Chemopreventive effects of beta-ionone and geraniol during rat hepatocarcinogenesis promotion: distinct actions on cell proliferation, apoptosis, HMGCoA reductase, and RhoA

Chemopreventive activities of the dietary isoprenoids beta-ionone (beta I) and geraniol (GOH) were evaluated during the promotion phase of hepatocarcinogenesis. Over 5 consecutive weeks, rats received daily 16 mg/100 g body weight (b.w.) of beta I (beta I group), 25 mg/100 g b.w. of GOH (GOH group), or only corn oil (CO group, controls). Compared to the CO group, the following was observed: only the beta I group showed a decrease in the mean number of visible hepatocyte nodules (P<.05); beta I and GOH groups had reduced mean number of persistent preneoplastic lesions (pPNLs) (P<.05), but no differences regarding number of remodeling PNL (rPNLs) were observed; only the beta I group exhibited smaller rPNL size and percentage of liver sections occupied by pPNLs (P<.05), whereas the GOH group displayed a smaller percentage of liver sections occupied by rPNLs (P<.05); a trend was observed in the beta I group, which showed reduced cell proliferation of pPNLs (P<.10), and the GOH group had increased apoptosis in pPNLs and rPNLs (P<.05); only the beta I group displayed reduced total plasma cholesterol concentrations (P<.05) and increased hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase mRNA levels (P<.05): only the GOH group had lower hepatic membrane RhoA protein levels (P<.05); both the beta I- and GOH-treated groups had higher hepatic concentrations of beta I and GOH, respectively (P<.05). Given these data, beta I and GOH show promising chemopreventive effects during promotion of hepatocarcinogenesis by acting through distinct mechanism of actions: beta I may inhibit cell proliferation and modulate HMGCoA reductase, and GOH can induce apoptosis and inhibit RhoA activation. (C) 2011 Elsevier Inc. All rights reserved.

Use of virtual screening, flexible docking, and molecular interaction fields to design novel HMG-CoA reductase inhibitors for the treatment of hypercholesterolemia

Dietary changes associated with drug therapy can reduce high serum cholesterol levels and dramatically decrease the risk of coronary artery disease, stroke, and overall mortality. Statins are hypolipemic drugs that are effective in the reduction of cholesterol serum levels, attenuating cholesterol synthesis in liver by competitive inhibition regarding the substrate or molecular target HMG-CoA reductase. We have herewith used computer-aided molecular design tools, i.e., flexible docking, virtual screening in large data bases, molecular interaction fields to propose novel potential HMG-CoA reductase inhibitors that are promising for the treatment of hypercholesterolemia.

Effects of ascorbic acid supplementation in ileum myenteric neurons of streptozotocin-induced diabetic rats

The exacerbation of the oxidative stress and of the polyol pathway which impair damage myenteric plexus are metabolic characteristics of diabetes. The ascorbic acid (AA) is an antioxidant and an aldose reductase inhibitor, which may act as neuroprotector. The effects of AA supplementation on the density and cellular body profile area (CP) of myenteric neurons in STZ-induced diabetes in rats were assessed. Four groups with five animals each were formed: normoglycemic (C); diabetic (D); AA-treated diabetic (DS) and AA-treated normoglycemic (CS). Dosagen of 50mg of AA were given, three times a week, for each animal (group DS and CS). Ninety days later and after euthanasia, the ileum was collected and processed for the NADPH-diaphorase technique. There were no differences (P>0.05) in the neuronal density among the groups. The CP area was lower (P<0.05) in the DS and CS groups, with a higher incidence of neurons with a CP area exceeding 200µm² for groups C and D. The AA had no influence on the neuronal density in the ileum but had a neuroprotective effect, preventing the increase in the CP area and allowing a higher number of neurons with a CP area with less than 200µm².

Diversity and identification of methanogenic archaea and sulphate-reducing bacteria in sediments from a pristine tropical mangrove

Mangrove sediments are anaerobic ecosystems rich in organic matter. This environment is optimal for anaerobic microorganisms, such as sulphate-reducing bacteria and methanogenic archaea, which are responsible for nutrient cycling. In this study, the diversity of these two functional guilds was evaluated in a pristine mangrove forest using denaturing gradient gel electrophoresis (DGGE) and clone library sequencing in a 50 cm vertical profile sampled every 5.0 cm. DGGE profiles indicated that both groups presented higher richness in shallow samples (0-30 cm) with a steep decrease in richness beyond that depth. According to redundancy analysis, this alteration significantly correlated with a decrease in the amount of organic matter. Clone library sequencing indicated that depth had a strong effect on the selection of dissimilatory sulphate reductase (dsrB) operational taxonomic units (OTUs), as indicated by the small number of shared OTUs found in shallow (0.0 cm) and deep (40.0 cm) libraries. On the other hand, methyl coenzyme-M reductase (mcrA) libraries indicated that most of the OTUs found in the shallow library were present in the deep library. These results show that these two guilds co-exist in these mangrove sediments and indicate important roles for these organisms in nutrient cycling within this ecosystem.

Increased class A scavenger receptor and glomerular lipid precede mesangial matrix expansion in the bGH mouse model

Objective: Elevated neutral lipid content and mRNA expression of class A scavenger receptor (SRA) have been found in the renal cortex of the bovine growth hormone (bGH) mouse model of progressive glomerulosclerosis (GS). We hypothesize that the increased expression of SRA precedes glomerular scarring in this model. Design: Real time RT-PCR and immunofluorescence were employed to measure SRA and collagen types I and IV in the bGH transgenic and control mice at 5 and 12 weeks (wk) of age to determine the chronology of change in SRA expression in relation to glomerular scarring. Alternative mechanisms for increasing glomerular lipid were assessed by measuring mRNA expression levels of low-density lipoprotein receptor (LDL-r), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), and ATP-binding cassette transporter A1 (ABCA1). In addition, the involvement of macrophages in early GS was assessed by CD68 mRNA expression in kidney cortex. Results: Both mRNA and protein levels of SRA were significantly increased in 5-wk bGH compared with control mice, whereas the expression of collagen I and IV was unaltered. Unchanged levels of LDL-r and HMGR mRNA indicate that neither regulated cholesterol uptake via LDL-r nor the cholesterol synthetic pathway played a role in the early lipid increase. The finding of increased ABCA1 expression was an indicator of excess intracellular lipid in the renal cortex of bGH mice at 5 wk. CD68 expression in bGH did not differ significantly from that of controls at 5 wk suggesting that cortical macrophage infiltration was not increased in bGH mice at this time point. Conclusion: An early increase in SRA mRNA and protein expression in the bGH kidney precedes glomerular scarring and is independent of macrophage influx. Published by Elsevier Ltd. on behalf of Growth Hormone Research Society.

Anti-inflammatory effect of atorvastatin ameliorates insulin resistance in monosodium glutamate-treated obese mice

Considering that inflammation contributes to obesity-induced insulin resistance and that statins have been reported to have other effects beyond cholesterol lowering, the present study aimed to it whether atorvastatin treatment has anti-inflammatory action in white adipose tissue of obese mice, consequently improving insulin sensitivity. Insulin sensitivity in vivo (by insulin tolerance test); metabolic-hormonal profile; plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and adiponectin; adipose tissue immunohistochemistry; glucose transporter (GLUT) 4; adiponectin; INF-alpha; IL-1 beta; and IL-6 gene expression; and I kappa B kinase (IKK)-alpha/beta activity were assessed in 23-week-old monosodium glutamate induced obese mice untreated or treated with atorvastatin for 4 weeks. Insulin-resistant obese mice had increased plasma triglyceride, insulin, TNF-alpha, and IL-6 plasma levels. Adipose tissue of obese animals showed increased macrophage infiltration, IKK-alpha (42%, P < .05) and IKK-beta (73%, P < .05) phosphorylation, and INF-alpha and IL-6 messenger RNA (mRNA) (similar to 15%, P < .05) levels, and decreased GLUT4 mRNA and protein (30%, P < .05) levels. Atorvastatin treatment lowered cholesterol, triglyceride, insulin, INF-alpha, and IL-6 plasma levels, and restored whole-body insulin sensitivity. In adipose tissue, atorvastatin decreased macrophage in and normalized IKK-alpha/beta phosphorylation; INF-alpha, IL-6, and GLUT4 mRNA; and GLUT4 protein to control levels. The present findings demonstrate that atorvastatin has anti-inflammatory effects on adipose tissue of obese mice, which may be important to its local and whole-body insulin-sensitization effects. (C) 2010 Published by Elsevier Inc.

Docking and molecular dynamics simulation of quinone compounds with trypanocidal activity

In this work, two different docking programs were used, AutoDock and FlexX, which use different types of scoring functions and searching methods. The docking poses of all quinone compounds studied stayed in the same region in the trypanothione reductase. This region is a hydrophobic pocket near to Phe396, Pro398 and Leu399 amino acid residues. The compounds studied displays a higher affinity in trypanothione reductase (TR) than glutathione reductase (GR), since only two out of 28 quinone compounds presented more favorable docking energy in the site of human enzyme. The interaction of quinone compounds with the TR enzyme is in agreement with other studies, which showed different binding sites from the ones formed by cysteines 52 and 58. To verify the results obtained by docking, we carried out a molecular dynamics simulation with the compounds that presented the highest and lowest docking energies. The results showed that the root mean square deviation (RMSD) between the initial and final pose were very small. In addition, the hydrogen bond pattern was conserved along the simulation. In the parasite enzyme, the amino acid residues Leu399, Met400 and Lys402 are replaced in the human enzyme by Met406, Tyr407 and Ala409, respectively. In view of the fact that Leu399 is an amino acid of the Z site, this difference could be explored to design selective inhibitors of TR.

Rapid and slow modulation of nitrate reductase activity in the red macroalga Gracilaria chilensis (Gracilariales, Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.

Short-Term effect of COX-2 selective inhibitor as an adjunct for the treatment of periodontal disease: a clinical double-blind study in humans

Adjunctive therapeutic strategies that modulate the inflammatory mediators can play a significant role in periodontal therapy. In this double-blind, placebo-controlled study, 60 subjects diagnosed as periodontitis patients were evaluated for 28 days after periodontal treatment combined with selective cyclooxygenase-2 (COX-2) inhibitor. The experimental group received scaling and root planning (SRP) combined with the Loxoprofen antiinflammatory drug (SRP+Loxoprofen). The control group received SRP combined with placebo (SRP+placebo). Plaque index (PI), probing pocket depth (PD) and bleeding on probing (BOP) were monitored with an electronic probe at baseline and after 14 and 28 days. Both groups displayed clinical improvement in PD, PI and BOP. They also showed statistically similar values (p>0.05) of PD reduction on day 14 (0.4 mm) and on day 28 (0.6 mm). At the baseline, few deeper sites (>7 mm) from SRP+Loxoprofen group were responsible and most PD reduction was observed after 14 days (p<0.05). The percentage of remaining deep pockets (>7 mm) after 14 days in the SRP+Loxoprofen group was significantly lower (p<0.05) than in the SRP+placebo group. Loxoprofen presents potential effect as an adjunct of periodontal disease treatment, but long-term clinical trials are necessary to confirm its efficacy.

Effect of nitric oxide inhibitor and donor substances on the infammatory process caused by endodontic irrigants

Nitric oxide (NO) has been considered a key molecule in infammation. OBJECTIVE: The aim of this study was to evaluate the effect of treatment with L-NAME and sodium nitroprussiate, substances that inhibit and release NO, respectively, on tissue tolerance to endodontic irrigants. MATERIAL AND METHODS: The vital dye exudation method was used in a rat subcutaneous tissue model. Injections of 2% Evans blue were administered intravenously into the dorsal penial vein of 14 male rats (200-300 g). The NO inhibitor and donor substances were injected into the subcutaneous tissue in the dorsal region, forming two groups of animals: G1 was inoculated with L-NAME and G2 with sodium nitroprussiate. Both groups received injections of the test endodontic irrigants: acetic acid, 15% citric acid, 17% EDTA-T and saline (control). After 30 min, analysis of the extravasated dye was performed by light absorption spectrophotometry (620 nm). RESULTS: There was statistically signifcant difference (p<0.05) between groups 1 and 2 for all irrigants. L-NAME produced a less intense infammatory reaction and nitroprussiate intensifed this process. CONCLUSIONS: Independently of the administration of NO inhibitors and donors, EDTA-T produced the highest irritating potential in vital tissue among the tested irrigating solutions.

A new bianthron glycoside as inhibitor of Trypanosoma cruzi glyceraldehyde 3-phosphate dehydrogenase activity

A phytochemical investigation of the ethanolic extract of stalks of Senna martiana Benth. (Leguminoseae), native specie of northeast Brazil, resulted in the isolation and spectroscopic characterization of a new bianthrone glycoside, martianine 1 (10,10'-il-chrysophanol-10-oxi10,10'-bi-glucosyl). Its identification was established by HRMS, IR and 2D NMR experiments. The evaluation of martianine trypanocidal activity was carried out against gliceraldehyde 3-phosphate dehydrogenase enzyme from Trypanosoma cruzi. Its inhibitory constant (Ki) is in the low micromolar concentration and it was determined by isothermal titration calorimetry to be 27.3 ± 2.47 µmol L-1. The non-competitive mechanism is asserted to be putative of the mode of action martianine displays against T. cruzi GAPDH. Results show that martianine has a great potential to become new lead molecule by inhibiting this key enzyme and for the development of new drugs against Chagas disease.

Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and high plasma homocysteine in chronic hepatitis C (CHC) infected patients from the Northeast of Brazil

Background/Aim: Hyperhomocysteinemia due to Methylenetetrahydrofolate Reductase (MTHFR) gene, in particular the C677T (Ala222Val) polymorphism were recently associated to steatosis and fibrosis. We analyzed the frequency of MTHFR gene in a cross-sectional study of patients affected by Chronic Hepatitis C (CHC) from Northeast of Brazil. Method: One hundred seven-four untreated patients with CHC were genotyped for the C677T MTHFR. Genomic DNA was extracted from peripheral blood cells and the C677T MTHFR polymorphism was identified by PCR-RFLP. The homocysteine (Hcy) levels were determined by chemiluminescence method. All patients were negative for markers of Wilson's disease, hemochromatosis and autoimmune diseases and have current and past daily alcohol intake less than 100 g/week. Results: Among subjects infected with CHC genotype non-1 the frequency of MTHFR genotypes TT was 9.8% versus 4.4% genotype 1 (p = 0.01). Nevertheless, association was found between the MTHFR genotype TT x CT/CC polymorphism and the degree of steatosis and fibrosis in both hepatitis C genotype (p < 0.05). A significant difference was found on plasma Hcy levels in patients with steatosis regardless of HCV genotype (p = 0.03). Conclusion: Our results indicate that plasma Hcy levels is highly prevalent in subjects with chronic hepatits C with steatosis regardless of HCV genotype and vitamin deficiency. The presence of genotype TT of MTHFR C677T polymorphism was more common in CHC genotype non-1 infected patient regardless of histopathological classification and genotype TT+CT frequencies were significant in the presence of fibrosis grade 1+2 and of steatosis in CHC infected patients from the northeast of Brazil regardless of HCV genotype. The genetic susceptibility of MTHFR C677T polymorphism should be confirmed in a large population.

Expression of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in medullary thyroid carcinoma: Prognostic implications

Background: Metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) participate in the degeneration of the extracellular matrix and are associated with carcinogenesis. MMP-2 is one of the main metalloproteinases active in neoplasia and is a marker of the malignant phenotype. Since the biological behavior of medullary thyroid carcinoma (MTC) varies widely, the present study was undertaken to determine if there is a correlation between the clinical evolution of MTC and the immunohistochemically detected expression of these enzymes in thyroid surgical specimens containing MTC. If so, their expression would be a novel indicator of the prognosis of MTC. Methods: Thirty-seven patients with MTC who had undergone thyroid surgery were followed for an average of 73 months. Immunohistochemical staining for metalloproteinase-related enzymes was performed in surgical paraffin blocks. The clinical status of the patients after surgery and at the end of the study period was characterized to determine correlations between these and the immunohistochemical markers. A value of p < 0.05 was considered statistically significant. Results: At the end of the study period, 15 patients (40.5%) were alive and without evidence of MTC, 17 (45.9%) had persistent MTC, and 5 (13.5%) had a relapse of their neoplasia. Four patients (10.8%) died during the course of the study. There was a significant correlation (p = 0.0005) between the immunohistochemical staining for MMP-2 and the clinical condition of the patients at the end of the study period, and a correlation between the state of apparent cure compared to persistence of MTC after thyroid surgery (p = 0.0207). No significant correlations were observed between either TIMP-2 expression or immune marking of metastatic lymph nodes and the clinical variables studied. Conclusion: Immunohistochemical expression of MMP-2 in thyroid surgical specimens from patients with MTC is a novel indicator of the prognosis of this cancer.

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k(2)=21 +/- 1 s(-1)) was much higher than the HNE deacylation step (k(3)=0.57 +/- 0.05 s(-1)). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k(1) 2.4-fold and reducing k(-1) 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k(2) value, whereas the k(3) value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs.

Profiles of xylose reductase, xylitol dehydrogenase and xylitol production under different oxygen transfer volumetric coefficient values

BACKGROUND: Xylitol is a sugar alcohol (polyalcohol) with many interesting properties for pharmaceutical and food products. It is currently produced by a chemical process, which has some disadvantages such as high energy requirement. Therefore microbiological production of xylitol has been studied as an alternative, but its viability is dependent on optimisation of the fermentation variables. Among these, aeration is fundamental, because xylitol is produced only under adequate oxygen availability. In most experiments with xylitol-producing yeasts, low oxygen transfer volumetric coefficient (K(L)a) values are used to maintain microaerobic conditions. However, in the present study the use of relatively high K(L)a values resulted in high xylitol production. The effect of aeration was also evaluated via the profiles of xylose reductase (XR) and xylitol clehydrogenase (XD) activities during the experiments. RESULTS: The highest XR specific activity (1.45 +/- 0.21 U mg(protein)(-1)) was achieved during the experiment with the lowest K(L)a value (12 h(-1)), while the highest XD specific activity (0.19 +/- 0.03 U mg(protein)(-1)) was observed with a K(L)a value of 25 h(-1). Xylitol production was enhanced when K(L)a was increased from 12 to 50 h(-1), which resulted in the best condition observed, corresponding to a xylitol volumetric productivity of 1.50 +/- 0.08 g(xylitol) L(-1) h(-1) and an efficiency of 71 +/- 6.0%. CONCLUSION: The results showed that the enzyme activities during xylitol bioproduction depend greatly on the initial KLa value (oxygen availability). This finding supplies important information for further studies in molecular biology and genetic engineering aimed at improving xylitol bioproduction. (C) 2008 Society of Chemical Industry