Antimicrobial activity of kaurane diterpenes against oral pathogens

Two kaurane diterpenes, ent-kaur-16(17)-en-19-oic acid (KA) and 15-beta-isovaleryloxy-ent-kaur-16(17)-en-19-oic acid (KA-Ival), isolated from Aspilia foliacea, and the methyl ester derivative of KA (KA-Me) were evaluated against oral pathogens. KA was the most active compound, with MIC values of 10 mu g mL(-1) against the following microorganisms: Streptococcus sobrinus, Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, and Lactobacillus casei. However, KA did not show significant activity against Streptococcus salivarius and Enterococcus faecalis, with MIC values equal to 100 and 200 mu g mL(-1), respectively. Our results show that KA has potential to be used as a prototype for the discovery of new effective anti-infection agents against microorganisms responsible for caries and periodontal diseases. Moreover, these results allow to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to studies on the structure-activity relationship of this type of metabolites with respect to caries and periodontal diseases.

The juvenile hormone (JH) epoxide hydrolase gene in the honey bee (Apis mellifera) genome encodes a protein which has negligible participation in JH degradation

Epoxide hydrolases are multifunctional enzymes that are best known in insects for their role in juvenile hormone (JH) degradation. Enzymes involved in JH catabolism can play major roles during metamorphosis and reproduction, such as the JH epoxide hydrolase (JHEH), which degrades JH through hydration of the epoxide moiety to form JH diol, and JH esterase (JHE), which hydrolyzes the methyl ester to produce JH acid. In the honey bee, JH has been co-opted for additional functions, mainly in caste differentiation and in age-related behavioral development of workers, where the activity of both enzymes could be important for JH titer regulation. Similarity searches for jheh candidate genes in the honey bee genome revealed a single Amjheh gene. Sequence analysis, quantification of Amjheh transcript levels and Western blot assays using an AmJHEH-specific antibody generated during this study revealed that the AmJHEH found in the fat body shares features with the microsomal JHEHs from several insect species. Using a partition assay we demonstrated that AmJHEH has a negligible role in JH degradation, which, in the honey bee, is thus performed primarily by JHE. High AmJHEH levels in larvae and adults were related to the ingestion of high loads of lipids, suggesting that AmJHEH has a role in dietary lipid catabolism. (C) 2010 Elsevier Ltd. All rights reserved.

Adsorption of phenylalanine on layered double hydroxides: effect of temperature and ionic strength

In this work we report the adsorption of phenylalanine (Phe) on Magnesium Aluminum Layered Double Hydroxides (Mg-Al-CO(3)-LDH) at two different temperatures (298 and 310 K) and under two distinct ionic strength conditions (with and without the addition 0.1 M of NaCl). The adsorption isotherms exhibit the same profile in all conditions, and they only differ in the amount of removed Phe. At lower ionic strength, the isotherms are almost identical at both temperatures, except for the last points, where the increase in temperature causes a decrease in the amount of adsorbed Phe. An increase in ionic strength results in a decrease in Phe adsorption. The electrokinetic potential decreases as the amount of adsorbed Phe increases, and only positive values are observed. This indicates that the surface of the adsorbent is not totally neutralized and suggests that more Phe could be removed by adsorption. The presence of Phe on the solid is confirmed by FTIR spectra, which present the specific bands assigned to Phe. The hydrophobicity of the amino acid probably contributes to its extraction, thus enabling the removal of a great amount of Phe. In conclusion, LDH is potentially applicable in the removal of Phe from wastewater.

Effects of chronic L-NAME treatment lung tissue mechanics, eosinophilic and extracellular matrix responses induced by chronic pulmonary inflammation

The importance of lung tissue in asthma pathophysiology has been recently recognized. Although nitric oxide mediates smooth muscle tonus control in airways, its effects on lung tissue responsiveness have not been investigated previously. We hypothesized that chronic nitric oxide synthase (NOS) inhibition by N-omega-nitro-L-arginine methyl ester (L-NAME) may modulate lung tissue mechanics and eosinophil and extracellular matrix remodeling in guinea pigs with chronic pulmonary inflammation. Animals were submitted to seven saline or ovalbumin exposures with increasing doses (1 similar to 5 mg/ml for 4 wk) and treated or not with L-NAME in drinking water. After the seventh inhalation (72 h), animals were anesthetized and exsanguinated, and oscillatory mechanics of lung tissue strips were performed in baseline condition and after ovalbumin challenge (0.1%). Using morphometry, we assessed the density of eosinophils, neuronal NOS (nNOS)- and inducible NOS (iNOS)-positive distal lung cells, smooth muscle cells, as well as collagen and elastic fibers in lung tissue. Ovalbumin-exposed animals had an increase in baseline and maximal tissue resistance and elastance, eosinophil density, nNOS- and iNOS-positive cells, the amount of collagen and elastic fibers, and isoprostane-8-PGF(2 alpha) expression in the alveolar septa compared with controls (P < 0.05). L-NAME treatment in ovalbumin-exposed animals attenuated lung tissue mechanical responses (P < 0.01), nNOS- and iNOS-positive cells, elastic fiber content (P < 0.001), and isoprostane-8-PGF(2 alpha) in the alveolar septa (P < 0.001). However, this treatment did not affect the total number of eosinophils and collagen deposition. These data suggest that NO contributes to distal lung parenchyma constriction and to elastic fiber deposition in this model. One possibility may be related to the effects of NO activating the oxidative stress pathway.

Nitric oxide modulates lipopolysaccharide-induced endothelial platelet endothelial cell adhesion molecule expression via interleukin-10

We have shown previously that nitric oxide (NO) controls platelet endothelial cell adhesion molecule (PECAM-1) expression on both neutrophils and endothelial cells under physiological conditions. Here, the molecular mechanism by which NO regulates lipopolysaccharide (LPS)-induced endothelial PECAM-1 expression and the role of interleukin (IL)-10 on this control was investigated. For this purpose, N-(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg/day for 14 days dissolved in drinking water) was used to inhibit both constitutive (cNOS) and inducible nitric oxide (iNOS) synthase activities in LPS-stimulated Wistar rats (5 mg/kg, intraperitoneally). This treatment resulted in reduced levels of serum NO. Under this condition, circulating levels of IL-10 was enhanced, secreted mainly by circulating lymphocytes, dependent on transcriptional activation, and endothelial PECAM-1 expression was reduced independently on reduced gene synthesis. The connection between NO, IL-10 and PECAM-1 expression was examined by incubating LPS-stimulated (1 mu g/ml) cultured endothelial cells obtained from naive rats with supernatant of LPS-stimulated lymphocytes, which were obtained from blood of control or L-NAME-treated rats. Supernatant of LPS-stimulated lymphocytes obtained from L-NAME-treated rats, which contained higher levels of IL-10, reduced LPS-induced PECAM-1 expression by endothelial cells, and this reduction was reversed by adding the anti-IL-10 monoclonal antibody. Therefore, an association between NO, IL-10 and PECAM-1 was found and may represent a novel mechanism by which NO controls endothelial cell functions.

Role of nitric oxide in hyperpnea-induced bronchoconstriction and airway microvascular permeability in guinea pigs

The present study aimed to evaluate the role of nitric oxide (NO) on hyperpnea-induced bronchoconstriction (HIB) and airway microvascular hyperpermeability (AMP). Sixty-four guinea pigs were anesthetized, tracheotonnized, cannulated, and connected to animal ventilator to obtain pulmonary baseline respiratory system resistance (Rrs). Animals were then submitted to 5 minutes hyperpnea and Rrs was evaluated during 15 minutes after hyperpnea. AMP was evaluated by Evans blue dye (25 mg/kg) extravasation in airway tissues. Constitutive and inductible NO was evaluated by pretreating animals with N(G)-nitro-1-arginine methyl ester (I-NAME) (50 mg/kg), aminoguadinine (AG) (50 mg/kg), and I-arginine (100 mg/kg) and exhaled NO (NOex) was evaluated before and after drug administration and hyperpnea. The results show that I-NAME potentiated (57%) HIB and this effect was totally reversed by I-arginine pretreatment, whereas AG did not have effect on HIB. I-NAME decreased basal AMP (48%), but neither I-NAME nor AG had any effect on hyperpnea-induced AMP. NOex levels were decreased by 50% with I-NAME, effect that was reversed by I-arginine treatment. These results suggest that constitutive but not inducible NO could have a bronchoprotective effect on HIB in guinea pigs. The authors also observed that neither constitutive nor inducible NO seems to have any effect on hyperpnea-induced AMP.

Photodynamic therapy for acne vulgaris: A critical review from basics to clinical practice Part II. Understanding parameters for acne treatment with photodynamic therapy

Photodynamic therapy requires a photosensitizer, oxygen, and activating light. For acne, pilosebaceous units are ""target"" structures. Porphyrins are synthesized in vivo from 5-aminolevulinic acid (ALA), particularly in pilosebaceous units. Different photosensitizers and drug delivery methods have been reported for acne treatment. There are a variety of porphyrin precursors with different pharmacokinetic properties. Among them, ALA and methyl-ester of ALA (MAT.) are available for possible off-label treatment of acne vulgaris. In addition, various light sources, light dosimetry, drug incubation time, and pre- and posttreatment care also change efficacy and side effects. None of these variables has been optimized for acne treatment, but a number of clinical trials provide helpful guidance. In this paper, we critically analyze clinical trials, case reports, and series of cases published through 2009. (J Am Acad Dermatol 2010;63:195-211.)

Development of microemulsions to topically deliver 5-aminolevulinic acid in photodynamic therapy

The aim of this study was to obtain and to characterize microemulsions containing 5-aminolevulinic acid (5-ALA) and to investigate the influence of these systems in drug skin permeation for further topical photodynamic therapy (PDT). 5-ALA was incorporated in water-in-oil (W/O), bicontinuous (Bc), and oil-in-water (O/W) microemulsions obtained by the titration of ethyl oleate and PEG-8 caprylic/capric glycerides:polyglyceryl-6 dioleate (3:1) mixtures with water. Selected systems were characterized by conductivity, viscosity, size of the droplets, and drug release. The stability of the drug in the microemulsions was also assessed. Moreover, the in vitro and in vivo skin permeation of 5-ALA was investigated using diffusion cells and confocal scanning laser microscopy (CSLM), respectively. Despite the fact that the O/W microemulsion decreased the 5-ALA diffusion coefficient and retarded the drug release, it also significantly increased the in vitro drug skin permeation when compared to other 5-ALA carriers. It was observed by CSLM that the red fluorescence of the skin increased homogeneously in the deeper skin layers when the 5-ALA microemulsion was applied in vivo, probably due to the formation of the photoactive protoporphyrin IX. The microemulsion developed carried 5-ALA to the deeper skin layers, increasing the red fluorescence of the skin and indicating the potentiality of the system for topical 5-ALA-PDT. (C) 2010 Elsevier B.V. All rights reserved.

EFFECT OF THE TEMPERATURE AND pH ON METHANOL RELEASE IN COFFEE BREW SWEETENED WITH ASPARTAME

Methanol is an alcohol which is metabolized to folmaldehyde in humans. It is a very toxic substance, responsible for blindness in cases of methanol intoxication. This study shows the production of methanol when aspartame is used to sweeten coffee brew. The temperature versus pH binomium was also tested. When assayed at 90 degrees C coffee brew+aspartame and aspartame solution showed an increase in methanol release of 17.8 and 19.3%, respectively, as compared with coffee brew. At 180 degrees C, the increase was 32.5 and 26.3%, respectively. Our data revealed a protective effect of the pH of coffee on the degradation of aspartame and formation of methanol; an important fact, mainly for specific populations that use aspartame, like diabetics.

Sildenafil inhibits duodenal contractility via activation of the NO-K+ channel pathway

Phosphodiesterase type-5 (PDE5) specifically cleaves cyclic guanosine monophosphate (cGMP), a key intracellular secondary messenger. The PDE5 inhibitor sildenafil is a well-known vasodilator that also has gastrointestinal myorelaxant properties. In the present study, we further investigated sildenafil-induced myorelaxation in rat isolated duodenum, assessing its interaction with nitric oxide (NO) synthase and K+ channel opening. The spontaneous contractions of duodenal strips were reversibly inhibited by sildenafil (0.1-300 mu M) in a concentration-dependent manner [mean (95% confidence interval); EC50 = 6.8 (2.7-17.3) mu M]. The sildenafil-induced myorelaxation was significantly decreased by the NO synthase inhibitor N-nitro-L-arginine methyl ester [increasing the EC50 value to 41.9 (26.1-67.3) mu M]. Sodium nitroprusside or forskolin pretreatments enhanced the sildenafil-induced myorelaxation. In isolated strips pretreated with BaCl2 (0.2 mM), 4-aminopyridine (4-AP, 3 mM), or glybenclamide (1 mu M), the sildenafil-induced EC50 value was significantly increased to 32.8 (19.1-56.4), 27.1 (15.2-48.3) and 20.1 (16.4-24.7) mu M, respectively. Minoxidil (50 mu M) or diazoxide (100 mu M) also significantly attenuated the sildenafil-induced potency. In conclusion, the NO synthase/cyclic nucleotide pathway activation is involved in sildenafil-induced inhibition of spontaneous duodenal contractions. Its pharmacological action seems to be influenced by K+ channel opening, especially the voltage-sensitive ones, being inhibited by 4-AP and K-ATP channels, sensitive to glybenclamide.

Effects of nitric oxide on neutrophil influx depends on the tissue: role of leukotriene B(4) and adhesion molecules

Background and purpose: We investigated the effect of nitric oxide synthase (NOS) inhibition on polymorphonuclear cell (PMN) influx in zymosan or lipopolysaccharide (LPS)-induced arthritis and peritonitis. Experimental approach: Wistar rats received intra-articular (i.art.) zymosan (30-1000 mu g) or LPS (1-10 mu g). Swiss C57/Bl6 mice genetically deficient in intercellular adhesion molecule-1 (ICAM-1(-/-)) or in beta(2)-integrin (beta(2)-integrin(-/-)) received zymosan either i.art. or i.p. PMN counts, leukotriene B(4) (LTB(4)), tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) levels were measured in joint and peritoneal exudates. Groups received the NOS inhibitors N(G)-nitro-L-arginine methyl ester (LN), nitro-L-arginine, N-[3-(aminomemethyl) benzyl] acetamide or aminoguanidine, prior to zymosan or LPS, given i.p. or s.c. in the arthritis and peritonitis experiments respectively. A group of rats received LN locally (i.art. or i.p.), 30 min prior to 1 mg zymosan i.art. Key results: Systemic or local NOS inhibition significantly prevented PMN migration in arthritis while increasing it in peritonitis, regardless of stimuli, concentration of NOS inhibitors and species. NOS inhibition did not alter TNF-alpha and IL-10 but decreased LTB(4) in zymosan-induced arthritis. LN administration significantly inhibited PMN influx into the joints of ICAM-1(-/-) and beta(2)-integrin(-/-) mice with zymosan-arthritis, while not altering PMN influx into the peritoneum of mice with zymosan-peritonitis. Conclusions and implications: Nitric oxide has a dual modulatory role on PMN influx into joint and peritoneal cavities that is stimulus-and species-independent. Differences in local release of LTB(4) and in expression of ICAM-1 and beta(2)-integrin account for this dual role of NO on PMN migration.

Murine and rat cavernosal responses to endothelin-1 and urotensin-II Vasoactive Peptide Symposium

Endothelin-1 (ET-1) and urotensin-II (U-II) are the most potent constrictors of human vessels. Although the cavernosal tissue is highly responsive to ET-1, no information exists on the effects of U-II on cavernosal function. The aim of this study was to characterize ET-1 and U-II responses in corpora cavernosa from rats and mice. Male Wistar rats and C57/BL6 mice were used at 13 weeks. Cumulative concentration-response curves to ET-1, U-II, and IRL-1620, an ET(B) agonist, were performed. ET-1 increased force generation in cavernosal strips from mice and rats, but no response to U-II was observed in the presence or absence of N(omega)-nitro-L-arginine methyl ester (L-NAME), or in strips prestimulated with 20 mM KCI. IRL-1620 did not induce cavernosal contraction even in presence of L-NAME, but induced a cavernosal relaxation that was greater in rats than mice. No relaxation responses to U-II were observed in cavernosal strips precontracted with phenylephrine. mRNA expression of ET-1, ET(A), ET(B), and U-II receptors, but not U-II was observed in cavernosal strips. ET-1, via ET(A) receptors activation, causes contractile responses in cavernosal strips from rats and mice, whereas ET(B) receptor activation produces relaxation. Although the cavernosal tissue expresses U-II receptors, U-II does not induce contractile responses in corpora cavernosa from mice or rats. J Am Soc Hypertens 2008;2(6): 439-447. Published by Elsevier Inc. on behalf of the American Society of Hypertension.

P2X(7) receptor activation amplifies lipopolysaccharide-induced vascular hyporeactivity via interleukin-1 beta release

Lipopolysaccharide (LPS) stimulates cytoplasmic accumulation of pro-interleukin (IL)-1 beta. Activation of P2X(7) receptors stimulates conversion of pro-IL-1 beta into mature IL-1 beta, which is then secreted. Because both LPS (in vivo) and IL-1 beta (in vitro) decrease vascular reactivity to contractile agents, we hypothesized the following: 1) P2X(7) receptor activation contributes to LPS-induced vascular hyporeactivity, and 2) IL-1 beta mediates this change. Thoracic aortas were obtained from 12-week-old male C57BL/6 mice. The aortic rings were incubated for 24 h in Dulbecco`s modified Eagle`s medium, LPS, benzoylbenzoyl-ATP (BzATP; P2X(7) receptor agonist), LPS plus BzATP, oxidized ATP (oATP; P2X(7) receptor antagonist), or oATP plus LPS plus BzATP. After the treatment, the rings were either mounted in a myograph for evaluation of contractile activity or homogenized for IL-1 beta and inducible nitric-oxide synthase (iNOS) protein measurement. In endothelium-intact aortic rings, phenylephrine (PE)-induced contractions were not altered by incubation with LPS or BzATP, but they significantly decreased in aortic rings incubated with LPS plus BzATP. Treatment with oATP or IL-1ra (IL-1 beta receptor antagonist) reversed LPS plus BzATP-induced hyporeactivity to PE. In the presence of N(G)-nitro-L-arginine methyl ester or N-([3-(aminomethyl) phenyl] methyl) ethanimidamide (selective iNOS inhibitor), the vascular hyporeactivity induced by LPS plus BzATP on PE responses was not observed. BzATP augmented LPS-induced IL-1 beta release and iNOS protein expression, and these effects were also inhibited by oATP. Moreover, incubation of endothelium-intact aortic rings with IL-1 beta induced iNOS protein expression. Thus, activation of P2X 7 receptor amplifies LPS-induced hyporeactivity in mouse endothelium-intact aorta, which is associated with IL-1 beta-mediated release of nitric oxide by iNOS.

Mechanisms underlying the biphasic effect of vitamin K-1 (phylloquinone) on arterial blood pressure

Phylloquinone (vitamin K-1, VK1) is widely used therapeutically and intravenous administration of this quinone can induce hypotension. We aimed to investigate the mechanisms underlying the effects induced by VK1 on arterial blood pressure. With this purpose a catheter was inserted into the abdominal aorta of male Wistar rats for blood pressure and heart rate recording. Bolus intravenous injection of VK1 (0.5-20 mg kg(-1)) produced a transient increase in blood pressure followed by a fall. Both the pressor and depressor response induced by VK1 were dose-dependent. On the other hand, intravenous injection of VK1 did not alter heart rate. The nitric oxide synthase (NOS) inhibitor N-G-nitro-L-arginine methyl ester (L-NAME, 10 and 20 mg kg(-1)) reduced both the increase and decrease in blood pressure induced by VK1 (5 mgkg(-1)). On the other hand, indometacin (10 mg kg(-1)), a non-selective cyclooxygenase inhibitor, did not alter the increase in mean arterial pressure (MAP) induced by VK1. However, VK1-induced fall in MAP was significantly attenuated by indometacin. We concluded that VK1 induces a dose-dependent effect on blood pressure that consists of an acute increase followed by a more sustained decrease in MAP. The hypotension induced by VK1 involves the activation of the nitric oxide (NO) pathway and the release of vasodilator prostanoid(s).

Nitric oxide inhibition in paraventricular nucleus on cardiovascular and autonomic modulation after exercise training in unanesthetized rats

It is well known that regular physical exercise alter cardiac function and autonomic modulation of heart rate variability (HRV). The paraventricular nucleus of hypothalamus (PVN) is an important site of integration for autonomic and cardiovascular responses, where nitric oxide (NO) plays an important role. The aim of our study was to evaluate the cardiovascular parameters and autonomic modulation by means of spectral analysis after nitric oxide synthase (NOS) inhibition in the PVN in conscious sedentary (S) or swimming trained (ST) rats. After swimming training protocol, adult male Wistar rats, instrumented with guide cannulas to PVN and femoral artery and vein catheters were submitted to mean arterial pressure (MAP) and heart rate (HR) recording. At baseline, the physical training induced a resting bradycardia (S: 374 +/- 5, ST: 346 +/- 1 bpm) and promoted adaptations in HRV characterized by an increase in high-frequency oscillations (HF; 26.43 +/- 6.91 to 88.96 +/- 244) and a decrease in low-frequency oscillations (LF; 73.57 +/- 6.91 to 11.04 +/- 2.44) in normalized units. The microinjection of N(omega)-nitro-L-arginine methyl ester (L-NAME) in the PVN of sedentary and trained rats promoted increase in MAP and HR. L-NAME in the PVN did not significantly alter the spectral parameters of HRV of sedentary animals, however in the trained rats increased LF oscillations (11.04 +/- 2.44 to 27.62 +/- 6.97) and decreased HF oscillations (88.96 +/- 2.44 to 72.38 +/- 6.97) in normalized units compared with baseline. Our results suggest that NO in the PVN may collaborate to cardiac autonomic modulation after exercise training. (c) 2010 Elsevier B.V. All rights reserved.