Inhibitory effects of 7-epiclusianone on glucan synthesis, acidogenicity and biofilm formation by Streptococcus mutans

The aim of this study was to examine the effects of 7-epiclusianone, a new prenylated benzophenone isolated from the plant Rheedia gardneriana, on some of the virulence properties of Streptococcus mutans associated with biofilm development and acidogenicity. The synthesis of glucans by glucosyltransferases B (GTF B) and C (GTF C) was markedly reduced by 7-epiclusianone showing more than 80% inhibition of enzymatic activity at a concentration of 100 microg mL(-1). Double-reciprocal analysis (Lineweaver-Burk plots) revealed that the inhibition of GTF B activity was noncompetitive (mixed) while GTF C was inhibited uncompetitively. The glycolytic pH drop by S. mutans cells was also disrupted by 7-epiclusianone without affecting the bacterial viability, an effect that can be attributed, in part, to inhibition of F-ATPase activity (61.1+/-3.0% inhibition at 100 microg mL(-1)). Furthermore, topical applications (1-min exposure, twice daily) of 7-epiclusianone (at 250 microg mL(-1)) disrupted biofilm formation and physiology. The biomass (dry-weight), extracellular insoluble polysaccharide concentration and acidogenicity of the biofilms were significantly reduced by the test agent (P<0.05). The data show that 7-epiclusianone disrupts the extracellular and intracellular sugar metabolism of S. mutans, and holds promise as a novel, naturally occurring compound to prevent biofilm-related oral diseases.     

Vascular pro-oxidant effects secondary to the autoxidation of gallic acid in rat aorta

Gallic acid autoxidation was monitored by absorption spectroscopy and H₂O₂ production; vascular effects related to the autoxidation process were studied on intact and rubbed aortic rings from WKY rats. Gallic acid autoxidation in an oxygenated physiological salt solution (37°C, pH=7.4) mostly occurred in a 2-h time period. Superoxide anions, H₂O₂ and gallic acid quinones were produced during gallic acid autoxidation. In rings partially precontracted with phenylephrine, 0.1–3 μM gallic acid induced marked and largely endothelium-dependent contractions, 10–30 μM gallic acid induced endothelium-independent contractions and 0.1–0.3 mM gallic acid induced complete, fast-developing, endothelium-independent relaxations. Superoxide dismutase (SOD) shifted the endothelium-dependent gallic acid contractions to the right, and N^G-nitro-l-arginine abolished them. Indomethacin suppressed the endothelium-independent gallic acid contractions, and catalase abolished the endothelium-independent contractions and relaxations. Gallic acid (30 μM) inhibited the relaxant effects of acetylcholine and sodium nitroprusside. In rings maximally precontracted with KCl, 0.1–100 μM gallic acid did not modify the tone, whereas 0.3 mM induced complete, slow-developing, endothelium-independent relaxations. Moreover, 0.3 mM gallic acid induced an irreversible impairment of ring reactivity and the release of lactate dehydrogenase. Catalase and N-acetyl cysteine suppressed the deleterious effects induced by gallic acid in the rings. In conclusion: (a) gallic acid is rapidly and nonenzymatically oxidized in physiological solutions, generating superoxide anions, H₂O₂ and quinones; (b) superoxide anions (by destroying NO) and low H₂O₂ levels (by activating cyclooxygenase) both increase vascular tone; (c) moderate H₂O₂ levels decrease vascular tone; (d) high H₂O₂ and quinone levels cause irreversible relaxations due to cellular damage.     

Vascular effects of caffeic acid phenethyl ester (CAPE) on isolated rat thoracic aorta

This study was aimed to investigate the vascular activity of caffeic acid phenethylester (CAPE), one of the major components of honeybee propolis. Experiments were performed on rat thoracic aortic rings, mounted in an isolated organ bath and connected to an isometric force transducer. The effect of CAPE (0.1-300 microM) was evaluated on tissue pre-contracted with phenylephrine (PE, 1 microM) or with KCl (100 mM). In another set of experiments, tissue was incubated with CAPE (1-100 microM) and responses to PE (0.01-3 microM) or KCl (60 mM) were evaluated. The effect of CAPE on cytosolic Ca(2+) concentration in aortic smooth muscle cells stimulated with PE or KCl was also evaluated. CAPE (0.1-300 microM) caused a concentration-dependent relaxation (pEC(50) 4.99 +/- 0.19; Emax 100.75 +/- 1.65%; n = 4) of tissue pre-contracted with PE that was reduced by endothelium removal or by incubation with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM). CAPE also relaxed KCl-precontracted tissue (pEC(50) 4.40 +/- 0.08; n = 4). CAPE inhibited contractile responses to PE or to KCl, and also inhibited the contractile response to PE obtained in a Ca(2+)-free medium. In addition, CAPE inhibited the increase in cytosolic Ca(2+) concentration triggered by stimulation of aortic smooth muscle cells with PE or KCl. Our results demonstrate a vascular activity for CAPE, that is only partially dependent on nitric oxide. Indeed, at high concentrations, CAPE vasorelaxant effect occurs also in absence of endothelium and it is likely due to an inhibitory effect on calcium movements through cell membranes.     

Vascular renin-like activity in aorta and mesenteric artery of the rat

Vascular renin-like enzymatic activity (VRLA) has been measured in the artery wall of control and experimental rats. The following groups have been studied: 1-normal salt diet; 2-high salt diet; 3-low salt diet; 4-bilateral nephrectomy (Nx); 5-sham operated for Nx. VRLA was evaluated in the aorta (ARLA) and in the mesenteric arteries (MRLA). Blood samples were obtained for plasma renin activity (PRA) determination. High salt diet decreased PRA, ARLA and MRLA whereas low salt diet increased PRA, did not change ARLA and decreased MRLA. PRA was almost undetectable in Nx animals while ARLA showed a 40% reduction and MRLA was unchanged in these animals. These results would indicate that the changes in PRA induced different variations in the renin-like content of the aorta and the mesenteric artery. The differences could be mainly due to two factors: 1) the capacity of the vascular tissue to bind circulating renin and 2) the capacity of each tissue to synthetize renin-like material in situ.     

Characterization of the sequence and expression of a Ykt6 prenylated SNARE from rat

The Ykt6 protein represents a novel soluble N-ethylmaleimide-sensitive fusion protein receptor (SNARE), as it is the only one known without a hydrophobic transmembrane region at the carboxy terminus. For this SNARE, however, membrane interaction is thought to be mediated through a cysteine/aliphatic/aliphatic/methionine or histidine (CAAX) C-terminal motif, a consensus sequence involved in prenylated membrane anchoring. To date, two full-length Ykt6 cDNAs have been reported, these being in yeast and human, with a further protein predicted from a Caenorhabditis elegans cosmid. Using a mouse EST clone identified as having 65% homology with the human Ykt6, we isolated a cDNA clone encoding the rat Ykt6 homolog (rYkt6). Sequence analysis of rYkt6 demonstrated that a high level of species conservation exists between the rat and human prenylated SNAREs, as both the nucleotide and amino acid sequences share >90% homology. Mammalian Ykt6 is shown here for the first time to be constitutively expressed in a variety of tissues. The species conservation and ubiquitous expression of prenylated SNAREs hence may be indicative of an important and central role for these proteins in cellular protein trafficking.     

The toxic effects of bis (tributyltin) oxide on the rat thoracic aorta.

The toxic effects of bis (tributyltin) oxide (TBTO) on the ultrastructure and permeability of rat thoracic aorta were studied electron microscopically and the accumulation sites of tin were determined with an X-ray microanalyzer. Male Wistar rats received 0.05ml/kg of TBTO as an emulsion in 1 ml of distilled water through a stomach tube. After time intervals of 2, 4, 6, 8, 10, 12 h after intubation, thoracic aortae were isolated and prepared for electron microscopy. Marked swelling of mitochondria in the aortic endothelial cells appeared at 4 h after TBTO treatment. By x-ray microanalysis, tin L-alpha peaks (3.44 keV) were obtained from these swollen mitochondria. Subendothelial edema progressed between 6 and 8 h after TBTO treatment. By tracer experiment, it was seen that large amounts of peroxidase reaction products filled the expanded subendothelial space. At 12 h after TBTO treatment, degenerative changes of the endothelial cells were prominent. These results indicated that orally administered TBTO accumulated in the mitochondria of the endothelial cells of thoracic aorta. The direct toxic effects of TBTO on mitochondria might induce severe damage to the endothelial cells and cause disturbance of the permeability barrier function of the endothelial layer and subendothelial edema.     

Permeability of the post-ischemic rat aorta.

The time course of the vascular permeability disorder following upon acute hypoxia and the fate of plasma substances entering the vessel wall in consequence of increased permeability were studied on the rat abdominal aorta rendered hypoxic for one hour by double ligature and recirculated for periods ranging from one hour to 30 days. The distribution and quantity of mural plasma inhibition were determined histochemically by means of a colloidal iron tracer and Prussian blue reaction and by photometric analysis, respectively. Plasma imbibition reached its maximum after recirculation for 24 to 48 hours and fell to an almost normal level after 10 days. Administration of the colloidal iron tracer on the second day of recirculation, when the permeability disorder was at its peak, showed plasma imbibition in every layer of the vessel wall. At seven days it was restricted to the outer third of the media and the adventitia. The endothelium is acting as the main barrier to mural plasma imbibition and in the case of a permeability disorder only the elastic lamellae constitute a temporary mechanical obstacle to the ingression of plasma.     

Effects of isoproterenol treatment for 7 days on inflammatory mediators in the rat aorta

The aim of the present study was to evaluate the effect of overstimulation of beta-adrenoceptors on vascular inflammatory mediators. Wistar rats were treated with the beta-adrenoceptor agonist isoproterenol (0.3 mg.kg(-1).day(-1) sc) or vehicle (control) for 7 days. At the end of treatment, the right carotid artery was catheterized for arterial and left ventricular (LV) hemodynamic evaluation. Isoproterenol treatment increased LV weight but did not change hemodynamic parameters. Aortic mRNA and protein expression were quantified by real-time RT-PCR and Western blot analysis, respectively. Isoproterenol enhanced aortic mRNA and protein expression of IL-1beta (124% and 125%) and IL-6 (231% and 40%) compared with controls but did not change TNF-alpha expression. The nuclear-to-cytoplasmatic protein expression ration of the NF-kappaB p65 subunit was increased by isoproterenol treatment (51%); in addition, it reduced the cytoplasmatic expression of IkappaB-alpha (52%) in aortas. An electrophoretic mobility shift assay was performed using the aorta, and increased NF-kappaB DNA binding (31%) was observed in isoproterenol-treated rats compared with controls (P < 0.05). Isoproterenol treatment increased phenylephrine-induced contraction in aortic rigs (P < 0.05), which was significantly reduced by superoxide dismutase (150 U/ml) and sodium salicylate (5 mM). Cotreatment with thalidomide (150 mg.kg(-1).day(-1) for 7 days) also reduced hyperreactivity to phenylephrine induced by isoproterenol. In conclusion, overstimulation of beta-adrenoceptors increased proinflammatory cytokines and upregulated NF-kappaB in the rat aorta. Moreover, local oxidative stress and the proinflammatory state seem to play key roles in the altered vascular reactivity of the rat aorta induced by chronic beta-adrenergic stimulation.     

Relaxant effects of selected sildenafil analogues in the rat aorta

A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine with chiral amino group has been synthesized and characterized. The compounds were tested for their relaxant effects in the rat aorta. Evaluation of prepared derivatives demonstrated that compound (8a) is probably a non-selective phosphodiesterase (PDE) inhibitor, as it induced aortic relaxation through endothelium-independent mechanism.     

Cyclolobatriene, a novel prenylated germacrene diterpene, from the soft coral Lobophytum pauciflorum

A new 10-membered-ring diterpene, cyclolobatriene (1), along with three other known diterpenes, lobatriene (2), eunicol (3), and fuscol (4), were isolated from the Okinawan soft coral Lobophytum pauciflorum. Their structures were established by extensive NMR spectroscopic analyses. Cyclolobatriene (1) is an additional example of rare prenylated germacrenes. Although 1, due to a 10-membered-ring structure, exists as an equilibrium mixture of three conformers, the NMR measurement in CDCl(3) at 7°C enabled us to assign the NMR signals of the three, which is the first example of the complete NMR assignment of all the existing conformers of germacrene-type compounds. Cyclolobatriene (1) was thermally unstable and converted into 2 through Cope rearrangement upon heating at 70°C. Eunicol (3) also possesses the same prenylated germacrene structure as 1, showing similar physico-chemical properties to 1. All four compounds 1-4 showed cytotoxic effect with IC(50)'s of 0.64, 0.41, 0.35 and 0.52 μM, respectively, against human epidermoid carcinoma A431 cells.     

Vascular smooth muscle cell hypertrophy during maturation in rat thoracic aorta. Volumetric and morphometric studies

Rat thoracic smooth muscle cell volume increase was measured in normal Wistar rats at 3, 7, 12 and 18 weeks of age either by direct volumetric analysis of vascular smooth muscle single cell suspensions or by morphometric analysis of intact media layers. Results obtained by either method were similar and convergent: cell hypertrophy amounted to 100 % between 3 and 18 weeks of age, the largest part of this increase took place between 3 and 7 weeks of age.     

Isolation, characterisation, growth and culture of endothelial cells from the rat aorta

Rat aortic endothelial cells have been isolated by the explantation technique and grown in culture. They have been identified morphologically using standard staining techniques, biochemically by identification of angiotensin convertase and have been positively stained for Factor VIII-related antigen by immunofluorescence using both anti-human and anti-rat Factor VIII antibodies. The explantation technique is a successful alternative to enzyme digestion which is not applicable to rat aortic endothelial cells because of the nature of their attachment to the subendothelial layer.     

Opposing effects of ascorbate on collagen and elastin deposition in the neonatal rat aorta

Ascorbic acid plays an important role in connective tissue metabolism, where, among other effects, it acts as a reducing factor in the reactions catalyzed by prolyl and lysyl hydroxylases. In vitro, ascorbic acid has been shown to have a positive influence on collagen synthesis at pre- and/or post-translational levels and a negative effect on elastin production. In the present work, the effects of vitamin C on extracellular matrix deposition have been studied in vivo. Stereological analysis on electron micrographs showed, compared to age-matched controls, a 50 to 60% increase of collagen deposition in the media and in the adventitia of the aorta of rats treated for 30 days from the 18th day of life with 10% ascorbate in their drinking water. By contrast, elastin volume density was significantly reduced by the treatment at all ages examined. These morphological data were supported by in situ hybridization observations showing enhanced collagen type I mRNA and reduced elastin mRNA expression upon treatment. Although vitamin C did not inhibit lysyl oxidase activity in vivo, being only slightly higher than in controls, enzyme activity was significantly reduced, when high doses of ascorbate were added in vitro. Lysyl oxidase activity may be a function of enhanced collagen metabolism rather than a direct effect of the vitamin on the enzyme activity. These data indicate that ascorbate exerts opposite effects on the deposition of two major components of the extracellular matrix in vivo, at least during periods of rapid growth.     

Studies on the stereoselective effects of a novel 5-HT2 receptor antagonist on contractile responses of rat aorta

The effect of the enantiomers of a novel 5-HT2 receptor antagonist, (+/-)-(1R,3S)-1-[2-[4-[3-(p-fluorophenyl)-1-indanyl]-piperazinyl] ethyl]-2-imidazolidinone, was studied on serotonin (5-HT), noradrenaline (NA), potassium (K+), and calcium (Ca2+)-induced contractions in isolated rat thoracic aorta. The enantiomers shifted the 5-HT, NA, K+, and Ca2+ concentration-response curves to the right in a concentration-dependent manner and depressed the maximal contractile responses. The (+)-enantiomer was a far more potent inhibitor of 5-HT-induced contractions than the (-)-enantiomer. The (+)-enantiomer and phentolamine, both at 10(-6) M, had equal inhibitory effects on NA-evoked contractions. The (+)-enantiomer was again more potent inhibiting NA-induced contractions than the (-)-enantiomer. Both enantiomers had an equieffective inhibitory effect on K+ and Ca2(+)-induced contractions. The results show that the 5-HT and alpha-adrenoceptor antagonism of the two enantiomers is stereoselective, the (+)-enantiomer being more potent than the (-)-enantiomer. In contrast the enantiomers had equal, nonstereoselective inhibitory effects on K+ and Ca2(+)-evoked contractions.     

Effects of hirsutine, an antihypertensive indole alkaloid from Uncaria rhynchophylla, on intracellular calcium in rat thoracic aorta.

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla (MIQ.) Jackson, on cytosolic Ca2+ level ([Ca2+]cyt) were studied by using fura-2-Ca2+ fluorescence in smooth muscle of the isolated rat aorta. Noradrenaline and high K+ solution produced a sustained increase in [Ca2+]cyt. Application of hirsutine after the increases in [Ca2+]cyt induced by noradrenaline and high K+ notably decreased [Ca2+]cyt, suggesting that hirsutine inhibits Ca2+ influx mainly through a voltage-dependent Ca2+ channel. Furthermore, the effect of hirsutine on intracellular Ca2+ store was studied by using contractile responses to caffeine under the Ca(2+)-free nutrient condition in the rat aorta. When hirsutine was added at 30 microM before caffeine treatment, the agent slightly but significantly reduced the caffeine-induced contraction. When added during Ca2+ loading, hirsutine definitely augmented the contractile response to caffeine. These results suggest that hirsutine inhibits Ca2+ release from the Ca2+ store and increases Ca2+ uptake into the Ca2+ store, leading to a reduction of intracellular Ca2+ level. It is concluded that hirsutine reduces intracellular Ca2+ level through its effect on the Ca2+ store as well as through its effect on the voltage-dependent Ca2+ channel.     

Evaluation of the antioxidant and cytotoxic activity of arzanol, a prenylated alpha-pyrone-phloroglucinol etherodimer from Helichrysum italicum subsp.microphyllum

Various phenolics and (mero)terpenoids from Helichrysum italicum subsp. microphyllum, a plant endemic to Sardinia, were investigated for their capacity to inhibit non-enzymatic lipid peroxidation. These compounds were studied in simple in vitro systems, under conditions of autoxidation and of iron (EDTA)-mediated oxidation of linoleic acid at 37 degrees C. Arzanol, a pyrone-phloroglucinol etherodimer, and helipyrone, a dimeric pyrone, showed antioxidant activity, and could protect linoleic acid against free radical attack in assays of autoxidation and EDTA-mediated oxidation. Methylarzanol, as well as the sesquiterpene alcohol rosifoliol, showed a decreased, but still significant, protective effect against linoleic acid oxidation. Arzanol and helipyrone were also tested in an assay of thermal (140 degrees C) autoxidation of cholesterol, where arzanol showed significant antioxidant activity. The cytotoxicity of arzanol was further evaluated in VERO cells, a line of fibroblasts derived from monkey kidney. Arzanol, at non-cytotoxic concentrations, showed a strong inhibition of TBH-induced oxidative stress in VERO cells. The results of the present work suggest that the natural compound arzanol exerts useful antioxidant properties in different in vitro systems of lipid peroxidation.     

Reactivity of the aorta and mesenteric resistance arteries from the obese spontaneously hypertensive rat: effects of glitazones

The obese spontaneously hypertensive rat (SHROB) is a model of metabolic syndrome in which, to our knowledge, vascular function has never been studied. The actions of insulin sensitizers (glitazones) on vascular function have not been analyzed either. Our purpose was to characterize microvascular and macrovascular responses of the SHROB and to study the effects of glitazones on these responses. The reactivity of mesenteric resistance arteries (MRAs) and the aorta from SHROBs and control rats to cumulative concentrations of phenylephrine, ACh, and sodium nitroprusside (SNP) was myographically analyzed. Some animals were orally treated with rosiglitazone (3 mg·kg(-1)·day(-1), 3 wk), and myography was performed. Phenylephrine, ACh, and SNP dose-response curves were impaired to different extents in arteries of SHROBs. Incubation with N-nitro-L-arginine methyl ester caused little effects on phenylephrine and ACh curves in MRAs but enhanced phenylephrine contractions and abolished ACh-induced relaxations of aortae. Incubation with indomethacin reduced phenylephrine reactivity and improved ACh-induced relaxations of all vessels studied. NS-398 and tempol increased relaxations to ACh of MRAs. Incubation with pioglitazone or rosiglitazone (both 10(-5) M) or oral treatment with rosiglitazone improved, to different extents, ACh and SNP curves in all vessels. Glitazone incubation diminished aortic ACh sensitivity. The release of thromboxane A(2) and PGI(2) metabolites (thromboxane B(2) and 6-keto-PGF(1α)) was analyzed. ACh increased the MRA release of thromboxane B(2) from SHROBs but not control rats, and the former was prevented by rosiglitazone coincubation. In contrast, in aortae, ACh failed to alter the release of metabolites, and rosiglitazone treatment increased that of 6-keto-PGF(1α). Thus, SHROBs displayed microvascular and macrovascular dysfunction. MRAs, but not aortae, of SHROBs revealed an impaired endothelial nitric oxide pathway, whereas both, but especially MRAs, displayed an impaired cyclooxygenase pathway. Glitazones elicited beneficial effects on macrovascular and, especially, microvascular function of SHROBs.     

Endothelium-dependent relaxation by cilostazol, a phosphodiesteras III inhibitor, on rat thoracic aorta

The relaxation effect of cilostazol, a phosphodiesterase III inhibitor, on the thoracic aorta was investigated. Cilostazol induced the relaxation of the thoracic aorta precontracted by phenylephrine in a concentration-dependent manner. The concentration-dependent relaxation was shifted to the right in the endothelium denuded aorta compared with that of intact endothelium, suggesting that this relaxation was partly dependent on endothelium. Cilostazol-induced relaxation of thoracic aorta tone was reversed by treatment with N(G)-nitro L-arginine (L-NNA), a competitive inhibitor of nitric oxide (NO) synthase. Cilostazol also significantly increased the NO level in the porcine thoracic aorta. In rats treated with cilostazol, the urinary excretion of nitrites, a stable metabolite of NO, and basal production of NO of the aortic ring were significantly greater than in those without treatment. These findings indicate that cilostazol-induced vasodilation of the rat thoracic aorta was dependent on the endothelium, which released NO from aortic endothelial cells.     

Atrovirisidone B, a new prenylated depsidone with cytotoxic property from the roots of Garcinia atroviridis

A new prenylated depsidone, atrovirisidone B (2), together with naringenin (3) and 3,8"-binaringenin (4) were isolated from the roots of Garcinia atroviridis. Their structures were determined on the basis of spectral data interpretation. Compound 2 showed cytotoxic activity against human breast (MCF-7), human prostate (DU-145) and human lung (H-460) cancer cells.