Effect of Byrsonima crassa and Phenolic Constituents on Helicobacter pylori-Induced Neutrophils Oxidative Burst

Byrsonima crassa Niedenzu (Malpighiaceae) is used in Brazilian folk medicine for the treatment of diseases related mainly to gastric ulcers. In a previous study, our group described the gastric protective effect of the methanolic extract from the leaves of B. crassa. The present study was carried out to investigate the effects of methanolic extract and its phenolic compounds on the respiratory burst of neutrophils stimulated by H. pylori using a luminol-based chemiluminescence assay as well as their anti-H. pylori activity. The suppressive activity on oxidative burst of H. pylori-stimulated neutrophils was in the order of methyl gallate > (+)-catechin > methanol extract > quercetin 3-O-alpha-L-arabinopyranoside > quercetin 3-O-beta-D-galactopyranoside > amentoflavone. Methyl gallate, compound that induced the highest suppressive activity with IC50 value of 3.4 mu g/mL, did not show anti-H. pylori activity. B. crassa could be considered as a potential source of natural antioxidant in gastric ulcers by attenuating the effects on the damage to gastric mucosa caused by neutrophil generated reactive oxygen species, even when H. pylori displays its evasion mechanisms.

A Validated Chromatographic Method for the Determination of Flavonoids in Copaifera langsdorffii by HPLC

Hydroethanolic extracts of C. langsdorffii leaves have therapeutic potential. This work reports a validated chromatographic method for the quantification of polar compounds in the hydroethanolic extract of C. langsdorffii leaves. A reliable HPLC method was developed using two monolithic columns linked in series (100 x 4.6 mm - C-18), with nonlinear gradient elution, and UV detection set at 257 nm. A procedure for the extraction of flavonols was also developed, which involved the use of 70% aqueous ethanol and the addition of benzophenone as the internal standard. The developed method led to a good detection response as the values for linearity were between 10.3 and 1000 mu g/mL, and those for recovery between 84.2 and 111.1%. The detection limit ranged from 0.02 to 1.70 mu g/mL and the quantitation limit from 0.07 to 5.1 mu g/mL, with a maximum RSD of 5.24%. Five compounds, rutin, quercetin-3-O-alpha-L-rhamnopyranoside, kaempferol-3-O-alpha-L-rhamnopyranoside, quercetin and kaempferol, were quantified. This method could, therefore, be used for the quality control of hydroethanolic extracts of Copaifera leaves and their cosmetic and pharmaceutical products.

In Vitro Schistosomicidal Activity of Some Brazilian Cerrado Species and Their Isolated Compounds

Miconia langsdorffii Cogn. (Melastomataceae), Roupala montana Aubl. (Proteaceae), Struthanthus syringifolius (Mart.) (Loranthaceae), and Schefflera vinosa (Cham. & Schltdl.) Frodin (Araliaceae) are plant species from the Brazilian Cerrado whose schistosomicidal potential has not yet been described. The crude extracts, fractions, the triterpenes betulin, oleanolic acid, ursolic acid and the flavonoids quercetin 3-O-beta-D-rhamnoside, quercetin 3-O-beta-D-glucoside, quercetin 3-O-beta-D-glucopyranosyl-(1-2)-alpha-L-rhamnopyranoside and isorhamnetin 3-O-beta-D-glucopyranosyl-(1-2)-alpha-L-rhamnopyranoside were evaluated in vitro against Schistosoma mansoni adult worms and the bioactive n-hexane fractions of the mentioned species were also analyzed by GC-MS. Betulin was able to cause worm death percentage values of 25% after 120 h (at 100 mu M), and 25% and 50% after 24 and 120 h (at 200 mu M), respectively; besides the flavonoid quercetin 3-O-beta-D-rhamnoside promoted 25% of death of the parasites at 100 mu M. Farther the flavonoids quercetin 3-O-beta-D-glucoside and quercetin 3-O-beta-D-rhamnoside at 100 mu M exhibited significantly reduction in motor activity, 75% and 87.5%, respectively. Biological results indicated that crude extracts of R. montana, S. vinosa, and M. langsdorffii and some n-hexane and EtOAc fractions of this species were able to induce worm death to some extent. The results suggest that lupane-type triterpenes and flavonoid monoglycosides should be considered for further antiparasites studies.

Quercetin decreases inflammatory response and increases insulin action in skeletal muscle of ob/ob mice and in L6 myotubes

Quercetin is a potent anti-inflammatory flavonoid, but its capacity to modulate insulin sensitivity in obese insulin resistant conditions is unknown. This study investigated the effect of quercetin treatment upon insulin sensitivity of ob/ob mice and its potential molecular mechanisms. Obese ob/ob mice were treated with quercetin for 10 weeks, and L6 myotubes were treated with either palmitate or tumor necrosis factor-alpha (TNF alpha) plus quercetin. Cells and muscles were processed for analysis of glucose transporter 4 (GLUT4), TNF alpha and inducible nitric oxide synthase (iNOS) expression, and c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor-kappa B (NF-kappa B) kinase (I kappa K) phosphorylation. Myotubes were assayed for glucose uptake and NF-kappa B translocation. Chromatin immunoprecipitation assessed NF-kappa B binding to GLUT4 promoter. Quercetin treatment improved whole body insulin sensitivity by increasing GLUT4 expression and decreasing JNK phosphorylation, and TNF alpha and iNOS expression in skeletal muscle. Quercetin suppressed palmitate-induced upregulation of TNF alpha and iNOS and restored normal levels of GLUT4 in myotubes. In parallel, quercetin suppressed TNF alpha-induced reduction of glucose uptake in myotubes. Nuclear accumulation of NF-kappa B in myotubes and binding of NF-kappa B to GLUT4 promoter in muscles of ob/ob mice were also reduced by quercetin. We demonstrated that quercetin decreased the inflammatory status in skeletal muscle of obese mice and in L6 myotubes. This effect was followed by increased muscle GLUT4, with parallel improvement of insulin sensitivity. These results point out quercetin as a putative strategy to manage inflammatory-related insulin resistance. (C) 2012 Elsevier B.V. All rights reserved.

Schistosomicidal evaluation of flavonoids from two species of Styrax against Schistosoma mansoni adult worms

Context: Schistosomiasis is a major health problem worldwide. Thus, the search for new schistosomicidal agents from natural sources can provide prototypes for drug discovery. Objective: The present study investigated the chemical composition of the EtOAc fractions of Styrax pohlii Pohl (Styracaceae) (EF-SP) aerial parts and S. camporum A. DC. leaves (EF-SC), as well as schistosomicidal activities against Schistosoma mansoni adult worms, which have not yet been studied. Materials and methods: The crude ethanol extracts of S. camporum leaves and S. pohlii aerial parts (EE-SC and EE-SP) were partitioned with n-hexane, EtOAc, and n-BuOH. The EtOAc fractions were purified by preparative HPLC. The crude extracts, EtOAc fractions and pure compounds were tested against S. mansoni adult worms in vitro. Results: The purification procedure resulted in the isolation of kaempferol-3-O-(2 '',4 ''-di-O-(E)-p-coumaroyl)-beta-D-glucopyranoside (1), kaempferol-3-O-(2 '',6 ''-di-O-(E)-p-coumaroyl)-beta-D-glucopyranoside (2), quercetin (3), and kaempferol (4). The bioassay results indicated that EE-SC, EF-SC, EF-SP, and compounds 2 and 4 are able to separate coupled S. mansoni adult worms. Additionally, EE-SC, EF-SP, and compound 4 killed the adult schistosomes in vitro at 100 mu g/mL and 100 mu M. Discussion and conclusion: This is the first time that the presence of compounds 1-2 in S. pohlii and 3-4 in S. camporum has been reported. Additionally, biological results indicated that S. pohlii and S. camporum have great potential as a source of active compounds.

Current practices in the management of patients with ureteral calculi in the emergency room of a university hospital

OBJECTIVE: Urinary lithiasis is a common disease. The aim of the present study is to assess the knowledge regarding the diagnosis, treatment and recommendations given to patients with ureteral colic by professionals of an academic hospital. MATERIALS AND METHODS: Sixty-five physicians were interviewed about previous experience with guidelines regarding ureteral colic and how they manage patients with ureteral colic in regards to diagnosis, treatment and the information provided to the patients. RESULTS: Thirty-six percent of the interviewed physicians were surgeons, and 64% were clinicians. Forty-one percent of the physicians reported experience with ureterolithiasis guidelines. Seventy-two percent indicated that they use noncontrast CT scans for the diagnosis of lithiasis. All of the respondents prescribe hydration, primarily for the improvement of stone elimination (39.3%). The average number of drugs used was 3.5. The combination of nonsteroidal anti-inflammatory drugs and opioids was reported by 54% of the physicians (i. e., 59% of surgeons and 25.6% of clinicians used this combination of drugs) (p = 0.014). Only 21.3% prescribe alpha blockers. CONCLUSION: Reported experience with guidelines had little impact on several habitual practices. For example, only 21.3% of the respondents indicated that they prescribed alpha blockers; however, alpha blockers may increase stone elimination by up to 54%. Furthermore, although a meta-analysis demonstrated that hydration had no effect on the transit time of the stone or on the pain, the majority of the physicians reported that they prescribed more than 500 ml of fluid. Dipyrone, hyoscine, nonsteroidal anti-inflammatory drugs, and opioids were identified as the most frequently prescribed drug combination. The information regarding the time for the passage of urinary stones was inconsistent. The development of continuing education programs regarding ureteral colic in the emergency room is necessary.

Naturally acquired antibodies to Plasmodium vivax blood-stage vaccine candidates (PvMSP-1(19) and PvMSP-3 alpha(359-798)) and their relationship with hematological features in malaria patients from the Brazilian Amazon

An important step when designing a vaccine is identifying the antigens that function as targets of naturally acquired antibodies. We investigated specific antibody responses against two Plasmodium vivax vaccine candidates, PvMSP-1(19) and PvMSP-3 alpha(359-798). Moreover, we assessed the relationship between these antibodies and morbidity parameters. PvMSP-1(19) was the most immunogenic antigen and the frequency of responders to this protein tended to increase in P. vivax patients with higher parasitemia. For both antigens, IgG antibody responses tended to be lower in patients who had experienced their first bout of malaria. Furthermore, anemic patients presented higher IgG antibody responses to PvMSP-3 alpha(359-798). Since the humoral response involves a number of antibodies acting simultaneously on different targets, we performed a Principal Component Analysis (PCA). Anemic patients had, on average, higher first principal component scores (IgG1/IgG2/IgG3/IgG4 anti-MSP3 alpha), which were negatively correlated with hemoglobin levels. Since antibodies against PfMSP-3 have been strongly associated with clinical protection, we cannot exclude the possibility of a dual role of PvMSP-3 specific antibodies in both immunity and pathogenesis of vivax malaria. Our results confirm the high immunogenicity of the conserved C terminus of PvMSP-1 and points to the considerable immunogenicity of polymorphic PvMSP-3 alpha(359-798) during natural infection. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Lipophilicity as a determinant of binding of procaine analogs to rat alpha(3)beta(4) nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (nAChRs) have been studied in detail with regard to their interaction with therapeutic and drug addiction-related compounds. Using a structureactivity approach, we have examined the relationship among the molecular features of a set of eight para-R-substituted N,N-[(dimethylamino)ethyl] benzoate hydrochlorides, structurally related to procaine and their affinity for the a3 beta 4 nAChR heterologously expressed in KXa3 beta 4R2 cells. Affinity values (log[1/IC50]) of these compounds for the a3 beta 4 nAChR were determined by their competition with [3H]TCP binding. Log(1/IC50) values were analyzed considering different hydrophobic and electronic parameters and those related to molar refractivity. These have been experimentally determined or were taken from published literature. In accordance with literature observations, the generated cross-validated quantitative structureactivity relationship (QSAR) equations indicated a significant contribution of hydrophobic term to binding affinity of procaine analogs to the receptor and predicted affinity values for several local anesthetics (LAs) sets taken from the literature. The predicted values by using the QSAR model correlated well with the published values both for neuronal and for electroplaque nAChRs. Our work also reveals the general structure features of LAs that are important for interaction with nAChRs as well as the structural modifications that could be made to enhance binding affinity. (c) 2012 Wiley Periodicals, Inc.

Preservation of Alpha-3 Neuronal Nicotinic Acetylcholine Receptor Expression in Sympathetic Ganglia After Brain Death

The goal of this study was to evaluate if the immunohistochemical expression of alpha-3 neuronal nicotinic acetylcholine receptor subunit in sympathetic ganglia remains stable after brain death, determining the possible use of sympathetic thoracic ganglia from subjects after brain death as study group. The third left sympathetic ganglion was resected from patients divided in two groups: BD-organ donors after brain death and CON-patients submitted to sympathectomy for hyperhidrosis (control group). Immunohistochemical staining for alpha-3 neuronal nicotinic acetylcholine receptor subunit was performed; strong and weak expression areas were quantified in both groups. The BD group showed strong alpha-3 neuronal nicotinic acetylcholine receptor expression in 6.55% of the total area, whereas the CON group showed strong expression in 5.91% (p = 0.78). Weak expression was found in 6.47% of brain-dead subjects and in 7.23% of control subjects (p = 0.31). Brain death did not affect the results of the immunohistochemical analysis of sympathetic ganglia, and its use as study group is feasible.

Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Early Amyloid-like Oligomers and Their Implication in alpha-Synuclein Aggregation

Lewy bodies and Lewy neurites, neuropathological hallmarks of several neurological diseases, are mainly made of filamentous assemblies of alpha-synuclein. However, other macromolecules including Tau, ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, and glycosaminoglycans are routinely found associated with these amyloid deposits. Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that can form fibrillar aggregates in the presence of acidic membranes, but its role in Parkinson disease is still unknown. In this work, the ability of heparin to trigger the amyloid aggregation of this protein at physiological conditions of pH and temperature is demonstrated by infrared and fluorescence spectroscopy, dynamic light scattering, small angle x-ray scattering, circular dichroism, and fluorescence microscopy. Aggregation proceeds through the formation of short rod-like oligomers, which elongates in one dimension. Heparan sulfate was also capable of inducing glyceraldehyde-3-phosphate dehydrogenase aggregation, but chondroitin sulfates A, B, and C together with dextran sulfate had a negligible effect. Aided with molecular docking simulations, a putative binding site on the protein is proposed providing a rational explanation for the structural specificity of heparin and heparan sulfate. Finally, it is demonstrated that in vitro the early oligomers present in the glyceraldehyde-3-phosphate dehydrogenase fibrillation pathway promote alpha-synuclein aggregation. Taking into account the toxicity of alpha-synuclein prefibrillar species, the heparin-induced glyceraldehyde-3-phosphate dehydrogenase early oligomers might come in useful as a novel therapeutic strategy in Parkinson disease and other synucleinopathies.

Looking over Toxin-K+ Channel Interactions. Clues from the Structural and Functional Characterization of alpha-KTx Toxin Tc32, a Kv1.3 Channel Blocker

alpha-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif; including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an alpha/beta scaffold typical of the members of the alpha-KTx family. TdK2 and TdK3, all belonging to the same alpha-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.

Cocaine effects on mouse incentive-learning and human addiction are linked to alpha 2 subunit-containing GABA(A) receptors

Because GABA(A) receptors containing alpha 2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with alpha 2 gene deletion showed reduced synaptic GABA(A) receptor-mediated responses. Behaviorally, the deletion abolished cocaine`s ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of alpha 2-GABA(A) receptors (alpha 2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In alpha 2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of alpha 2-GABA(A) receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.

Epidural anesthesia and postoperatory analgesia with alpha-2 adrenergic agonists and lidocaine for ovariohysterectomy in bitches

The aim of this study was to determine the viability and cardiorespiratory effects of the association of epidural alpha-2 adrenergic agonists and lidocaine for ovariohysterectomy (OH) in bitches. Forty-two bitches were spayed under epidural anesthesia with 2.5 mg/kg body weight (BW) of 1% lidocaine with adrenaline (CON) or in association with 0.25 mg/kg BW of xylazine (XYL), 10 mu g/kg BW of romifidine (ROM), 30 mu g/kg BW of detomidine (DET), 2 mu g/kg BW of dexmedetomidine (DEX), or 5 mu g/kg BW of clonidine (CLO). Heart rate (HR), respiratory rate (f(R)) and arterial pressures were monitored immediately before and every 10 min after the epidural procedure. Blood gas and pH analysis were done before, and at 30 and 60 min after the epidural procedure. Animals were submitted to isoflurane anesthesia if they presented a slightest sign of discomfort during the procedure. Time of sensory epidural block and postoperative analgesia were evaluated. All animals in CON and DEX, 5 animals in ROM and CLO, 4 animals in XYL, and 3 in DET required supplementary isoflurane. All groups, except CLO, showed a decrease in HR. There was an increase in arterial pressures in all groups. Postoperative analgesia lasted the longest in XYL. None of the protocols were totally efficient to perform the complete procedure of OH; however, xylazine provided longer postoperative analgesia than the others.

CC chemokine ligand 3 and receptors 1 and 5 gene expression in recurrent aphthous stomatitis

Objective. The aim of this study was to investigate the local and systemic expression of CC-chemokine ligand 3 (CCL3) and its receptors (CCR1 and CCR5) in tissue samples and peripheral blood mononuclear cells of recurrent aphthous stomatitis (RAS) patients. Study Design. This case-control study enrolled 29 patients presenting severe RAS manifestations and 20 non-RAS patients proportionally matched by sex and age. Total RNA was extracted from biopsy specimens and peripheral blood mononuclear cells for quatitative reverse-transcription polymerase chain reaction. The data obtained by relative quantification were evaluated by the 2(-Delta Delta Ct) method, normalized by the expression of an endogenous control, and analyzed by Student t test. Results. The results demonstrated overexpression in RAS tissue samples of all of the chemokines evaluated compared with healthy oral mucosa, whereas the blood samples showed only CCR1 overexpression in RAS patients. Conclusions. These findings suggest that the increased expression of CCL3, CCR1, and CCR5 may influence the immune response in RAS by T(H)1 cytokine polarization. (Oral Surg Oral Med Oral Pathol Oral Radiol 2012;114:93-98)

Angiotensin-(3-4) counteracts the Angiotensin II inhibitory action on renal Ca2+-ATPase through a cAMP/PKA pathway

We recently demonstrated that Angiotensin-(3-4) [Ang-(3-4)], an Ang II-derived dipeptide, overcomes inhibition of plasma membrane Ca2+-ATPase promoted by nanomolar concentrations of Ang II in basolateral membranes of renal proximal tubule cells, with involvement of a so far unknown AT(2)R-dependent and NO-independent mechanism. The present study investigates the signaling pathway triggered by Ang-(3-4) that is responsible for counteracting the inhibitory effect of Ang II, and attempts to elucidate the functional interaction of the dipeptide with Ang II at the level of AT(2)R. Stimulation by cholera toxin of G(s)alpha protein structurally linked to AT(2)R as revealed by their co-immunoprecipitation mimicked the effect of Ang-(3-4) on Ca2+-ATPase activity. Furthermore, addition of dibutyril-cAMP (db-cAMP) mimicked Ang-(3-4), whereas the specific PKA inhibitor, PKAi((5-24)) peptide, suppressed the counter-regulatory effect of Ang-(3-4) and the AT(2)R agonist, CGP42112A. Membrane-associated PKA activity was stimulated by Ang-(3-4) or CGP42112A to comparable levels as db-cAMP, and the Ang-(3-4) effect was abrogated by the AT(2)R antagonist PD123319, whereas the AT(1)R antagonist Losartan had no effect. Ang-(3-4) stimulated PKA-mediated phosphorylation of Ca2+-ATPase and activated PKA to comparable levels. Binding assays demonstrated that Ang-(3-4) could not displace H-3-Ang II from HEK 293T cells expressing AT(2)R, but 10(-10) mol/L Ang-(3-4) resulted in the appearance of a probable higher-affinity site (picomolar range) for Ang II. The results presented herein demonstrate that Ang-(3-4), acting as an allosteric enhancer, suppresses Ang II-mediated inhibition of Ca2+-ATPase through an AT(2)R/cAMP/PKA pathway, after inducing conformational changes in AT(2)R that results in generation of higher-affinity sites for Ang II. (C) 2012 Elsevier B.V. All rights reserved.