preparation time.Purpose. The purpose of this study was to analyze pulpal temperature increases generated by an ultrasonic cavity preparation with chemical vapor deposition (CVD) tips, in comparison to preparation with a high-speed handpiece with a diamond rotary cutting instrument. The time required to complete the cavity preparation with each system was also evaluated.Material and methods. Thermocouples were positioned in the pulp chamber of 20 extracted human third molars. Slot-type cavities (3 x 3 x 2 mm) were prepared on the buccal and the lingual surfaces of each tooth. The test groups were: high-speed cavity preparation with diamond rotary cutting instruments (n = 20) and ultrasonic cavity preparation with CVD points (n = 20). During cavity preparation, the increases In pulpal temperature, and the time required for the preparation, were recorded and analyzed by Student's t test for paired samples (alpha = .05).Results. The average pulpal temperature increases were 4.3 degrees C for the high-speed preparation and 3.8 degrees C for the ultrasonic preparation, which were statistically similar (P = .052). However, significant differences were found (P < .001) for the time expended (3.3 minutes for the high-speed bur and 13.77 minutes for the ultrasound device).Conclusions. The intrapulpal temperatures produced during cavity preparation by ultrasonic tips versus high-speed bur preparation were similar. However, the use of the ultrasonic device required 4 times longer for the completion of a cavity preparation.

Quark clustering and chiral symmetry breaking in nuclear matter

Chiral symmetry breaking at finite baryon density is usually discussed in the context of quark matter, i.e. a system of deconfined quarks. Many systems like stable nuclei and neutron stars however have quarks confined within nucleons. In this paper we construct a Fermi sea of three-quark nucleon clusters and investigate the change of the quark condensate as a function of baryon density. We study the effect of quark clustering on the in-medium quark condensate and compare results with the traditional approach of modeling hadronic matter in terms of a Fermi sea of deconfined quarks.

Analytical calculation of the transition to complete phase synchronization in coupled oscillators

Here we present a system of coupled phase oscillators with nearest neighbors coupling, which we study for different boundary conditions. We concentrate at the transition to the total synchronization. We are able to develop exact solutions for the value of the coupling parameter when the system becomes completely synchronized, for the case of periodic boundary conditions as well as for a chain with fixed ends. We compare the results with those calculated numerically.

Transition to complete synchronization in phase-coupled oscillators with nearest neighbor coupling

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A note on the attenuation of the sommerfeld effect of a non-ideal system taking into account a MR damper and the complete model of a DC motor

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Complete Assignments of ¹H and 13C-NMR Spectra of the 3,4-seco-Triterpene Canaric Acid isolated from Rudgea jasminoides

O ácido canárico 1 foi isolado das folhas de Rudgea jasminoides. A substância isolada é um derivado triterpênico do tipo seco-lupano e teve sua estrutura elucidada com base nos dados espectrais, principalmente em experimentos de RMN a 1D e 2D. O sitosterol, o estigmasterol e os ácidos ursólico e oleanólico também foram isolados.