Simvastatin impairs murine melanoma growth

Background: Statins induces cell cycle arrest, apoptosis, reduction of angiogenic factors, inhibition of the endothelial growth factor, impairing tissue adhesion and attenuation of the resistance mechanisms. The aim of this study was evaluate the anti tumoral activity of simvastatin in a B16F10 melanoma-mouse model. Methods: Melanoma cells were treated with different concentrations of simvastatin and assessed by viability methods. Melanoma cells (5 x 10(4)) were implanted in two month old C57Bl6/J mice. Around 7 days after cells injection, the oral treatments were started with simvastatin (5 mg/kg/day, p.o.). Tumor size, hematological and biochemical analyses were evaluated. Results: Simvastatin at a concentration of 0.8 mu M, 1.2 mu M and 1.6 mu M had toxic effect. Concentration of 1.6 mu M induced a massive death in the first 24 h of incubation. Simvastatin at 0.8 mu M induces early cell cycle arrest in G0/G1, followed by increase of hypodiploidy. Tumor size were evaluated and the difference of treated group and control, after ten days, demonstrates that simvastatin inhibited the tumor expansion in 68%. Conclusion: Simvastatin at 1.6 mu M, presented cytototoxicity after 72 h of treatment, with an intense death. In vivo, simvastatin being potentially useful as an antiproliferative drug, with an impairment of growth after ten days.

Abnormal Neurovascular Control in Anabolic Androgenic Steroids Users

NUNES ALVES, M. J. N., M. R. DOS SANTOS, R. G. DIAS, C. A. AKIHO, M. C. LATERZA, M. U. P. B. RONDON, R. L. DE MORAES MOREAU, and C. E. NEGRAO. Abnormal Neurovascular Control in Anabolic Androgenic Steroids Users. Med. Sci. Sports Exerc., Vol. 42, No. 5, pp. 865-871, 2010. Purpose: Previous studies showed that anabolic androgenic steroids (AAS) increase vascular resistance and blood pressure (BP) in humans. In this study, we tested the hypotheses 1) that AAS users would have increased muscle sympathetic nerve activity (MSNA) and reduced forearm blood flow (FBF) compared with AAS nonusers and 2) that there would be an association between MSNA and 24-h BP. Methods: Twelve AAS users aged 31 +/- 2 yr (means +/- SE) and nine age-matched AAS nonusers aged 29 T 2 yr participated in the study. All individuals were involved in strength training for at least 2 yr. AAS was determined by urine test (chromatography-mass spectrometry). MSNA was directly measured by microneurography technique. FBF was measured by venous occlusion plethysmography. BP monitoring consisted of measures of BP for 24 h. Results: MSNA was significantly higher in AAS users than that in AAS nonusers (29 +/- 3 vs 20 +/- 1 bursts per minute, P = 0.01). FBF (1.92 +/- 0.17 vs 2.77 +/- 0.24 mL.min(-1).100 mL(-1), P = 0.01) and forearm vascular conductance (2.01 +/- 0.17 vs 2.86 +/- 0.31 U, P = 0.02) were significantly lower in AAS users than that in AAS nonusers. Systolic (131 +/- 4 vs 120 +/- 3 mm Hg, P = 0.001), diastolic (74 +/- 4 vs 68 +/- 3 mm Hg, P = 0.02), and mean BP (93 +/- 4 vs 86 +/- 3 mm Hg, P = 0.005) and heart rate (74 +/- 3 vs 68 +/- 3 bpm, P = 0.02) were significantly higher in AAS users when compared with AAS nonusers. Further analysis showed that there was a significant correlation between MSNA and 24-h mean BP (r = 0.75, P = 0.002). Conclusions: AAS increases MSNA and reduces muscle blood flow in young individuals. In addition, the increase in BP levels in AAS users is associated with augmented sympathetic outflow. These findings suggest that AAS increases the susceptibility for cardiovascular disease in humans.

RECK-Mediated Inhibition of Glioma Migration and Invasion

RECK is an anti-tumoral gene whose activity has been associated with its inhibitory effects regulating MMP-2, MMP-9, and MT1-MMP. RECK level decreases as gliobastoma progresses, varying from less invasive grade II gliomas to very invasive human glioblastoma multiforme (GBM). Since RECK expression and glioma invasiveness show an inverse correlation, the aim of the present study is to investigate whether RECK expression would inhibit glioma invasive behavior. We conducted this study to explore forced RECK expression in the highly invasive T98G human GBM cell line. Expression levels as well as protein levels of RECK, MMP-2, MMP-9, and MT1-MMP were assessed by qPCR and immunoblotting in T98G/RECK+ cells. The invasion and migration capacity of RECK+ cells was inhibited in transwell and wound assays. Dramatic cytoskeleton modifications were observed in the T98G/RECK+ cells, when compared to control cells, such as the abundance of stress fibers (contractile actin-myosin II bundles) and alteration of lamellipodia. T98G/RECK+ cells also displayed phosphorylatecl focal adhesion kinase (P-FAK) in mature focal adhesions associated with stress fibers; whereas P-FAK in control cells was mostly associated with immature focal complexes. Interestingly, the RECK protein was predominantly localized at the leading edge of migrating cells, associated with membrane ruffles. Unexpectedly, introduced expression of RECK effectively inhibited the invasive process through rearrangement of actin filaments, promoting a decrease in migratory ability. This work has associated RECK tumor-suppressing activity with the inhibition of motility and invasion in this GBM model, which are two glioma characteristics responsible for the inefficiency of current available treatments. J. Cell. Biochem. 110: 52-61, 2010. (C) 2010 Wiley-Liss. Inc.

Effects of isoflavone-supplemented soy yogurt on lipid parameters and atherosclerosis development in hypercholesterolemic rabbits: a randomized double-blind study

Background: There is increasing interest in natural treatments to control dyslipidemia and reduce the risk of cardiovascular disease. Previous studies have demonstrated the beneficial effects of soy yogurt fermented with Enterococcus faecium CRL 183 and of dietary isoflavones on the lipid profile. The purpose of the present study was to investigate the effects of isoflavone-supplemented soy yogurt, fermented with E. faecium CRL183, on lipid parameters and atherosclerosis development in rabbits with induced hypercholesterolemia. Methods: Forty-eight rabbits were randomly assigned to eight groups fed on the following diets for 60 days: C - control; IY - isoflavone-supplemented soy yogurt; H - hypercholesterolemic (1.0% cholesterol wt/wt diet); HY - hypercholesterolemic plus soy yogurt; HIY - hypercholesterolemic plus isoflavone-supplemented soy yogurt; HP - hypercholesterolemic plus placebo; HI hypercholesterolemic plus isoflavone and HE - hypercholesterolemic plus pure culture of E. faecium CRL 183. Serum lipids and autoantibodies against oxLDL (oxLDL Ab) were analyzed on days 0, 30 and 60 of the treatment and the atherosclerotic lesions were quantified at the end of the experiment. Results: Soy yogurt, soy yogurt supplemented with isoflavones and placebo promoted significant reductions in total cholesterol level (38.1%, 27.0% and 26.6%, respectively). Significant increases in serum HDL-C concentration relative to group H were detected in animals that ingested soy yogurt, with or without the isoflavone supplement (55.2%), E. faecium culture (43.3%) or placebo (35.8%). Intake of soy yogurt and soy yogurt supplemented with isoflavones prevented the rise of oxLDL Ab during the study period. The extent of atherosclerosis in the thoracic and abdominal aortas was reduced in the HIY, HY and HP groups. However, when the whole aorta was analyzed, animals treated with soy yogurt supplemented with isoflavones exhibited the greatest reduction (51.4%, P < 0.05) in atherosclerotic lesion area, compared to group H. Conclusion: Soy yogurt could be consumed as an alternative means of reducing the risk of cardiovascular disease by improving the lipid profile and inhibiting oxLDL Ab formation. Our findings also suggest that isoflavone supplementation may enhance the antiatherosclerotic effect of soy yogurt.

Detection of the TLR4 1196C > T Polymorphism by Mismatched-Polymerase Chain Reaction Using Plasmid DNA as Internal Control in Restriction Fragment Length Polymorphism Assays

Background: Restriction fragment length polymorphism (RFLP) is a common molecular assay used for genotyping, and it requires validated quality control procedures to prevent mistyping caused by impaired endonuclease activity. We have evaluated the usefulness of a plasmid-based internal control in RFLP assays. Results: Blood samples were collected from 102 individuals with acute myocardial infarction (AMI) and 108 non-AMI individuals (controls) for DNA extraction and laboratory analyses. The 1196C> T polymorphism in the toll-like receptor 4 (TLR4) gene was amplified by mismatched-polymerase chain reaction (PCR). Amplicons and pBluescript II SK-plasmid were simultaneously digested with endonuclease HincII. Fragments were separated on 2% agarose gels. Plasmid was completely digested using up to 55.2 nmL/L DNA solutions and 1 mu L PCR product. Nevertheless, plasmid DNA with 41.4 nM or higher concentrations was incompletely digested in the presence of 7 mL PCR product. In standardized conditions, TLR4 1196C> T variant was accurately genotyped. TLR4 1196T allele frequency was similar between AMI (3.1%) and controls (2.0%, p = 0.948). TLR4 SNP was not associated with AMI in this sample population. In conclusion, the plasmid-based control is a useful approach to prevent mistyping in RFLP assays, and it is validate for genetic association studies such as TLR4 1196C> T.

Influence of ammonium sulphate feeding time on fed-batch Arthrospira (Spirulina) platensis cultivation and biomass composition with and without pH control

Previous work demonstrated that a mixture of NH(4)Cl and KNO(3) as nitrogen source was beneficial to fed-batch Arthrospira (Spirulina) platensis cultivation, in terms of either lower costs or higher cell concentration. On the basis of those results, this study focused on the use of a cheaper nitrogen source mixture, namely (NH(4))(2)SO(4) plus NaNO(3), varying the ammonium feeding time (T = 7-15 days), either controlling the pH by CO(2) addition or not. A. platensis was cultivated in mini-tanks at 30 degrees C, 156 mu mol photons m(-2) s(-1), and starting cell concentration of 400 mg L(-1), on a modified Schlosser medium. T = 13 days under pH control were selected as optimum conditions, ensuring the best results in terms of biomass production (maximum cell concentration of 2911 mg L(-1), cell productivity of 179 mg L(-1) d(-1) and specific growth rate of 0.77 d(-1)) and satisfactory protein and lipid contents (around 30% each). (C) 2011 Elsevier Ltd. All rights reserved.

Sterilization of Medical Devices by Ethylene Oxide, Determination of the Dissipation of Residues, and Use of Green Fluorescent Protein as an Indicator of Process Control

Ethylene oxide (EO) is used to sterilize Oxygenator and Tubing applied to heart surgery. Residual levels of EO and its derivatives, ethylene chlorohydrin (ECH) and ethylene glycol (EG), may be hazardous to the patients. Therefore, it must be removed by the aeration process. This study aimed to estimate the minimum aeration time for these devices to attain safe limits for use (avoiding excessive aeration time) and to evaluate the Green Fluorescent Protein (GFP) as a biosensor capable of best indicating the distribution and penetration of EO gas throughout the sterilization chamber. Sterilization cycles of 2, 4, and 8 h were monitored by Bacillus atrophaeus ATCC 9372 as a biological indicator (131) and by the GFP. Residual levels of EO, ECH, and EG were determined by gas chromatography (GC), and the residual dissipation was studied. Safe limits were reached right after the sterilization process for Oxygenator and after 204 h of aeration for Tubing. In the 2 h cycle, the GFP concentration decreased from 4.8 (+/- 3.2)% to 7.5 (+/- 2.5)%. For the 4 h cycle, the GFP concentration decreased from 17.4 (+/- 3.0)% to 21.5 (+/- 6.8)%, and in the 8 h cycle, it decreased from 22.5 (+/- 3.2)% to 23.9 (+/- 3.9)%. This finding showed the potentiality for GFP applications as an EO biosensor. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 9113: 626-630, 2009

Ultra-Fast Gradient LC Method for Omeprazole Analysis Using a Monolithic Column: Assay Development, Validation, and Application to the Quality Control of Omeprazole Enteric-Coated Pellets

A method was optimized for the analysis of omeprazole (OMZ) by ultra-high speed LC with diode array detection using a monolithic Chromolith Fast Gradient RP 18 endcapped column (50 x 2.0 mm id). The analyses were performed at 30 degrees C using a mobile phase consisting of 0.15% (v/v) trifluoroacetic acid (TFA) in water (solvent A) and 0.15% (v/v) TFA in acetonitrile (solvent B) under a linear gradient of 5 to 90% B in 1 min at a flow rate of 1.0 mL/min and detection at 220 nm. Under these conditions, OMZ retention time was approximately 0.74 min. Validation parameters, such as selectivity, linearity, precision, accuracy, and robustness, showed results within the acceptable criteria. The method developed was successfully applied to OMZ enteric-coated pellets, showing that this assay can be used in the pharmaceutical industry for routine QC analysis. Moreover, the analytical conditions established allow for the simultaneous analysis of OMZ metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in the same run, showing that this method can be extended to other matrixes with adequate procedures for sample preparation.

Antinociceptive and anti-inflammatory properties of 7-hydroxycoumarin in experimental animal models: potential therapeutic for the control of inflammatory chronic pain

Objectives In the present study we investigated the anti nociceptive, anti-inflammatory and antipyretic effects of 7-hydroxycoumarin (7-HC) in animal models. Methods The effects of oral 7-HC were tested against acetic acid-induced writhing, formalin test, tail flick test, complete Freund`s adjuvant (CFA)-induced hypemociception, carrageenan-induced paw oedema, lipopolysaccharide-induced fever and the rota rod test. Key findings 7-HC (3-60 mg/kg) produced a dose-related antinociception against acetic acid-induced writhing in mice and in the formalin test. In contrast, treatment with 7-HC did not prevent thermal nociception in the tail flick test. A single treatment with 7-HC, 60 mg/kg, produced a long-lasting antinociceptive effect against CFA-induced hypernociception, a chronic inflammatory pain stimulus. Notably, at 60 mg/kg per day over 4 days the administration of 7-HC produced a continuous antinociceptive effect against CFA-induced hypernociception. 7-HC (30-120 mg/kg) produced anti-inflammatory and antipyretic effects against carrageenan-induced inflammation and lipopolysaccharide-induced fever, respectively. Moreover, 7-HC was found to be safe with respect to ulcer induction. In the rota rod test, 7-HC-treated mice did not show any motor performance alterations. Conclusions The prolonged antinociceptive and anti-inflammatory effects of 7-HC, in association with its low ulcerogenic activity, indicate that this molecule might be a good candidate for development of new drugs for the control of chronic inflammatory pain and fever.

Evaluation of the genotoxic and cytotoxic effects of crude extracts of Cordia ecalyculata and Echinodorus grandiflorus

Ethnopharmacological relevance: Cordia ecalyculata Veil. and Echinodorus grandiflorus (Cham. & Schltdl) Micheli are extensively used in Brazil as therapeutic preparations for indigenous groups and the general population. These plants have been used in the folk medicine as: tonic, diuretic, anti-inflammatory, appetite suppressants, for the treatment of snake bites, and weight loss. Aim of the study: In this study, it was verified the possible cytotoxic and genotoxic effects of the crude extracts of. Cordia ecalyculata and Echinodorus grandiflorus, as well as their effectiveness in treating obesity. Materials and methods: The Micronucleus Test was used for the evaluation of possible clastogenic and aneugenic effects, and the Comet Assay was used for the evaluation of single-strand and double-strand DNA breaks. The cytotoxic effects of the crude extracts were verified by PCE/NCE ratio. Swiss mice (Mus musculus) were used as the experimental model. Results: It was observed a significant (P < 0.05) increase, dose-independent, in the average frequency of micronucleated erythrocytes in peripheral blood in mice treated with either the Cordia ecalyculata or Echinodorus grandiflorus extracts, in comparison with the negative control. There were no significant differences (P > 0.05) in the frequency of micronucleated polychromatic erythrocytes for both extract treatment. We observed that treatment with the Cordia ecalyculata extract at concentrations of 1000 and 2000 mg/kg bw resulted in a PCE/NCE ratio that was larger (P < 0.05) than the negative control. After 15 days of daily treatment. a dose of 2000 mg/kg bw of either phytotherapeutic did not reduce body mass gain or the amount of food consumed by Swiss mice when compared with the negative control (P > 0.05). Conclusion: The results of this study allowed us to infer that the crude extracts of Cordia ecalyculata and Echinodorus grandiflorus do not display cytotoxic or genotoxic activities. However, they do possess weak clastogenic activity (without significance) on peripheral blood cells. Contrary to commonly held beliefs it was also found in this study that the extracts are not effective for obesity treatments. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Microbiological quality of ready-to-eat minimally processed vegetables consumed in Brazil

Minimally processed leafy vegetables are ready-to-eat (RTE) products very attractive to consumers looking for healthy and convenient meals. However, the microbiological safety of these foods is of special concern due to the absence of lethal treatments during processing. In the present study, indicator microorganisms, Listeria spp. and Salmonella spp. were determined for 162 samples of minimally processed leafy vegetables commercialized in Brazil. Psychrotrophic aerobic bacterial populations >5 log CFU/g were found in 96.7% of the samples, while total and thermotolerant coliforms were detected respectively in 132 (81.5%) and 107 (66%) of vegetables analyzed. Escherichia coil was present in 86 (53.1%) samples analyzed and Listeria spp. and Salmonella spp. were detected respectively in 6 (3.7%) and 2 (1.2%) samples. These results indicate the need of implementing quality programs in the production chain of RTE vegetables to improve shelf life and microbiological safety. (C) 2011 Elsevier Ltd. All rights reserved.

Variability in UVB Tolerances of Melanized and Nonmelanized Cells of Cryptococcus neoformans and C-laurentii

Solar radiation is one of the major factors responsible for the control of fungus populations in the environment. Inactivation by UVA and UVB radiation is especially important for the control of fungi that disperse infective units through the air, including fungi such as Cryptococcus spp. that infect their vertebrate hosts by inhalation. Cryptococcus neoformans produces melanin in the presence of certain exogenous substrates such as l-3,4 dihydroxyphenylalanine and melanization may protect the fungus against biotic and abiotic environmental factors. In the present study, we investigated the effect of exposure to an UVB irradiance of 1000 mW m(-2) (biologically effective weighted irradiance) on the survival of melanized and nonmelanized cells of four strains of C. neoformans and four strains of C. laurentii. The relative survival (survival of cells exposed to radiation in relation to cells not exposed) of cells grown 2, 4, 6 or 8 days on medium with or without L-dopa was determined after exposure to UVB doses of 1.8 and 3.6 kJ m(-2). Both the irradiance spectrum and the intensities of those doses are environmentally realistic, and, in fact, occur routinely during summer months in temperate regions. Differences in tolerance to UVB radiation were observed between the C. neoformans and C. laurentii strains. The C. neoformans strains were more susceptible to UVB radiation than the C. laurentii strains. In C. neoformans, differences in tolerance to radiation were observed during development of both melanized and nonmelanized cells. For most treatments (strain, time of growth and UVB dose), there were virtually no differences in tolerances between melanized and nonmelanized cells, but when differences occurred they were smaller than those previously observed with UVC. In tests with two strains of C. laurentii, there was no difference in tolerance to UVB radiation between melanized and nonmelanized cells during 8 days of culture; and in tests with four strains for less culture time (4 days) there were no significant differences in tolerance between melanized and nonmelanized cells of any strain of this species.

Suppressive action of melatonin on the TH-2 immune response in rats infected with Trypanosoma cruzi

Control of the acute phase of Trypanosoma cruzi infection is critically dependent on cytokine-mediated macrophage activation to intracellular killing, natural killer (NK) cells, CD4(+) T cells, CD8(+) T cells and B cells. Cell-mediated immunity in T. cruzi infection is also modulated by cytokines, but in addition to parasite-specific responses, autoimmunity can be also triggered. Importantly, cytokines may also play a role in the cell-mediated immunity of infected subjects. Here we studied the role of cytokines in the regulation of innate and adaptive immunity during the acute phase of T. cruzi infection in Wistar rats. Melatonin is an effective regulator of the immune system. Macrophages and T lymphocytes, which have melatonin receptors, are target cells for the immunomodulatory function of melatonin. In this paper melatonin was orally given via two protocols: prior to and concomitant with infection. Both treatments were highly effective against T. cruzi with enhanced action for the concomitant treatment. The data suggest an up-regulation of the TH-1 immune response as all analyzed parameters, interleukin (IL)-4, IL-10, transforming growth factor-beta 1 and splenocyte proliferation, displayed reduced levels as compared with the untreated counterparts. However, the direct effects of melatonin on immune cells have not been fully investigated during T. cruzi infection. We conclude that in light of the current results, melatonin exerted important therapeutic benefits through its immune regulatory effects.

Melatonin enhances pro-inflammatory cytokine levels and protects against Chagas disease

Pro-inflammatory and modulatory cytokines have an essential role in host defense against human and murine Trypanosoma cruzi infection. Control of T. cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. Melatonin has been proposed to regulate the immune system by affecting cytokine production in immunocompetent cells, enhancing the production of several T helper (Th)1 cytokines. The aims of this work were to evaluate in rats, the influences of exogenous melatonin treatment on T. cruzi-infected host`s immune responses. With this in mind, several immunological parameters were analyzed, including tumor necrosis factor-alpha, gamma-interferon, interleukin-12, nitric oxide (NO) and macrophage count. The melatonin therapy was provided in one of two different treatment regimens, that is, either beginning 7 days prior to infection or concomitant with the infection. Both treatments triggered an up-regulation of the immune response, with the concomitant treatment being more effective; in this case all cytokines studied, with exception of NO, displayed enhanced concentrations and there was a higher number of peritoneal macrophages, which displayed reduced concentrations under melatonin therapy. We conclude that melatonin plays a pivotal role in up-regulating the Th1 immune response thus controlling parasite replication.