Salivary immunoglobulin levels and periodontal indices in Brazilian children with and without type 1 diabetes.

This cross-sectional study evaluated the association between salivary immunoglobulins, plaque index, and gingival index in Brazilian children with and without type 1 diabetes mellitus (DM1). The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist for the reporting of observational studies was followed. The DM1 group had 38 children, and an equal number of volunteers matched by sex and age were recruited as controls. Clinical examination was performed for plaque index and gingival index determination. Non-stimulated whole saliva was collected. Concentrations of IgA, IgG, and IgM were determined by ELISA test. Data were tested by the Kolmogorov-Smirnov, Mann-Whitney, and Spearman tests and a multiple linear regression model (p<0.05) was performed. Gingival index was higher in the Control (DM1: 0.16±0.17; Control: 0.24±0.23, p=0.040). In DM1, there was a correlation between IgA and age (rho=0.371, p=0.024), IgM and IgG (rho=0.459, p=0.007), and IgM and gingival index (rho=0.394, p=0.014). In DM1, multiple linear regression showed that age (p=0.041; ß=0.363), gingival index (p=0.041; ß=0.398), and plaque index (p=0.008; ß=-0.506) were good predictors of IgA levels in saliva. Thus, IgA was the only researched immunoglobulin that was directly associated with plaque and gingival indices in Brazilian children with DM1, but not in control subjects.

Rutin ameliorates nitrergic and endothelial dysfunction on vessels and corpora cavernosa of diabetic animals.

Endothelial dysfunction is an early complication of diabetes and it is related to both micro- and macroangiopathies. In addition, >70% of diabetic patients develop autonomic neuropathies. Increased oxidative stress has a major role in the development of both nitrergic and endothelial dysfunction. The aim of this work is to evaluate whether rutin, a potent antioxidant, could ameliorate nitrergic and/or endothelial dysfunction in diabetic animals. Primary and secondary treatment protocols with rutin were investigated on rat aortic rings and the mesenteric arteriolar bed, and on rabbit aortic rings and corpora cavernosa (RbCC) from both euglycemic and alloxan-diabetic animals. Acetylcholine endothelium-dependent and sodium nitroprusside endothelium-independent relaxations were compared in tissues from euglycemic or diabetic animals. Electrical field stimulation (EFS)-induced relaxation was performed only in the RbCC. Endothelial-dependent relaxations were blunted by 40% in vessels and neuronal relaxation was blunted by 50% in RbCC taken from diabetic animals when compared to euglycemic animals. Pre-treatment with rutin restored both neuronal and endothelial dependent relaxations in diabetic animals towards the values achieved in control euglycemic tissues. Rutin was able to ameliorate both endothelial dysfunction and nitrergic neuropathy in animal experimental models. Rutin could be a lead compound in the primary or secondary preventive ancillary treatment of endothelial and nitrergic dysfunction in the course of diabetes.

Renal protective effect of pinitol in experimental diabetes.

Pinitol is a natural antidiabetic agent shown to prevent or ameliorate metabolic and overall vascular and neural function. In the present study we have evaluated the potential benefits of pinitol on renal function of streptozotocin (STZ)-induced diabetic rats. Both euglycemic or 8-week or 16-week diabetic rats were treated with either saline (1 ml/kg/12h; p.o) or pinitol (20 mg/kg/12h; p.o). The renal function was evaluated by using metabolic cages, renal hemodynamic and tubular parameters measurements. Histological examination and evaluation of the protein expression of renal markers such as nephrin, TGFß and pERK were also performed. Pinitol decreased by 50% the increased urinary albumin/creatinine ratio in both 8-week and 16 week diabetic rats. In addition, the glomerular volume of 16-week rats increased by 55% and this increase was blunted by pinitol. Remarkably, pressure-natriuresis was completely blunted in both 8 and 16-week diabetic rats but this impairment was prevented by pinitol in both treatment regimens. Pinitol ameliorated renal lesions and also prevented the decrease in nephrin expression and the increase of pERK and TGFß expression in both diabetic groups. Natriuresis due to high renal perfusion pressure increased 7-fold in control animals but was blocked in 16-week diabetic rats and remarkably pinitol partially restored pressure natriuresis (3-fold increase in sodium excretion during pressure natriuresis). Pinitol prevents and ameliorates albuminuria, glomerular expansion, impairment of pressure-natriuresis, renal structural alterations and changes of renal markers and has the potential to be tested for the prevention of diabetic kidney disease.

Antihypertensive potential of cis-[Ru(bpy)2(ImN)(NO)]3+, a ruthenium-based nitric oxide donor.

The aim of this study was to investigate the antihypertensive properties of cis-[Ru(bpy)2ImN(NO)]3+ (FOR0811) in normotensive and in Nω-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats. Vasorelaxant effects were analyzed by performing concentration response curve to FOR0811 in rat aortic rings in the absence or presence of 1H-[1,2,4]-oxadiazolo-[4,3,-a]quinoxalin-1-one (ODQ), L-cysteine or hydroxocobalamin. Normotensive and L-NAME-hypertensive rats were treated with FOR0811 and the effects in blood pressure and heart rate variability in the frequency domain (HRV) were followed. FOR0811 induced relaxation in rat aortic rings. Neither endothelium removal nor L-cysteine altered the FOR0811 effects. However, the incubation with ODQ and hydroxocobalamin completely blunted FOR0811 effects. FOR0811 administered intravenously by bolus infusion (0.01-1 mg/bolus) or chronically by using subcutaneous implanted osmotic pumps significantly reduced the mean arterial blood pressure. The effect was long lasting and did not induce reflex tachycardia. FOR0811 prevented both LF and VLF increases in L-NAME hypertensive rats and has antihypertensive properties. This new ruthenium complex compound might be a promising nitric oxide donor to treat cardiovascular diseases.

Early consumption of high-fat diet worsens renal damage in spontaneously hypertensive rats in adulthood.

The association between hypertension and obesity has been shown to be an important cause of kidney disease. We aimed to investigate the impact of a high-fat diet (HFD) administered in spontaneously hypertensive rats (SHR) after weaning in renal morphology and functional parameters. Male post-weaned SHR were divided into two groups: standard control diet (CD) (3% lipids; n = 8) or HFD (30% lipids; n = 8) during 8 weeks. The group HFD showed an increase in serum triglycerides (HFD: 96 ± 7 vs. CD: 33 ± 2 mg/dL) and glucose intolerance (HFD: 185 ± 7 vs. CD: 149 ± 4 mg/dL/min). Moreover, the HFD also showed an increase in almost 90% of the periepididymal and retroperitoneal adiposity. There was no difference in arterial blood pressure between groups. Renal morphofunctional parameters were decreased in HFD group for glomerular tuft area and diameter (4733 ± 65 µm2 and 82 ± 1 µm, respectively) when compared with CD group (5289 ± 171 µm2 and 88 ± 2 µm, respectively). HFD also showed a decrease of 50% of the renal function, which was associated with higher renal extracellular matrix and lipid deposition. Therefore, our data suggest that HFD since early period of life may contribute to renal damage in adults with hypertension, and this impairment can be associated with increased renal lipid accumulation.

Effect of co-administration of two different phosphodiesterase inhibitors and a ß3-adrenoceptor agonist in an experimental model of detrusor overactivity.

The purpose of this study was to evaluate in vitro the effect of the combination of BRL 37344 (ß3-adrenoceptor agonist) with tadalafil (phosphodiesterase type 5 inhibitor) or rolipram (phosphodiesterase type 4 inhibitor) in an experimental model of detrusor overactivity. The experiments were carried out in two phases using bladder strips of mice. In the first phase, on the top of 40 mM potassium-induced contraction, strips isolated from control mice were exposed to increasing concentrations of each study drug. In another series of experiments, prior to contraction, strips were incubated with either tadalafil or rolipram, followed by the addition of increasing concentrations of BRL 37344. In the second phase, the same protocols were performed with animals previously treated with L-NAME for 30 days. Chronic L-NAME administration leads to detrusor overactivity due to nitric oxide synthase inhibition. In phase one, preincubation with tadalafil enhanced relaxation response to BRL 37344 at two concentrations. Pretreatment with rolipram had no effect on BRL 37344-induced relaxation. In L-NAME-treated mice, rolipram induced more relaxation than the other drugs, enhancing relaxation response to BRL 37344 at almost all concentrations, but no synergistic effect with tadalafil was observed. The relaxant effect of BRL 37344 was enhanced by rolipram but not by tadalafil, suggesting that PDE4 inhibition, especially when associated with ß3-adrenoceptor stimulation, could represent a potential treatment for overactive bladder.

Salivary immunoglobulin levels in juvenile autoimmune hepatitis.

AIMS: The aim of the present study was to investigate the presence of immunoglobulins (Ig) in whole saliva from patients affected by autoimmune hepatitis (AIH). DESIGN: Twelve individuals with AIH and 12 healthy individuals without (CON) autoimmune hepatitis, aged 8-18 years, participated in this study. Non-stimulated whole saliva was collected and centrifuged. Supernatants were separated and lyophilized. Salivary pH was measured and immunoglobulins were analyzed through ELISA technique. RESULTS: Salivary pH (CON, 7.17 ±â€¯0.45; AIH, 6.92 ±â€¯0.43) did not differ between groups (p = 0.183). Measurable levels of IgG, IgA, IgM and IgE were detected on all patients. IgG levels were higher in AIH individuals (CON, 1.058 ±â€¯0.386; AIH, 1.635 ±â€¯0.373; p = 0.001), whereas IgA (CON, 0.915 ±â€¯0.187; AIH, 0.995 ±â€¯0.235; p = 0.362), IgM (CON, 0.683 ±â€¯0.147, AIH, 0.646 ±â€¯0.161; p = 0.561) and IgE levels (CON, 1.241 ±â€¯0.378; AIH, 1.312 ±â€¯0.412; p = 0.664) did not present differences between groups. CONCLUSIONS: The results of the present study suggest differences in salivary IgG levels between individuals with and without AIH. Thus, saliva has the potential of becoming an important diagnostic tool for the assessment of AIH.

High Salt Intake Promotes Different Responses to Urodilatin and Uroguanylin in the Isolated Rat Kidney.

Urodilatin (UD) and uroguanylin (UGN) have been implicated in the regulation of salt and water homeostasis, particularly in the balance handling of salt intake. In this sense, the aim of the present work was to study the main effects of these peptides in kidneys from animals subjected to high NaCl (2%) intake, during 10 days in metabolic cages. The control group received only normal water, whereas the treated group drank 2% solution of NaCl (NaCl 2%). In addition, we studied effect of subthreshold UD (0.14 nM) and UGN (0.06 µM) doses in NaCl 2% after a 30-min control period. Kidney perfusion was performed with Krebs-Henseleit containing 6 g% bovine albumin previously dialyzed. The effects of UD (0.14 nM) promoted reduction of PP, RVR, and UF in the NaCl 2% group. We also observed an increase in %TNa+ and %TCl-. The main effects of UGN in NaCl 2% were increase in PP, UF, and GFR, followed by a reduction in %TNa+ and %TCl-. After an increased intake of salt, physiological pathways are activated and regulated in order to eliminate excess sodium. In this study, we observed that in a subthreshold dose, UD does not promotes natriuresis and diuresis, suggesting that UGN is an important hormone in inducing salt excretion in a chronic salt overload. Therefore, the effects herein described may play a contributory role in the regulation of kidney function after ingestion of salty meals.

The effect of Cratylia floribunda lectin on renal hemodynamics and ion transport

Lectins have been described as glycoproteins that reversibly and specifically bind to carbohydrates. Legume lectins isolated from the subtribe Diocleinae (Canavalia, Dioclea andCratylia) are structurally homologous with respect to their primary structures. The Diocleinae lectins of Canavalia brasiliensis, Dioclea guianensis andCanavalia ensiformis have been shown to distinctly alter physiological parameters in isolated rat kidneys. Thus, the aim of this study was to investigate the effect of Cratylia floribunda lectin (CFL) on renal hemodynamics and ion transport in rats. In isolated perfused kidneys, CFL (10 mg/mL, n=5) increased RPP, RVR and decreased %TK+, but did not change urinary flow, glomerular filtration rate, sodium or chloride tubular transport. In isolated perfused mesenteric bed, CFL (3 and 10 mg/mL/min; n=4) did not alter tissue basal tonus or tissue contraction by phenylephrine (1 mM/mL/min). In conclusion, the seed lectin of Cratylia floribunda increased renal hemodynamic parameters showing a kaliuretic effect. This effect could be of tubular origin, rather than a result from haemodynamic alterations.

As lectinas são descritas como (glico)proteínas que se ligam, especificamente e reversivelmente, a carboidratos. Lectinas de leguminosas isoladas da subtribo Diocleinae (Canavalia, Dioclea eCratylia) são estruturalmente homólogas em relação às suas estruturas primárias. Demonstrou-se que as lectinas de DiocleinaeCanavalia brasiliensis, Dioclea guianensis eCanavalia ensiformis alteram diferentemente parâmetros fisiológicos em rins isolados de ratos. Dessa maneira, o objetivo deste estudo foi investigar o papel da lectina de Cratylia floribunda (CFL) na hemodinâmica renal e no transporte de íons em ratos. Em rins isolados perfundidos, CFL (10 mg/mL, n=5) aumentou a pressão de perfusão renal, a resistência vascular renal e reduziu o percentual do transporte tubular de K+, mas não alterou o fluxo urinário, a taxa de filtração glomerular e o percentual de transporte tubular dos íons sódio e cloreto. No leito mesentérico isolado perfundido, CFL (3 e 10 mg/mL/min, n=4) não alterou o tônus basal ou a contração do tecido induzida por fenilefrina (1 mM/mL/min). Em conclusão, a lectina de sementes de Cratylia floribunda altera parâmetros hemodinâmicos renais, provavelmente de origem tubular, e não por alterações hemodinâmicas.

Effects of the essential oil of Croton zehntneri and its major components, anethole and estragole, on the rat corpora cavernosa.

AIMS: The effects of the essential oil of Croton zehntneri (EOCz) and its major components anethole, estragole and methyl eugenol were evaluated in phenylephrine precontracted rat corpora cavernosa (RCC). MAIN METHODS: RCC strips were mounted in 5 ml organ baths for isometric recordings of tension, precontracted with 10 µM phenylephrine and exposed to test drugs. KEY FINDINGS: All major compounds relaxed RCC. The order of potency was estragole>anethole>methyl eugenol. The maximal relaxation to EOCz and methyl eugenol was 62.67% (IC50 of 1.67 µM) and 45.8% (IC50 of 1.7 µM), respectively. Estragole relaxed RCC with an IC50 of 0.6 µM (maximal relaxation-76.6%). The maximal relaxation to estragole was significantly reduced by L-NAME (43.46%-IC50 of 1.4 µM), ODQ (53.11%-IC50 of 0.83 µM) and indomethacin (24.41%-IC50 of 1.3 µM). On the other hand, anethole relaxed RCC by 66.73% (IC50 of 0.96 µM) and this relaxation was blunted by indomethacin (35.65%-IC50 of 1.6 µM). Both estragole and anethole increased the relaxation achieved upon electrical stimulation. Both compounds increased the levels of cAMP (estragole by 3-fold and anethole by 2-fold when compared to controls). Estragole also increased the levels of cGMP (0.5-fold). SIGNIFICANCE: The higher potency of these compounds to relax corpora cavernosa smooth muscle may form the pharmacological basis for the use of such substances as leading compounds in the search of alternative treatments of erectile dysfunction.

Salivary peptide profile and its association with early childhood caries.

BACKGROUND: As dietary management during early childhood is a great barrier in caries control, there is a need for the identification of intrinsic risk factors, capable of allowing the use of a more cost-effective approach to early childhood caries (ECC). OBJECTIVE: To evaluate the salivary peptide profile of children with and without ECC and its association with caries experience. METHODS: One hundred and six 10- to 71-month-old children participated in the study. Caries experience was determined through the visual/tactile method, based on the number of decayed, missing, and filled teeth, and surface scores (dmft/dmfs). Whole saliva was collected for mutans streptococci (MS) detection and peptide analysis. RESULTS: Chromatograms from CF (children without caries experience, n = 58) and CE (children with caries experience, n = 48) saliva pools expressed different patterns. Identification of molecular masses suggested the presence of nine peptides. Three of them were significantly related with caries experience. HNP-3 (α-defensin 3) (P = 0.019) and HBD-3 (ß-defensin 3) (P = 0.034) reduced the chances of experiencing ECC. Proline-rich peptides IB-4 significantly increased caries experience (P = 0.035). Age (P = 0.020) and MS counts (P = 0.036) increased caries experience; however, gender was not associated with dental caries (P = 0.877). CONCLUSION: Specific salivary peptides of CF or CE children in early childhood predispose to a higher or lower risk of caries experience.

Comparative proteomic analysis of human whole saliva of children with protein-energy undernutrition.

OBJECTIVE: The aim of the present study was to investigate the protein profile of children with different levels of protein-energy undernutrition (PEU) through a proteomic approach of human whole saliva. METHODS: Initially, saliva samples of children with mild, moderate, and severe PEU were collected and lyophilized. Saliva samples of healthy children were used as controls. Samples were analyzed for total protein using the Bradford method. Saliva samples were analyzed by two-dimensional electrophoresis according to their isoelectric point (pI) and their molecular weights (MWs). RESULTS: Comparisons of protein bands among the healthy and mildly, moderately, and severely undernourished children showed significant differences in the MWs (P = 0.001) and pI values (P = 0.03). In total 159 spots were identified in the healthy children; 156, 168, and 221 spots were observed in mildly, moderately, and severely undernourished children, respectively. Mildly undernourished children presented with the spot with the highest MW of 293 kDa (pI = 7.77) and the lowest MW of 5 kDa (pI = 4.83). Moderately undernourished children were the only ones who did not present with a protein band with an MW of 30 kDa. The presence of a protein band with an MW of 123 kDa (pI = 516), possibly a cyclin-dependent protein kinase, was also observed only in this group. CONCLUSION: The protein profile in saliva varies according to the presence or absence of PEU, and these variations are specifically expressed in different grades of undernutrition. Thus, saliva may be an important diagnostic tool for the assessment of PEU.

Guanylin peptide family: history, interactions with ANP, and new pharmacological perspectives.

The guanylin family of peptides has 3 subclasses of peptides containing either 3 intramolecular disulfide bonds found in bacterial heat-stable enterotoxins (ST), or 2 disulfides observed in guanylin and uroguanylin, or a single disulfide exemplified by lymphoguanylin. These peptides bind to and activate cell-surface receptors that have intrinsic guanylate cyclase (GC) activity. These hormones are synthesized in the intestine and released both luminally and into the circulation, and are also produced within the kidney. Stimulation of renal target cells by guanylin peptides in vivo or ex vivo elicits a long-lived diuresis, natriuresis, and kaliuresis by both cGMP-dependent and independent mechanisms. Uroguanylin may act as a hormone in a novel endocrine axis linking the digestive system and kidney as well as a paracrine system intrarenally to increase sodium excretion in the postprandial period. This highly integrated and redundant mechanism allows the organism to maintain sodium balance by eliminating excess sodium in the urine. In addition, small concentrations of the atrial natriuretic peptide (ANP) can synergize with low concentrations of both guanylin or uroguanylin, which do not induce natriuresis per se, to promote significant natriuresis. Interestingly, the activation of the particulate guanylate cyclase receptors by natriuretic peptides can promote relaxation of animal and human penile erectile tissue and increase intracavernosal pressure to induce penile erection. These peptides can be prototypes for new drugs to treat erectile dysfunction, especially in patients with endothelial and nitrergic dysfunction, such as in diabetes.

The extract of the jellyfish Phyllorhiza punctata promotes neurotoxic effects.

Phyllorhiza punctata (P. punctata) is a jellyfish native to the southwestern Pacific. Herewith we present the biochemical and pharmacological characterization of an extract of the tentacles of P. punctata. The tentacles were subjected to three freeze-thaw cycles, homogenized, ultrafiltered, precipitated, centrifuged and lyophilized to obtain a crude extract (PHY-N). Paralytic shellfish poisoning compounds such as saxitoxin, gonyautoxin-4, tetrodotoxin and brevetoxin-2, as well as several secretory phospholipase A(2) were identified. PHY-N was tested on autonomic and somatic neuromuscular preparations. In mouse vas deferens, PHY-N induced phasic contractions that reached a peak of 234 ± 34.7% of control twitch height, which were blocked with either 100 µ m of phentolamine or 1 m m of lidocaine. In mouse corpora cavernosa, PHY-N evoked a relaxation response, which was blocked with either L-N(G) -Nitroarginine methyl ester (0.5 m m) or 1 m m of lidocaine. PHY-N (1, 3 and 10 µg ml(-1) ) induced an increase in tonus of the biventer-cervicis neuromuscular preparation that was blocked with pre-treatment of galamine (10 µ m). Administration of 6 mg kg(-1) PHY-N intramuscularly produced death in broilers by spastic paralysis. In conclusion, PHY-N induces nerve depolarization and nonspecifically increases neurotransmitter release.

Renal and cardiovascular effects of Bothrops marajoensis venom and phospholipase A2.

Bothrops marajoensis is found in the savannah of Marajó Island in the State of Pará and regions of Amapá State, Brazil. The aim of the work was to study the renal and cardiovascular effects of the B. marajoensis venom and phospholipase A(2) (PLA(2)). The venom was fractionated by Protein Pack 5PW. N-terminal amino acid sequencing of sPLA(2) showed amino acid identity with other lysine K49 sPLA(2)s of snake venom. B. marajoensis venom (30 microg/mL) decreased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate and sodium tubular transport. PLA(2) did not change the renal parameters. The perfusion pressure of the mesenteric bed did not change after infusion of venom. In isolated heart, the venom decreased the force of contraction and increased PP but did not change coronary flow. In the arterial pressure, the venom and PLA(2) decreased mean arterial pressure and cardiac frequency. The presence of atrial flutter and late hyperpolarisation reversed, indicating QRS complex arrhythmia and dysfunction in atrial conduction. In conclusion, B. marajoensis venom and PLA(2) induce hypotension and bradycardia while simultaneously blocking electrical conduction in the heart. Moreover, the decrease in glomerular filtration rate, urinary flow and electrolyte transport demonstrates physiological changes to the renal system.

1,8-Cineole induces relaxation in rat and guinea-pig airway smooth muscle.

OBJECTIVES: 1,8-Cineole is a monoterpene with anti-inflammatory, vascular and intestinal smooth muscle relaxant activity. We have evaluated the potential bronchodilatatory activity of this compound. METHODS: 1,8-Cineole was tested against carbachol, histamine, K+ 80 mM and ovalbumin-induced bronchial contractions in Wistar rat or guinea-pig tissues. Some of the guinea-pigs had been previously sensitized with an intramuscular injection of 5% (w/v) ovalbumin/saline solution. Control animals received 0.3 ml saline. In separate experimental groups the response to 1,8-cineole (1-30 mg/kg), phenoterol (0.05-5 mg/kg) or vehicle (0.3% Tween in saline) was studied. KEY FINDINGS: 1,8-Cineole decreased, in vivo, rat bronchial resistance with similar efficacy as phenoterol (66.7 +/- 3.2% vs 72.1 +/- 5.3%). On the other hand, the maximal relaxant response to 1,8-cineole in carbachol-precontracted rat tracheas was 85.5 +/- 5.7% (IC50 = 408.9 (328-5196) microg/ml) compared with 80.2 +/- 4.8% (IC50 = 5.1 (4.3-6.1) microg/ml) with phenoterol. The addition of 1,8-cineole to guinea-pig tracheal rings tonically contracted with K+ 80 mM induced a concentration-related relaxation. The maximal relaxation elicited by 1,8-cineole was 113.6 +/- 11.7% (IC50 127.0 (115.9-139.2) microg/ml) compared with 129.7 +/- 14.6% (IC50 0.13 (0.12-0.14) microg/ml) achieved after phenoterol administration. In addition, the incubation of tracheal rings with 1,8-cineole (100, 300 or 1000 microg/ml), 15 min before inducing phasic contractions with K+ 80 mM, decreased the maximal amplitude of the contraction by 31.6 +/- 4.6, 75.7 +/- 2.7 and 92.2 +/- 1.5%, respectively. In another set of experiments, neither the maximal response nor the IC50 for the 1,8-cineole-induced relaxation were different between normal and ovalbumin-sensitized tissues. Moreover, the relaxation of bronchial rings contracted after exposure to 1 microg/ml ovalbumin occurred at a faster rate in rings pre-incubated with 1,8-cineole when compared with rings pre-incubated with vehicle only (Tween 0.3%). Therefore, in the first minute after the antigen challenge, the tracheal tissue relaxed after the peak contraction by 6.5, 21.4 (P < 0.05 vs control) and 66.9% (P < 0.05 vs control) in the presence of 100, 300 or 1000 microg/ml 1,8-cineole, respectively. CONCLUSIONS: 1,8-Cineole relaxed rat and guinea-pig (nonsensitized and ovalbumin-sensitized) airway smooth muscle by a nonspecific mechanism.

Purification and renal effects of phospholipase A(2) isolated from Bothrops insularis venom.

Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A(2) protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A(2) (PLA(2)) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as IV-1 to IV-5, from which IV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA(2) of Bothrops jararaca and Bothrops asper. Phospholipase A(2) isolated from B. insularis (Bi PLA(2) ) venom (10 microg/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n=6). Bi PLA(2) increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa(+)) and chloride tubular reabsorption (%TCl(-)) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA(2) isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney.

Purification and biological effects of L-amino acid oxidase isolated from Bothrops insularis venom.

Bothrops insularis is a snake from Ilha da Queimada Grande, an island located about 20 miles away from the Southeastern coast of Brazil. Compared with other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood, and so far, no fraction from this venom with amino acid oxidase activity had been isolated or its biological activity tested. We investigated the biochemical and biological effects of one l-amino acid oxidase enzyme isolated from B. insularis snake venom (BiLAO), which was purified using HPLC and sequence grade. We also evaluated the renal effects induced by BiLAO. Chromatographic profile of B. insularis whole venom disclosed seven main fractions (I, II, III, IV, V, VI and VII) and the main LAO enzymatic activity was detected in fraction II. The group treated with BiLAO showed a decrease in perfusion pressure (C(120)=110.28+/-3.69; BiLAO(120)=82.2+/-5.6 mmHg*); renal vascular resistance (C(120)=5.48+/-0.53; BiLAO(120)=4.12+/-0.42 mmHg/mL/g/min*), urinary flow (C(120)=0.160+/-0.020; BiLAO(120)=0.064+/-0.012 mL/g/min*), glomerular filtration rate (C(120)=0.697+/-0.084; BiLAO(120)=0.176+/-0.017 mL/g/min*), sodium (C(120)=79.76+/-0.56; BiLAO(120)=65.39+/-6.19%*), potassium (C(120)=69.94+/-6.86; BiLAO(120)=60.26+/-2.24%*) and chloride tubular reabsortion (C(120)=78.53+/-2.33; BiLAO(120)=64.58+/-6.68%*). Acute tubular necrosis foci were observed in the group treated with the LAO fraction of the B. insularis snake venom. Some findings have the same morphological aspect of apoptosis, more evident cortically; otherwise, reversible degenerative phenomena represented by hydropic ballooning with extensive cytoplasmic vacuolization and discontinuity of the cell brush borders in the proximal tubular epithelium were observed; furthermore, necrotic detachment of these cells into the tubular lumina, and increased amount of protein deposits in the distal and proximal tubules were observed. In conclusion, the slowness of blood flow and of glomerular filtration resulted in more time for filtration and tubular reabsorption, with elevation of the total percentage of sodium and chlorine reabsorption. The maintenance of the decrease in glomerular filtration rate would determine the subsequent decreases, which were noticed in these parameters. The necrosis observed was the result of damage cell induced by l-amino acid oxidase isolated from B. insularis venom.

Purification and biological activity of the thrombin-like substance isolated from Bothrops insularis venom.

The venom of Bothrops insularis snake, known in Brazil as jararaca ilhoa, contains a variety of proteolytic enzymes such as a thrombin-like substance that is responsible for various pharmacological effects. B. insularis venom chromatography profile showed an elution of seven main fractions. The thrombin-like activity was detected in fractions I and III, the latter being subjected to two other chromatographic procedures, so to say DEAE and Hi Trap Benzamidine. The purity degree of this fraction was confirmed by analytical reverse phase HPLC, which displayed only one main fraction confirmed by SDS-PAGE constituting fraction III. About 5 microg of fraction III protein potentiated the secretion of insulin induced by 2.8 mM of glucose in rats isolated pancreatic beta-cells treated; the increase being around 3-fold higher than its respective control. B. insularis lectin (BiLec; 10 microg/mL) was also studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. BiLec increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, the thrombin-like substance isolated from B. insularis venom induced an increase in insulin secretion, in vitro, and transiently altered vascular, glomerular and tubular parameters in the isolated rat kidney.

Purification and biological effects of C-type lectin isolated from Bothrops insularis venom.

Bothrops insularis is a snake from Queimada Grande Island, which is an island located about 20 miles away from the southeastern coast of Brazil. Compared to other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood. Its C-type lectin is involved in several biological processes including anticoagulant and platelet-modulating activities. We purified the C-type lectin (BiLec) from Bothrops insularis venom and investigated its effect in the isolated kidney. BiLec was purified after two chromatographic steps; firstly, the whole venom was submitted to an HPLC molecular exclusion chromatography followed by a second purification through affinity chromatography. B. insularis lectin (BiLec) was studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. The concentration of 10mug/mL increased perfusion pressure (PP; control(60)=108.27+/-4.9; BiLec(60)=112.9+/-5.4 mmHg; *p<0.05) and renal vascular resistance (RVR; control(60)=5.38+/-0.51; BiLec(60)=6.01+/-0.57 mmHg; *p<0.05). The urinary flow reduced significantly at 90 and 120 min of perfusion (UF; control(120)=0.160+/-0.020; BiLec(120)=0.082+/-0.008 mL g(-1) min(-1); *p<0.05). Glomerular filtration rate (GFR; control(120)=0.697+/-0.084; BiLec(120)=0.394+/-0.063 mL g(-1) min(-1); *p<0.05) diminished only at 120 min. BiLec did not change the percentage of sodium (TNa(+)), potassium (TK(+)) and chloride tubular transport (TCl(-)). The histological alterations probably reflected direct injury on glomerular and tubular renal cells, as demonstrated by the rise in permeability of glomerular endothelial cells, revealed by the presence of a proteinaceous material in the Bowman space. We postulate that the C-type lectin B. insularis promoted its effects probably through interactions with endothelial cells or through the release of other mediators by tubular, mesangial and endothelial cells.