Inhibition of cannabinoid CB1 receptor upregulates Slc2a4 expression via nuclear factor-kappa B and sterol regulatory element-binding protein-1 in adipocytes

Evidences have suggested that the endocannabinoid system is overactive in obesity, resulting in enhanced endocannabinoid levels in both circulation and visceral adipose tissue. The blockade of cannabinoid receptor type 1 (CB1) has been proposed for the treatment of obesity. Besides loss of body weight, CB1 antagonism improves insulin sensitivity, in which the glucose transporter type 4 (GLUT4) plays a key role. The aim of this study was to investigate the modulation of GLUT4-encoded gene (Slc2a4 gene) expression by CB1 receptor. For this, 3T3-L1 adipocytes were incubated in the presence of a highly selective CB1 receptor agonist (1 mu M arachidonyl-2'-chloroethylamide) and/or a CB1 receptor antagonist/inverse agonist (0.1, 0.5, or 1 mu M AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide). After acute (2 and 4 h) and chronic (24 h) treatments, cells were harvested to evaluate: i) Slc2a4, Cnr1 (CB1 receptor-encoded gene), and Srebf1 type a (SREBP-1a type-encoded gene) mRNAs (real-time PCR); ii) GLUT4 protein (western blotting); and iii) binding activity of nuclear factor (NF)-kappa B and sterol regulatory element-binding protein (SREBP)-1 specifically in the promoter of Slc2a4 gene (electrophoretic mobility shift assay). Results revealed that both acute and chronic CB1 receptor antagonism greatly increased (similar to 2.5-fold) Slc2a4 mRNA and protein content. Additionally, CB1-induced upregulation of Slc2a4 was accompanied by decreased binding activity of NF-kappa B at 2 and 24 h, and by increased binding activity of the SREBP-1 at 24 h. In conclusion, these findings reveal that the blockade of CB1 receptor markedly increases Slc2a4/GLUT4 expression in adipocytes, a feature that involves NF-kappa B and SREBP-1 transcriptional regulation. Journal of Molecular Endocrinology (2012) 49, 97-106

ANTI-TRYPANOSOMAL ACTIVITY OF 1,2,3,4,6-PENTA-O-GALLOYL-beta-D-GLUCOSE ISOLATED FROM Plectranthus barbatus Andrews (Lamiaceae)

MeOH extract from the leaves of Plectranthus barbatus Andrews (Lamiaceae), showed in vitro anti-trypanosomal activity. The bioassay-guided fractionation resulted in the isolation of a gallic acid derivative, identified as 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG), after thorough NMR and MS spectral analysis. Finally, this compound was tested against trypomastigote forms of T. cruzi and displayed an EC50 value of 67 mu M, at least 6.6-fold more effective than the standard drug benznidazole. This is the first occurrence of PGG in the Plectranthus genus and the first anti-parasitic activity described for PGG in the literature.

Schistosoma mansoni: In vitro schistosomicidal activity and tegumental alterations induced by piplartine on schistosomula

Schistosomiasis is one of the most important parasitic infections in humans that occur in many tropical and subtropical countries. Currently, the control of schistosomiasis rests with a single drug, praziquantel, which is effective against adult worms but not the larval stages. Recent studies have shown that piplartine, an amide isolated from plants of the genus Piper (Piperaceae), reveals interesting antischistosomal properties against Schistosoma mansoni adult worms. Here, we report the in vitro antischistosomal activity of piplartine on S. mansoni schistosomula of different ages (3 h old and 1, 3, 5, and 7 days old), and examine alterations on the tegumental surface of worms by means of confocal laser scanning microscopy. Piplartine at a concentration of 7.5 mu M caused the death of all schistosomula within 120 h. The lethal effect occurred in a dose-dependent manner and was also dependent on the age of the parasite. Microscopy observation revealed extensive tegumental destruction, including blebbing, granularity, and a shorter body length. This report provides the first evidence that piplartine is able to kill schistosomula of different ages and reinforce that piplartine is a promising compound that could be used for the development of new schistosomicidal agent. (c) 2012 Elsevier Inc. All rights reserved.

Chronic Stress Improves the Myocardial Function without Altering L-type Ca+2 Channel Activity in Rats

Background: Chronic stress is associated with cardiac remodeling; however the mechanisms have yet to be clarified. Objective: The purpose of this study was test the hypothesis that chronic stress promotes cardiac dysfunction associated to L-type calcium Ca2+ channel activity depression. Methods: Thirty-day-old male Wistar rats (70 - 100 g) were distributed into two groups: control (C) and chronic stress (St). The stress was consistently maintained at immobilization during 15 weeks, 5 times per week, 1h per day. The cardiac function was evaluated by left ventricular performance through echocardiography and by ventricular isolated papillary muscle. The myocardial papillary muscle activity was assessed at baseline conditions and with inotropic maneuvers such as: post-rest contraction and increases in extracellular Ca2+ concentration, in presence or absence of specific blockers L-type calcium channels. Results: The stress was characterized for adrenal glands hypertrophy, increase of systemic corticosterone level and arterial hypertension. The chronic stress provided left ventricular hypertrophy. The left ventricular and baseline myocardial function did not change with chronic stress. However, it improved the response of the papillary muscle in relation to positive inotropic stimulation. This function improvement was not associated with the L-type Ca2+ channel. Conclusion: Chronic stress produced cardiac hypertrophy; however, in the study of papillary muscle, the positive inotropic maneuvers potentiated cardiac function in stressed rats, without involvement of L-type Ca2+ channel. Thus, the responsible mechanisms remain unclear with respect to Ca2+ influx alterations. (Arq Bras Cardiol 2012;99(4):907-914)

Matrix metalloproteinases, tissue inhibitors of metalloproteinases, and growth factors regulate the aggressiveness and proliferative activity of keratocystic odontogenic tumors

Objective. The objective of this preliminary study was to evaluate the expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and growth factors in keratocystic odontogenic tumors (KOTs). Study Design. The expression of MMPs, TIMPs, growth factors, and the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway were assessed by immunohistochemistry in 15 cases of KOT and 4 cases of calcifying cystic odontogenic tumor (CCOT). Results. KOT samples expressed significantly higher amounts of MMPs, TIMPs, growth factors, epidermal growth factor receptor (EGFR), and ERK compared with CCOT samples, with the exception of MMP-2 and TIMP-1. Conclusions. MMP-9, TIMP-2, EGF and transforming growth factor alpha act together and likely regulate the proliferation and aggressiveness of KOT. ERK-1/2 serves as the transducer of signals generated by these proteins, which signal through the common receptor, EGFR. This process may be related to the increased proliferation and aggressiveness observed in KOT. (Oral Surg Oral Med Oral Pathol Oral Radiol 2012;114:487-496)

1,3-Azoles from ortho-naphthoquinones: Synthesis of aryl substituted imidazoles and oxazoles and their potent activity against Mycobacterium tuberculosis

Twenty-three naphthoimidazoles and six naphthoxazoles were synthesised and evaluated against susceptible and rifampicin- and isoniazid-resistant strains of Mycobacterium tuberculosis. Among all the compounds evaluated, fourteen presented MIC values in the range of 0.78 to 6.25 mu g/mL against susceptible and resistant strains of M. tuberculosis, Five structures were solved by X-ray crystallographic analysis. These substances are promising antimycobacterial prototypes. (C) 2012 Elsevier Ltd. All rights reserved.

Inspiratory Muscle Training Reduces Sympathetic Nervous Activity and Improves Inspiratory Muscle Weakness and Quality of Life in Patients With Chronic Heart Failure A CLINICAL TRIAL

PURPOSE: To evaluate the effect of inspiratory muscle training (IMT) on cardiac autonomic modulation and on peripheral nerve sympathetic activity in patients with chronic heart failure (CHF). METHODS: Functional capacity, low-frequency (LF) and high-frequency (HF) components of heart rate variability, muscle sympathetic nerve activity inferred by microneurography, and quality of life were determined in 27 patients with CHF who had been sequentially allocated to 1 of 2 groups: (1) control group (with no intervention) and (2) IMT group. Inspiratory muscle training consisted of respiratory exercises, with inspiratory threshold loading of seven 30-minute sessions per week for a period of 12 weeks, with a monthly increase of 30% in maximal inspiratory pressure (PImax) at rest. Multivariate analysis was applied to detect differences between baseline and followup period. RESULTS: Inspiratory muscle training significantly increased PImax (59.2 +/- 4.9 vs 87.5 +/- 6.5 cmH(2)O, P = .001) and peak oxygen uptake (14.4 +/- 0.7 vs 18.9 +/- 0.8 mL.kg(-1).min(-1), P = .002); decreased the peak ventilation (V. E) +/- carbon dioxide production (V-CO2) ratio (35.8 +/- 0.8 vs 32.5 +/- 0.4, P = .001) and the (V) over dotE +/-(V) over dotCO(2) slope (37.3 +/- 1.1 vs 31.3 +/- 1.1, P = .004); increased the HF component (49.3 +/- 4.1 vs 58.4 +/- 4.2 normalized units, P = .004) and decreased the LF component (50.7 +/- 4.1 vs 41.6 +/- 4.2 normalized units, P = .001) of heart rate variability; decreased muscle sympathetic nerve activity (37.1 +/- 3 vs 29.5 +/- 2.3 bursts per minute, P = .001); and improved quality of life. No significant changes were observed in the control group. CONCLUSION: Home-based IMT represents an important strategy to improve cardiac and peripheral autonomic controls, functional capacity, and quality of life in patients with CHF.

Lateral septal area alpha(1)-and alpha(2)-adrenoceptors differently modulate baroreflex activity in unanaesthetized rats

The lateral septal area (LSA) is a limbic structure involved in autonomic, neuroendocrine and behavioural responses. An inhibitory influence of the LSA on baroreflex activity has been reported; however, the local neurotransmitter involved in this modulation is still unclear. In the present study, we verified the involvement of local LSA adrenoceptors in modulating cardiac baroreflex activity in unanaesthetized rats. Bilateral microinjection of the selective a1-adrenoceptor antagonist WB4101 (10 nmol in a volume of 100 nl) into the LSA decreased baroreflex bradycardia evoked by blood pressure increases, but had no effect on reflex tachycardia evoked by blood pressure decreases. Nevertheless, bilateral administration of the selective a2-adrenoceptor antagonist RX821002 (10 nmol in 100 nl) increased baroreflex tachycardia without affecting reflex bradycardia. Treatment of the LSA with a cocktail containing WB4101 and RX821002 decreased baroreflex bradycardia and increased reflex tachycardia. The non-selective beta-adrenoceptor antagonist propranolol (10 nmol in 100 nl) did not affect either reflex bradycardia or tachycardia. Microinjection of noradrenaline into the LSA increased reflex bradycardia and decreased the baroreflex tachycardic response, an opposite effect compared with those observed after double blockade of a1- and a2-adrenoceptors, and this effect of noradrenaline was blocked by local LSA pretreatment with the cocktail containing WB4101 and RX821002. The present results provide advances in our understanding of the baroreflex neural circuitry. Taken together, data suggest that local LSA a1- and a2-adrenoceptors modulate baroreflex control of heart rate differently. Data indicate that LSA a1-adrenoceptors exert a facilitatory modulation on baroreflex bradycardia, whereas local a2-adrenoceptors exert an inhibitory modulation on reflex tachycardia.

Lasiodiplodan, an exocellular (1 -> 6)-beta-d-glucan from Lasiodiplodia theobromae MMPI: production on glucose, fermentation kinetics, rheology and anti-proliferative activity

Lasiodiplodan, an exopolysaccharide of the (1 -> 6)-beta-d-glucan type, is produced by Lasiodiplodia theobromae MMPI when grown under submerged culture on glucose. The objective of this study was to evaluate lasiodiplodan production by examining the effects of carbon (glucose, fructose, maltose, sucrose) and nitrogen sources (KNO3, (NH4)(2)SO4, urea, yeast extract, peptone), its production in shake flasks compared to a stirred-tank bioreactor, and to study the rheology of lasiodiplodan, and lasiodiplodan's anti-proliferative effect on breast cancer MCF-7 cells. Although glucose (2.05 +/- A 0.05 g L-1), maltose (2.08 +/- A 0.04 g L-1) and yeast extract (2.46 +/- A 0.06 g L-1) produced the highest amounts of lasiodiplodan, urea as N source resulted in more lasiodiplodan per unit biomass than yeast extract (0.74 +/- A 0.006 vs. 0.22 +/- A 0.008 g g(-1)). A comparison of the fermentative parameters of L. theobromae MMPI in shake flasks and a stirred-tank bioreactor at 120 h on glucose as carbon source showed maximum lasiodiplodan production in agitated flasks (7.01 +/- A 0.07 g L-1) with a specific yield of 0.25 +/- A 0.57 g g(-1) and a volumetric productivity of 0.06 +/- A 0.001 g L-1 h(-1). A factorial 2(2) statistical design developed to evaluate the effect of glucose concentration (20-60 g L-1) and impeller speed (100-200 rpm) on lasiodiplodan production in the bioreactor showed the highest production (6.32 g L-1) at 72 h. Lasiodiplodan presented pseudoplastic behaviour, and the apparent viscosity increased at 60A degrees C in the presence of CaCl2. Anti-proliferative activity of lasiodiplodan was demonstrated in MCF-7 cells, which was time- and dose-dependent with an IC50 of 100 mu g lasiodiplodan mL(-1).

The meristematic activity of the endodermis and the pericycle and its role in the primary thickening of stems in monocotyledonous plants

Background: It had long been thought that a lateral meristem, the so-called primary thickening meristem (PTM) was responsible for stem thickening in monocotyledons. Recent work has shown that primary thickening in the stems of monocotyledons is due to the meristematic activity of both the endodermis and the pericycle. Aims: The aim of this work is to answer a set of questions about the developmental anatomy of monocotyledonous plants: (1) Do the stem apices of monocots have a special meristematic tissue, the PTM? (2) Are the primary tissues of the stem the same as those of the root? (3) Is there good evidence for the formation of both the cortex and the vascular tissue from a single meristem, the PTM, in the shoot and from two distinguishable meristems in the root? (4) If the PTM forms only the cortex, what kind of meristem forms the vascular tissue? Methods: Light microscopy was used to examine stem and root anatomy in 16 species from 10 monocotyledonous families. Results: It was observed that radially aligned cortical cells extend outwards from endodermal initial cells in the cortex of the roots and the stems in all the species. The radial gradation in size observed indicates that the cortical cells are derivatives of a meristematic endodermis. In addition, perfect continuity was observed between the endodermis of the root and that of the stem. Meristematic activity in the pericycle gives rise to cauline vascular bundles composed of metaxylem and metaphloem. Conclusion: No evidence was obtained for the existence in monocotyledons of a PTM. Monocotyledons appear to resemble other vascular plants in this respect.

Baroreceptor-mediated activation of sympathetic nerve activity to salivary glands

Salivary gland function is regulated by both the sympathetic and parasympathetic nervous systems. Previously we showed that the basal sympathetic outflow to the salivary glands (SNA(SG)) was higher in hypertensive compared to normotensive rats and that diabetes reduced SNA(SG) discharge at both strains. In the present study we sought to investigate how SNA(SG) might be modulated by acute changes in the arterial pressure and whether baroreceptors play a functional role upon this modulation. To this end, we measured blood pressure and SNA(SG) discharge in Wistar-Kyoto rats (WRY-intact) and in WRY submitted to sinoaortic denervation (WRY-SAD). We made the following three major observations: (i) in WRY-intact rats, baroreceptor loading in response to intravenous infusion of the phenylephrine evoked an increase in SNA(SG) spike frequency (81%, p<0.01) accompanying the increase mean arterial pressure ((sic)MAP: +77 +/- 14 mmHg); (ii) baroreceptor unloading with sodium nitroprusside infusion elicited a decrease in SNA(SG) spike frequency (17%, p<0.01) in parallel with the fall in arterial blood pressure ((sic)MAP: 30 3 mmHg) in WRY-intact rats; iii) in the WRY-SAD rats, phenylephrine-evoked rises in the arterial pressure ((sic)MAP: +56 +/- 6 mmHg) failed to produce significant changes in the SNA(SG) spike frequency. Taken together, these data show that SNA(SG) increases in parallel with pharmacological-induced pressor response in a baroreceptor dependent way in anaesthetised rats. Considering the key role of SNA(SG) in salivary secretion, this mechanism, which differs from the classic cardiac baroreflex feedback loop, strongly suggests that baroreceptor signalling plays a decisive role in the regulation of salivary gland function. (C) 2012 Elsevier Inc. All rights reserved.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1β maturation

Abstract Background Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1β and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-). Results Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE2 and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1β and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)α and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1β and PGE2 did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-α and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1β was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE2 production in the paw of WT mice, but was reduced in casp1-/- mice. Conclusion These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1β maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE2 production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

Brain activity and medical diagnosis: an EEG study

Abstract Background Despite new brain imaging techniques that have improved the study of the underlying processes of human decision-making, to the best of our knowledge, there have been very few studies that have attempted to investigate brain activity during medical diagnostic processing. We investigated brain electroencephalography (EEG) activity associated with diagnostic decision-making in the realm of veterinary medicine using X-rays as a fundamental auxiliary test. EEG signals were analysed using Principal Components (PCA) and Logistic Regression Analysis Results The principal component analysis revealed three patterns that accounted for 85% of the total variance in the EEG activity recorded while veterinary doctors read a clinical history, examined an X-ray image pertinent to a medical case, and selected among alternative diagnostic hypotheses. Two of these patterns are proposed to be associated with visual processing and the executive control of the task. The other two patterns are proposed to be related to the reasoning process that occurs during diagnostic decision-making. Conclusions PCA analysis was successful in disclosing the different patterns of brain activity associated with hypothesis triggering and handling (pattern P1); identification uncertainty and prevalence assessment (pattern P3), and hypothesis plausibility calculation (pattern P2); Logistic regression analysis was successful in disclosing the brain activity associated with clinical reasoning success, and together with regression analysis showed that clinical practice reorganizes the neural circuits supporting clinical reasoning.

Discovery and Pharmacological Characterization of a Novel Small Molecule Antagonist of Integrin α4β1: Focus on Antiallergic Activity

Allergy is a common hypersensitivity disorder that affects 15% to 20% of the population and its prevalence is increasing worldwide. Its severity correlates with the degree of eosinophil infiltration into the conjunctiva, which is mediated by chemokines that stimulate the production of adhesion molecules like intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cell surface. The α4β1 and α4β7 integrins are expressed in eosinophils and contribute to their activation and infiltration in AC through the binding to VCAM-1 or fibronectin, expressed on vascular endothelial cells. Blockade of α4 integrins might be a therapeutical achievement in allergic eye diseases. DS 70, that show an IC50 in the nanomolar range against α4β1 integrin in Jurkat cells and in the eosinophilic cell line EOL-1. This compound was able to prevent cell adhesion to VCAM-1 and FN in vitro. In a scintillation proximity assay DS70 displaced 125I-FN binding to human α4β1 integrin and, in flow cytometry analysis, it antagonized the binding of a primary antibody to α4β1 integrin expressed on the Jurkat cells surface as well. Furthermore, we analysed also its effects on integrin α4β1 signalling. In an vivo model of allergic conjunctivitis, topical DS70 reduced the clinical aspects of EPR (early phase reaction) and LPR (late phase reaction), by reducing clinical score, eosinophil accumulation, mRNA levels of cytochines and chemochines pro-inflammatory and the conjunctival expression of α4 integrin. In conclusion, DS70 seems a novel antiallergic ocular agent that has significant effects on both early and late phases of ocular allergy.