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.

The ACB technique: a biomagentic tool for monitoring gastrointestinal contraction directly from smooth muscle in dogs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Muscle-specific growth hormone receptor (GHR) overexpression induces hyperplasia but not hypertrophy in transgenic zebrafish

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

PHYSIOLOGICAL and NEUROMUSCULAR PROFILE DURING A BODYPUMP SESSION: ACUTE RESPONSES DURING A HIGH-RESISTANCE TRAINING SESSION

Oliveira, AS, Greco, CC, Pereira, MP, Figueira, TR, de Araujo Ruas, VD, Goncalves, M, and Denadai, BS. Physiological and neuromuscular profile during a Bodypump session: acute responses during a high-resistance training session. J Strength Cond Res 23(2): 579-586, 2009-The main purposes of this study were 1) to describe and to compare blood lactate ([La]), heart rate (HR), and electromyographic (EMG) parameters during high-repetition training sessions (HRTSs), 2) to analyze the influence of physical fitness levels in these parameters, and, 3) to analyze the relationship between metabolic ([La]) and neuromuscular (EMG) responses during the HRTS. Fifteen healthy untrained women (21.7 +/- 2.1 years) performed an HRTS called Bodypump for 1 hour, which incorporated the use of variable free weights and high repetitions in a group setting. This session involved 10 music selections (M1-M10) containing resistive exercises for different muscle groups. After music selections 2 (M2), 4 (M4), 6 (M6), 7 (M7), and 9 (M9), [La], HR, and EMG (vastus medialis [VM], vastus lateralis [VL], iliocostalis lumborum [IC], and longissimus thoracis <) were determined. The [La] (M2, 4.00 +/- 1.45 mM; M7, 5.02 +/- 1.73 mM) and HR (M2, 153.64 +/- 18.89 bpm; M7, 16.14 +/- 20.14 bpm) obtained at M2 and M7 were similar but were significantly higher than the other moments of the session. However, EMG (root mean square [RMS]) at M2 (VL, VM, and LT) was lower than at M7. There was no significant correlation of strength and aerobic physical fitness with [La], RMS. In the same way, there was no significant correlation of [La] with RMS at M2 and M7. on the basis of our data, we can conclude that metabolic, cardiovascular, and EMG variables present different and independent behavior during an HRTS. Accordingly, for neuromuscular conditions during HRTS, it seems to be enough to induce improvement in the muscular strength of inferior limbs in untrained subjects.

Psychological, biochemical and physiological responses of Brazilian soccer players during a training program

Aims of the study. - The aim of this study is to investigate the behavior of the physiological, biochemical and psychological parameters in Brazilian soccer players during a training program.Materials. - Fifteen athletes were evaluated at the beginning (T1), in the middle (T2) and at the end (T3) of the training program. on the first day, at 7:30 am, before the blood collecting at rest for the determination of serum creatine kinase (CK), serum creatinine and serum urea, the athletes had their psychological parameters assessed by the profile of mood state questionnaire (POMS). After 90 min, they performed a 250-m sprint. on the second day at 8:30 am, the athletes had their alactic anaerobic performance measured and, after 40 min, they completed the aerobic test. Friedman test was used to verify the behavior of overtraining markers.Results. - There was a decrease in vigor score in T3 (p=0.01) compared with T1 and T2. In T3 (p=0.01), the athletes also showed an increase in serum creatinine levels compared to T1. Furthermore, in the same period, we verified a diminishing in the team performance.Conclusion. - The training program developed between T2 and T3 led to the fall of the vigor score, the increase in serum creatinine concentrations and the diminishing in team performance. (c) 2007 Elsevier Masson SAS. All rights reserved.

The Association between Skipping Breakfast and Biochemical Variables in Sedentary Obese Children and Adolescents

Objective To investigate the relationship between skipping meals and biochemical variables in obese children and adolescents.Study design The sample was composed of 174 obese children and adolescents, aged between 6 and 16 years (80 male and 94 female). Body composition was assessed by dual-energy x-ray absorptiometry, fasting blood glucose, and lipid profile were measured after 12 hours fasting. The frequency of skipping breakfast, lunch, or dinner was assessed through a face-to-face interview carried out with the parents.Results The prevalence of eating breakfast daily was low in boys (47.5%) and girls (44.7%). A higher frequency of eating breakfast was negatively correlated with glucose (r = -0.16; P = .026), triglycerides (r = -0.19; P = .011), and very low density lipoprotein cholesterol (r = -0.21; P = .005). In the multivariate model, the weekly frequency of eating breakfast remained negatively associated with glucose (beta = -0.975; P = .017), triglycerides (beta = -7.792; P = .017), and very low density lipoprotein cholesterol (beta = -1.870; P = .009) independent of age, sex, trunk fatness, and parents' education.Conclusion Skipping meals, mainly breakfast, is associated with glucose and lipid levels in obese children and adolescents. (J Pediatr 2012;161:871-4).

Impact of early fructose intake on metabolic profile and aerobic capacity of rats

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

Effects of 60Co gamma radiation on crotamine

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3%, but did not eliminate it.

Chemical profile of Eriocaulon ligulatum (Vell.) LB Smith (Eriocaulaceae)

Two new and six known flavonoids and two known naphthopyranones have been isolated by chromatographic methods and spectrometrically identified in capitulae and scapes of Eriocaulon ligulatum (Vell.) L.B. Smith (Eriocaulaceac). The presence of naphthopyranones suggests approximation between Eriocaulon and Paepalanthus genus. (c) 2005 Elsevier Ltd. All rights reserved.

A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: Biochemical characterization, crystallization, myotoxic and edematogenic activity

A, M. Soares, V, M, Rodrigues, M. I. Homsi-Brandeburgo, M. H. Toyama, F, R, Lombardi, K. Arni and J. R, Giglio. A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: Biochemical characterization, crystallization, myotoxic and edematogenic activity. Toxicon 36, 503-514, 1998.-Bothrops moojeni snake venom was fractionated on a CM-Sepharose column which was previously equilibrated with 0.05 M ammonium bicarbonate buffer at pH 8.0 and subsequently eluted with an ammonium bicarbonate concentration gradient from 0.05 to 0.5 M at constant pH (8.0) and temperature (25 degrees C). The fraction which eluted last (M-VI) showed, after direct lyophilization, a single band by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE, indicating an approximate M,. of 14 000 and 77 000, in the presence and absence of dithiothreitol, respectively. Its amino acid composition revealed a high level of hydrophobic and basic amino acids as well as 13 half-cystine residues. Its isoelectric point and extinction coefficient (E-1.0cm(1.0mg/ml) at 278 nm and pH 7.0) were 8.2 and 1.170, respectively. M-VI was devoid of phospholipase A(2) (PLA(2)) activity on egg yolk, as well as of hemorrhagic, anticoagulant and coagulant activities, but could induce drastic necrosis on skeletal muscle fibres as well as rapid and transient edema on the rat paw. Its N-terminal sequence: SLFELGKMILQETGKNPAKSYGVYGCNCGVGGRGKPKDATDRCCYVHKCCYK.... revealed high homology with other Lys 49 PLA(2)-like myotoxins from other bothropic venoms. Orthorhombic crystals of M-VI? which diffracted to a maximal resolution of 1.6 Angstrom. were obtained and indicated the presence of a dimer in the asymmetrical unit. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Renal Evaluation of Aotus azarai infulatus by Ultrasonography and Serum Chemistry Profile

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol. Initially (experiment 1), we analyzed two antigen lots of two human isolates (Bt1 and Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh and Morton medium) in SDS-PAGE and in two immunological tests (immunodiffusion-ID and footpad swelling test-FPT). Afterwards (experiment 2), we compared the antigenic profile of three antigen hatches from three human isolates (Bt1, Bt2 and Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P. brasiliensis antigens. In experiment 1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media. The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT. In experiment 2, the reference system for P brasiliensis showed 26 antigen peaks. There were important differences between batches prepared from the same isolate and between batches from different isolates. Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P. brasiliensis isolate, under controlled incubation conditions.

Effects of olive oil and its minor constituents on serum lipids, oxidative stress, and energy metabolism in cardiac muscle

Recent lines of evidence suggest that the beneficial effects of olive oil are not only related to its high content of oleic acid, but also to the antioxidant potential of its polyphenols. The aim of this work was determine the effects of olive oil and its components, oleic acid and the polyphenol dihydroxyphenylethanol (DPE), on serum lipids, oxidative stress, and energy metabolism on cardiac tissue. Twenty four male Wistar rats, 200 g, were divided into the following 4 groups (n = 6): control (C), OO group that received extra-virgin olive oil (7.5 mL/kg), OA group was treated with oleic acid (3.45 mL/kg), and the DPE group that received the polyphenol DPE (7.5 mg/kg). These components were administered by gavage over 30 days, twice a week. All animals were provided with food and water ad libitum The results show that olive oil was more effective than its isolated components in improving lipid profile, elevating high-density lipoprotein, and diminishing low-density lipoprotein cholesterol concentrations. Olive oil induced decreased antioxidant Mn-superoxide dismutase activity and diminished protein carbonyl concentration, indicating that olive oil may exert direct antioxidant effect on myocardium. DPE, considered as potential antioxidant, induced elevated aerobic metabolism, triacylglycerols, and lipid hydroperoxides concentrations in cardiac muscle, indicating that long-term intake of this polyphenol may induce its undesirable pro-oxidant activity on myocardium. © 2006 NRC Canada.

Morphometry and histochemistry of the Rectus abdominis muscle fibers of tufted capuchin monkeys (Cebus apella Linnaeus, 1758)

The phylogenetic proximity of primates to humans, along with their behavioral, biochemical, and anatomical similarities, make such animals more interesting experimental models for biomedical researches, as compared to classical laboratory animals. Another aspect that has called the attention of researchers is the differentiated quadrupedalism present in some primates. The tufted capuchin monkey uses the ground and tree branches as its support for locomotion, showing various postures while performing this task. On the basis of this information, we have decided to study the rectus abdominis muscle of the tufted capuchin monkey, with the following goals: the frequency and area of fiber types; its possible compartmentalization; and identify if this muscle is better adapted to phasic or postural activities. To do this, samples were removed from 4 regions of the rectus abdominis muscle of 6 adult male tufted capuchin monkeys, and were submitted to reaction with m-ATPase, (with alkaline and acid pre-incubation), NADH, and H.E.. Results showed: a statistically significant difference (P<0.05) for both frequency and area, between fiber types FG and FOG and FG and SO, but did not show a statistically significant difference between fibers FOG and SO, in all studied regions; similarity in frequency and area of a same fiber type (FG, FOG, and SO) among the studied regions. Based on these data, it was concluded that: the rectus abdominis muscle of the tufted capuchin monkey does not show fiber compartmentalization, since the distribution and size patterns of the different fiber types are similar in the studied regions; there is a predominance of fast twitch fibers (FG + FOG) over slow twitch fibers (SO), for frequency and area, which characterizes the muscle as being more dedicated to phasic than to postural activities. © 2006 Sociedad Chilena de Anatom.

Clinical and nutritional profile of individuals with Chagas disease

Chagas disease (CD), caused by the protozoan Trypanossoma cruzi, affects approximately 18 million individuals in the Americas, 5 million of which five in Brazil. Most chronic sufferers have either the indeterminate form of the disease, without organic compromise, or the cardiac or digestive forms. Despite the importance of this disease, there is no information on the effect of nutrition on CD evolution. We evaluated the clinical-nutritional profile of individuals with CD treated at the Tropical Diseases Nutrition Out-Patient Clinic of the Botucatu School of Medicine, UNESP.A retrospective cohort study was performed between 2002 and 2006, on 66 patients with serum and parasitological diagnosis of CD. Epidemiological, clinical, nutritional, and biochemical data were collected, including gender, age, skin color, smoking, alcoholism, physical activity, weight, stature, body mass index, abdominal circumference, glycemia, and lipid profile. Fifty-three percent were mate and 47% female; 96% were white skinned. Mean age was 49.6±6.36 years. The predominant form was indeterminate in 71 %; smoking and drinking were recorded in 23% and 17%, respectively. Sedentariness predominated in 83%, and 55% presented increased abdominal circumference. Most, 94%, were overweight or obese. The biochemical exam revealed hyperglycemia in 12% and dyslipidemia in 74%. These findings suggest that the Chagas population presents co-morbidities and risk factors for developing chronic non-transmissible diseases, including cardiovascular diseases, making CD evolution even worse. © 2007 by The Brazilian Journal of Infectious Diseases and Contexto Publishing. All rights reserved.