Modulation of the pharmacological effects of enzymatically-active PLA 2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

Background. An interaction between lectins from marine algae and PLA 2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA 2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA 2 and its complex. Results. This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24-26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA 2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion. The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules. © 2008 Oliveira et al; licensee BioMed Central Ltd.

Effects of pentoxifylline and n-acetylcysteine on injuries caused by ischemia and reperfusion of splanchnic organs in rats

Aim. Occlusion and reperfusion of splanchnic arteries cause local and systemic changes due to the release of cytotoxic substances and the interaction between neutrophils and endothelial cells. This study evaluated the role of pentoxifylline (PTX) and n-acetylcysteine (NAC) in the reduction of ischemia, reperfusion shock and associated intestinal injury. Methods. Sixty rats were divided into 6 groups of 10 animals. Rats in three groups underwent mesenteric ischemia for 30 minutes followed by 120 minutes of reperfusion, and were treated with saline (SAL-5 mL/kg/ h), pentoxifylline (PTX-50 mg/kg) or n-acetylcysteine (NAC-430 mg/kg/h). The other 3 groups underwent sham ischemia and reperfusion (I/R) and received the same treatments. Hemodynamic, biochemical and histological parameters were evaluated. Results. No significant hemodynamic or intestinal histological changes were seen in any sham group. No histological changes were found in the lung or liver of animals in the different groups. There was a progressive decrease in mean arterial blood pressure, from mean of 111.53 mmHg (30 minutes of ischemia) to 44.30±19.91 mmHg in SAL-I/R. 34.52±17.22 mmHg in PTX-I/R and 33.81±8.39 mmHg in NAC-I/R (P<0.05). In all I/R groups, there was a progressive decrease in: aortic blood flow, from median baseline of 19.00 mL/min to 2.50±5.25 mL/min in SAL-I/ R; 2.95±6.40 mL/min in PTX-I/R and 3.35±3.40 mL/min in NAC-I/R (P<0.05); in the heart rate, from mean baseline of 311.74 bpm to 233.33±83.88 bpm in SAL-I/R, 243.20±73.25 bpm in PTX-I/R and 244.92±76.05 bpm in NAC-I/R (P<0.05); and esophageal temperature, from mean baseline of 33.68°C to 30.53±2.05°C in SAL-I/R, 30.69±2.21°C in PTX-I/R and 31.43±1.03°C in NAC-I/R (P<0.05). In the other hand, there was an attenuation of mucosal damage in the small intestine of the animals receiving PTX, and only in the ileum of the animals receiving NAC. No changes were found in ileum or plasma malondialdehyde levels in any group. Conclusion. PTX was more efficient in reducing histological lesions than NAC, but neither treatment prevented hemodynamic changes during splanchnic organs I/R.

Entropy variation in isothermal fluid flow considering real gas effects

The present paper concerns on the estimative of the pressure loss and entropy variation in an isothermal fluid flow, considering real gas effects. The 1D formulation is based on the isothermal compressibility module and on the thermal expansion coefficient in order to be applicable for both gas and liquid as pure substances. It is emphasized on the simple methodology description, which establishes a relationship between the formulation adopted for ideal gas and another considering real gas effects. A computational procedure has been developed, which can be used to determine the flow properties in duct with a variable area, where real gas behavior is significant. In order to obtain quantitative results, three virial coefficients for Helium equation of state are employed to determine the percentage difference in pressure and entropy obtained from different formulations. Results are presented graphically in the form of real gas correction factors, which can be applied to perfect gas calculations.

Effects of acute normovolemic anemia on hemodynamic parameters and acid-base balance in dogs

The aim of this study was to evaluate the hemodynamic and acid-base status of dogs subjected to acute normovolemic anemia. The dogs (n = 10) were evaluated 15 minutes and 24 hours after induction of anemia (hematocrit below 18) with blood withdrawal and simultaneously replacement of same volume of Ringer's lactate solution and hydroxyethyl starch-based solution in a 2:1 ratio. The cardiac output was measured by Doppler echocardiography and blood pressure by oscillometric device, and posteriorly hemodynamic parameters were calculated. The anemic groups had increase in cardiac index (P <.05) (3.82 ± 1.05 to 5.86 ± 1.49 and 5.81 ± 1.63 L/min m) and decreases (P <.05) in the indices of total peripheral resistance (6797.81 ± 3060.22 to 3220.14 ± 1275.02 and 3887.74 ± 1394.89 dinaseg/cm 5× m2) and oxygen delivery (7942.84 ± 3344.00 to 4021.68 ± 1627.00 and 4430.82 ± 1402.61 mL/min× m 2), respectively. There were no significant changes in pH, but PaO2 and SaO2 values were increased, and PaCO2 reduced in anemic dogs (P <.05). Therefore, acute normovolemic anemia can create significant hemodynamic changes and despite some hemogasometric changes, there were no changes in the acid-base status in dogs. Copyright © 2011 Tatiana Champion et al.

Control of breathing and blood pressure by parafacial neurons in conscious rats

New Findings: • What is the central question of this study? The main purpose of the present manuscript was to investigate the cardiorespiratory responses to hypoxia or hypercapnia in conscious rats submitted to neuronal blockade of the parafacial region. We clearly showed that the integrity of parafacial region is important for the respiratory responses elicited by peripheral and central chemoreflex activation in freely behavior rats. • What is the main finding and its importance? Since the parafacial region is part of the respiratory rhythm generator, they are essential for postnatal survival, which is probably due to their contribution to chemoreception in conscious rats. The retrotrapezoid nucleus (RTN), located in the parafacial region, contains glutamatergic neurons that express the transcriptor factor Phox2b and that are suggested to be central respiratory chemoreceptors. Studies in anaesthetized animals or in vitro have suggested that RTN neurons are important in the control of breathing by influencing respiratory rate, inspiratory amplitude and active expiration. However, the contribution of these neurons to cardiorespiratory control in conscious rats is not clear. Male Holtzman rats (280-300 g, n= 6-8) with bilateral stainless-steel cannulae implanted into the RTN were used. In conscious rats, the microinjection of the ionotropic glutamatergic agonist NMDA (5 pmol in 50 nl) into the RTN increased respiratory frequency (by 42%), tidal volume (by 21%), ventilation (by 68%), peak expiratory flow (by 24%) and mean arterial pressure (MAP, increased by 16 ± 4, versus saline, 3 ± 2 mmHg). Bilateral inhibition of the RTN neurons with the GABAA agonist muscimol (100 pmol in 50 nl) reduced resting ventilation (52 ± 34, versus saline, 250 ± 56 ml min-1 kg-1 with absolute values) and attenuated the respiratory response to hypercapnia and hypoxia. Muscimol injected into the RTN slightly reduced resting MAP (decreased by 13 ± 7, versus saline, increased by 3 ± 2 mmHg), without changing the effects of hypercapnia or hypoxia on MAP and heart rate. The results suggest that RTN neurons activate facilitatory mechanisms important to the control of ventilation in resting, hypoxic or hypercapnic conditions in conscious rats. © 2012 The Authors. Experimental Physiology © 2012 The Physiological Society.

Infliximab prevents increased systolic blood pressure and upregulates the AKT/eNOS pathway in the aorta of spontaneously hypertensive rats

High systolic blood pressure caused by endothelial dysfunction is a comorbidity of metabolic syndrome that is mediated by local inflammatory signals. Insulin-induced vasorelaxation due to endothelial nitric oxide synthase (eNOS) activation is highly dependent on the activation of the upstream insulin-stimulated serine/threonine kinase (AKT) and is severely impaired in obese, hypertensive rodents and humans. Neutralisation of circulating tumor necrosis factor-α (TNFα) with infliximab improves glucose homeostasis, but the consequences of this pharmacological strategy on systolic blood pressure and eNOS activation are unknown. To address this issue, we assessed the temporal changes in the systolic pressure of spontaneously hypertensive rats (SHR) treated with infliximab. We also assessed the activation of critical proteins that mediate insulin activity and TNFα-mediated insulin resistance in the aorta and cardiac left ventricle. Our data demonstrate that infliximab prevents the upregulation of both systolic pressure and left ventricle hypertrophy in SHR. These effects paralleled an increase in AKT/eNOS phosphorylation and a reduction in the phosphorylation of inhibitor of nuclear factor-κB (Iκβ) and c-Jun N-terminal kinase (JNK) in the aorta. Overall, our study revealed the cardiovascular benefits of infliximab in SHR. In addition, the present findings further suggested that the reduction of systolic pressure and left ventricle hypertrophy by infliximab are secondary effects to the reduction of endothelial inflammation and the recovery of AKT/eNOS pathway activation. © 2012 Elsevier B.V.

Effects of Jugular Vein Occlusion on Cardiovascular Parameters in Horses During Exercise on a Treadmill

The purpose of the present investigation was to examine the effects of unilateral and bilateral jugular vein occlusion by temporary surgical ligature on the heart rate and arterial and venous blood pressure in sedentary horses during progressive treadmill exercise. Six horses performed three exercise tests (ET). ET1, considered the control, was performed in horses without jugular occlusions. ET2 and ET3 were performed with unilateral and bilateral occlusion by temporary surgical ligature of the jugular veins, respectively. Heart rate, arterial pressure, and pressure of the occluded jugular vein were evaluated. Clinically, the horses presented apathy, head edema, congested mucous membranes, increased capillary refill time, and dysphagia. These signs were observed with the unilateral jugular vein occlusion and became more evident with the bilateral occlusion. Comparing ETs, no differences were observed in heart rate. However, jugular occlusions promoted a decrease in the mean arterial pressure and a severe increase in jugular pressure. Head edema caused by the jugular vein occlusion in the horses could interfere with the autonomic cardiovascular regulation of arterial blood pressure during exercise, likely leading to an impairment of tissue perfusion. Jugular occlusion, even unilateral, also causes severe head venous congestion, leading to venous hypertension that was aggravated by exercise, which could risk development of cerebral edema and neurological damage. The present results obtained from sedentary horses are preliminary data that lead us to suggest that sport horses presenting jugular occlusive thrombophlebitis, even unilateral, may be prevented from performing athletic activities. © 2013 Elsevier Inc. All rights reserved.

Low-temperature structural effects in the (TMTSF)2PF6 and AsF6 Bechgaard salts

We present a detailed low-temperature investigation of the statics and dynamics of the anions and methyl groups in the organic conductors (TMTSF) 2PF6 and (TMTSF)2AsF6 (TMTSF: tetramethyl-tetraselenafulvalene). The 4 K neutron-scattering structure refinement of the fully deuterated (TMTSF)2PF6-D12 salt allows locating precisely the methyl groups at 4 K. This structure is compared to the one of the fully hydrogenated (TMTSF)2PF6-H12 salt previously determined at the same temperature. Surprisingly, it is found that deuteration corresponds to the application of a negative pressure of 5×102 MPa to the H12 salt. Accurate measurements of the Bragg intensity show anomalous thermal variations at low temperature both in the deuterated PF 6 and AsF6 salts. Two different thermal behaviors have been distinguished. Small Bragg-angle measurements reflect the presence of low-frequency modes at characteristic energies θE = 8.3 K and θE = 6.7 K for the PF6-D12 and AsF6-D12 salts, respectively. These modes correspond to the low-temperature methyl group motion. Large Bragg-angle measurements evidence an unexpected structural change around 55 K, which probably corresponds to the linkage of the anions to the methyl groups via the formation of F...D-CD2 bonds observed in the 4 K structural refinement. Finally we show that the thermal expansion coefficient of (TMTSF)2PF6 is dominated by the librational motion of the PF6 units. We quantitatively analyze the low-temperature variation of the lattice expansion via the contribution of Einstein oscillators, which allows us to determine for the first time the characteristic frequency of the PF6 librations: θE ≈ 50 K and θE = 76 K for the PF6-D12 and PF6-H12 salts, respectively. © 2013 American Physical Society.

Optimisation of High Hydrostatic Pressure Processing of Pêra Rio Orange Juice

The influence of high hydrostatic pressure (HHP) on Pêra Rio orange juice was investigated using response surface methodology. A central composite design was used to evaluate the effects of three processing conditions (independent variables), namely pressure (100-600 MPa), temperature (30-60 °C) and time (30-360 s), on the native microflora and pectin methylesterase (PME) activity of orange juice. Analysis of variance showed that second-order polynomial models fitted well with the experimental data for PME residual activity (R2 = 0.9586, p < 0.001) and aerobic microorganism count (R2 = 0.9879, p < 0.001). The optimum HHP processing conditions to produce orange juice with PME residual activity of less than 20 % and low microorganism count (<2 log cycles CFU/mL) were 550 to 600 MPa, 55 to 60 °C and 330 to 360 s. © 2013 Springer Science+Business Media New York.

Air-particle abrasion on zirconia ceramic using different protocols: Effects on biaxial flexural strength after cyclic loading, phase transformation and surface topography

This study evaluated the effect of different air-particle abrasion protocols on the biaxial flexural strength and structural stability of zirconia ceramics. Zirconia ceramic specimens (ISO 6872) (Lava, 3M ESPE) were obtained (N=336). The specimens (N=118, n=20 per group) were randomly assigned to one of the air-abrasion protocols: Gr1: Control (as-sintered); Gr2: 50 μm Al2O3 (2.5 bar); Gr3: 50 μm Al2O3 (3.5 bar); Gr4: 110 μm Al2O3(2.5 bar); Gr5: 110 μm Al2O3 (3.5 bar); Gr6: 30 μm SiO2 (2.5 bar) (CoJet); Gr7: 30 μm SiO2(3.5 bar); Gr8: 110 μm SiO2 (2.5 bar) (Rocatec Plus); and Gr9: 110 μm SiO2 (3.5 bar) (duration: 20 s, distance: 10 mm). While half of the specimens were tested immediately, the other half was subjected to cyclic loading in water (100,000 cycles; 50 N, 4 Hz, 37 °°C) prior to biaxial flexural strength test (ISO 6872). Phase transformation (t→m), relative amount of transformed monoclinic zirconia (FM), transformed zone depth (TZD) and surface roughness were measured. Particle type (p=0.2746), pressure (p=0.5084) and cyclic loading (p=0.1610) did not influence the flexural strength. Except for the air-abraded group with 110 μm Al2O3 at 3.5 bar, all air-abrasion protocols increased the biaxial flexural strength (MPa) (Controlnon-aged: 1030±153, Controlaged: 1138±138; Experimentalnon-aged: 1307±184-1554±124; Experimentalaged: 1308±118-1451±135) in both non-aged and aged conditions, respectively. Surface roughness (Ra) was the highest with 110 μm Al2O3(0.84 μm. FM values ranged from 0% to 27.21%, higher value for the Rocatec Plus (110 μm SiO2) and 110 μm Al2O3 groups at 3.5 bar pressure. TZD ranged between 0 and 1.43 μm, with the highest values for Rocatec Plus and 110 μm Al2O3 groups at 3.5 bar pressure. © 2013 Elsevier Ltd.

Commissural nucleus of the solitary tract regulates the antihypertensive effects elicited by moxonidine

The rostral ventrolateral medulla (RVLM) contains the presympathetic neurons involved in cardiovascular regulation that has been implicated as one of the most important central sites for the antihypertensive action of moxonidine (an α2-adrenergic and imidazoline agonist). Here, we sought to evaluate the cardiovascular effects produced by moxonidine injected into another important brainstem site, the commissural nucleus of the solitary tract (commNTS). Mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic nerve activity (sSNA) and activity of putative sympathoexcitatory vasomotor neurons of the RVLM were recorded in conscious or urethane-anesthetized, and artificial ventilated male Wistar rats. In conscious or anesthetized rats, moxonidine (2.5 and 5. nmol/50. nl) injected into the commNTS reduced MAP, HR and sSNA. The injection of moxonidine into the commNTS also elicited a reduction of 28% in the activity of sympathoexcitatory vasomotor neurons of the RVLM. To further assess the notion that moxonidine could act in another brainstem area to elicit the antihypertensive effects, a group with electrolytic lesions of the commNTS or sham and with stainless steel guide-cannulas implanted into the 4th V were used. In the sham group, moxonidine (20. nmol/1. μl) injected into 4th V decreased MAP and HR. The hypotension but not the bradycardia produced by moxonidine into the 4th V was reduced in acute (1. day) commNTS-lesioned rats. These data suggest that moxonidine can certainly act in other brainstem regions, such as commNTS to produce its beneficial therapeutic effects, such as hypotension and reduction in sympathetic nerve activity. © 2013 IBRO.

Isolation, homology modeling and renal effects of a C-type natriuretic peptide from the venom of the Brazilian yellow scorpion (Tityus serrulatus)

Mammalian natriuretic peptides (NPs) have been extensively investigated for use as therapeutic agents in the treatment of cardiovascular diseases. Here, we describe the isolation, sequencing and tridimensional homology modeling of the first C-type natriuretic peptide isolated from scorpion venom. In addition, its effects on the renal function of rats and on the mRNA expression of natriuretic peptide receptors in the kidneys are delineated. Fractionation of Tityusserrulatus venom using chromatographic techniques yielded a peptide with a molecular mass of 2190.64Da, which exhibited the pattern of disulfide bridges that is characteristic of a C-type NP (TsNP, T. serrulatus Natriuretic Peptide). In the isolated perfused rat kidney assay, treatment with two concentrations of TsNP (0.03 and 0.1μg/mL) increased the perfusion pressure, glomerular filtration rate and urinary flow. After 60min of treatment at both concentrations, the percentages of sodium, potassium and chloride transport were decreased, and the urinary cGMP concentration was elevated. Natriuretic peptide receptor-A (NPR-A) mRNA expression was down regulated in the kidneys treated with both concentrations of TsNP, whereas NPR-B, NPR-C and CG-C mRNAs were up regulated at the 0.1μg/mL concentration. In conclusion, this work describes the isolation and modeling of the first natriuretic peptide isolated from scorpion venom. In addition, examinations of the renal actions of TsNP indicate that its effects may be related to the activation of NPR-B, NPR-C and GC-C. © 2013 Elsevier Ltd.

Hemodynamic and antiarrhythmic effects of lidocaine or amiodarone in dogs anesthetized with halothane

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

Effects of continuous rate infusion of butorphanol in isoflurane-anesthetized calves

PURPOSE:To assess the hemodynamic changes and bispectral index (BIS) following administration of a continuous rate infusion (CRI) of butorphanol in isoflurane-anesthetized calves.METHODS: Eight calves weighing 110 ± 12 kg were included in the study. Anesthesia was induced with 5% isoflurane in O2 delivered via face mask and maintained with end-tidal concentration of 1.4%. IPPV was set to a peak inspiratory airway pressure of 15 cmH2O and respiratory rate of six breaths minute-1. Forty minutes after the start of anesthetic maintenance, 0.1 mg kg-1butorphanol was administered intravenously, followed by a CRI of 20 µg kg-1 minute-1. Hemodynamic variables and BIS were recorded before butorphanol administration (T0), and at 10, 20, 40 and 80 minutes following the CRI. Anesthesia was discontinued after the last recording and the calves were allowed to recover. The time to sternal recumbency (SRE) and standing (ST) were evaluated.RESULTS: There were no significant differences between the moments in all hemodynamic variables and BIS. The time to SRE and ST was 9 ± 5 and 14 ± 7 minutes, respectively.CONCLUSION: The continuous rate infusion did not produce clinically relevant changes in hemodynamic or bispectral index values compared to baseline in mechanically ventilated and unstimulated calves anesthetized at 1.4% isoflurane.

Effects of propofol in lipid-based emulsion and in microemulsion on the incidence of endothelial lesion in rabbits

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