Oxidative stress, metabolism of ethanol and alcohol-related diseases

Alcohol-induced oxidative stress is linked to the metabolism of ethanol. Three metabolic pathways of ethanol have been described in the human body so far. They involve the following enzymes: alcohol dehydrogenase, microsomal ethanol oxidation system (MEOS) and catalase. Each of these pathways could produce free radicals which affect the antioxidant system. Ethanol per se, hyperlactacidemia and elevated NADH increase xanthine oxidase activity, which results in the production of superoxide. Lipid peroxidation and superoxide production correlate with the amount of cytochrome P450 2E1. MEOS aggravates the oxidative stress directly as well as indirectly by impairing the defense systems. Hydroxyethyl radicals are probably involved in the alkylation of hepatic proteins. Nitric oxide (NO) is one of the key factors contributing to the vessel wall homeostasis, an important mediator of the vascular tone and neuronal transduction, and has cytotoxic effects. Stable metabolites--nitrites and nitrates--were increased in alcoholics (34.3 +/- 2.6 vs. 22.7 +/- 1.2 micromol/l, p < 0.001). High NO concentration could be discussed for its excitotoxicity and may be linked to cytotoxicity in neurons, glia and myelin. Formation of NO has been linked to an increased preference for and tolerance to alcohol in recent studies. Increased NO biosynthesis also via inducible NO synthase (NOS, chronic stimulation) may contribute to platelet and endothelial dysfunctions. Comparison of chronically ethanol-fed rats and controls demonstrates that exposure to ethanol causes a decrease in NADPH diaphorase activity (neuronal NOS) in neurons and fibers of the cerebellar cortex and superior colliculus (stratum griseum superficiale and intermedium) in rats. These changes in the highly organized structure contribute to the motor disturbances, which are associated with alcohol abuse. Antiphospholipid antibodies (APA) in alcoholic patients seem to reflect membrane lesions, impairment of immunological reactivity, liver disease progression, and they correlate significantly with the disease severity. The low-density lipoprotein (LDL) oxidation is supposed to be one of the most important pathogenic mechanisms of atherogenesis, and antibodies against oxidized LDL (oxLDL) are some kind of epiphenomenon of this process. We studied IgG oxLDL and four APA (anticardiolipin, antiphosphatidylserine, antiphosphatidylethanolamine and antiphosphatidylcholine antibodies). The IgG oxLDL (406.4 +/- 52.5 vs. 499.9 +/- 52.5 mU/ml) was not affected in alcoholic patients, but oxLDL was higher (71.6 +/- 4.1 vs. 44.2 +/- 2.7 micromol/l, p < 0.001). The prevalence of studied APA in alcoholics with mildly affected liver function was higher than in controls, but not significantly. On the contrary, changes of autoantibodies to IgG oxLDL revealed a wide range of IgG oxLDL titers in a healthy population. These parameters do not appear to be very promising for the evaluation of the risk of atherosclerosis. Free radicals increase the oxidative modification of LDL. This is one of the most important mechanisms, which increases cardiovascular risk in chronic alcoholic patients. Important enzymatic antioxidant systems - superoxide dismutase and glutathione peroxidase - are decreased in alcoholics. We did not find any changes of serum retinol and tocopherol concentrations in alcoholics, and blood and plasma selenium and copper levels were unchanged as well. Only the zinc concentration was decreased in plasma. It could be related to the impairment of the immune system in alcoholics. Measurement of these parameters in blood compartments does not seem to indicate a possible organ, e.g. liver deficiency.     

Expression of human Fas ligand on mouse beta islet cells does not induce insulitis but is insufficient to confer immune privilege for islet grafts

Fas (CD95) and Fas ligand (FasL/CD95L) are involved in programmed cell death and the regulation of host immune responses. FasL has been shown to provide immune privilege, thus prolonging the survival of unmatched grafts in a variety of tissues, such as eyes and testis. In murine FasL (mFasL) transgenic mice, FasL provoked granulocyte infiltration and insulitis in the pancreas. We intended to study whether the expression of human FasL, instead of mFasL, on mouse beta islet cells could avoid granulocyte infiltration, and whether islet cells transgenic for FasL could be used in islet transplantation. We produced transgenic mice in which the human FasL transgene was driven by rat insulin promoter and was expressed exclusively in the pancreas islet cells in ICR mice. In contrast to mFasL transgenic mice, histochemical staining showed that the pancreas was intact in human FasL transgenic ICR mice. However, when human FasL transgenic islet cells were transplanted into allogeneic mice with streptozotocin-induced diabetes, human FasL appeared not to prolong graft survival. Intensive granulocyte infiltration into the islet grafts was observed in recipients (Balb/c mice) which received islet grafts from human FasL transgenic mice, but not from nontransgenic, allogeneic ICR mice on day 31. Our observations suggest that FasL alone is insufficient to confer immune protection, and that other environmental factors might contribute to the formation of immune privilege sites in vivo Copyright 2001 National Science Council, ROC and S. Karger AG, Basel.     

Serum profiling by MALDI-TOF mass spectrometry as a diagnostic tool for domoic acid toxicosis in California sea lions

Background

There are currently no reliable markers of acute domoic acid toxicosis (DAT) for California sea lions. We investigated whether patterns of serum peptides could diagnose acute DAT. Serum peptides were analyzed by MALDI-TOF mass spectrometry from 107 sea lions (acute DAT n = 34; non-DAT n = 73). Artificial neural networks (ANN) were trained using MALDI-TOF data. Individual peaks and neural networks were qualified using an independent test set (n = 20).

Results

No single peak was a good classifier of acute DAT, and ANN models were the best predictors of acute DAT. Performance measures for a single median ANN were: sensitivity, 100%; specificity, 60%; positive predictive value, 71%; negative predictive value, 100%. When 101 ANNs were combined and allowed to vote for the outcome, the performance measures were: sensitivity, 30%; specificity, 100%; positive predictive value, 100%; negative predictive value, 59%.

Conclusions

These results suggest that MALDI-TOF peptide profiling and neural networks can perform either as a highly sensitive (100% negative predictive value) or a highly specific (100% positive predictive value) diagnostic tool for acute DAT. This also suggests that machine learning directed by populations of predictive models offer the ability to modulate the predictive effort into a specific type of error.     

Cell-free derivatives from mesenchymal stem cells are effective in wound therapy

AIM:To compare the efficacy of cell-free derivatives from Bone marrow derived human mesenchymal stem cells(hMSCs) in wound therapy.METHODS:hMSCs have been shown to play an important role in wound therapy.The present study sought to compare efficacy of hMSCs and cell-free derivatives of hMSCs,which may be clinically more relevant as they are easier to prepare,formulate and transport.hMSCs were isolated from human bone marrow and cultured.Multi lineage differentiation of hMSCs was performed to confirm their identity.The ability of hMSCs to migrate was evaluated using in vitro and in vivo migration assays.Cell lysates and conditioned medium concentrate was prepared from hMSCs(see Methods for details).Wounds were induced in mice and wound areas were measure before and after cell and cell-free derivative treatment.RNA and proteins were extracted from the skin and cytokine levels were measured.RESULTS:Co-culture of hMSCs with keratinocytes resulted in increased expression of CXCL-12(SDF1) and ENA78(CXCL-5) in the conditioned media indicating that the hMSCs can respond to signals from keratinocytes.Accelerated wound closure was observed when hMSCs were injected near the site of excisional wounds in athymic as well as NOD/SCID mice.Interestingly,cell-free lysates prepared from hMSCs were also effective in inducing accelerated wound closure and increased expression of SDF1 and CXCL-5 at the wound bed.Additionally,concentrated media from hMSCs as well as an emulsion containing lysates prepared from hMSCs was also found to be more effective in rapid re-epithelialization than fibroblasts or vehicle-alone control.Use of cell-free derivatives may help replace expensive wound care approaches including use of growth factors,epidermal/dermal substitutes,synthetic membranes,cytokines,and matrix components,and most importantly avoid transmission of pathogens from human and animal products.CONCLUSION:These results encourage development of derivatives of hMSCs for wound care and re-epithelialization applications.     

ACUTE EFFECTS OF MOVEMENT VELOCITY ON BLOOD LACTATE AND GROWTH HORMONE RESPONSES AFTER ECCENTRIC BENCH PRESS EXERCISE IN RESISTANCE-TRAINED MEN

This study aimed to compare the effects of different velocities of eccentric muscle actions on acute blood lactate and serum growth hormone (GH) concentrations following free weight bench press exercises performed by resistance-trained men. Sixteen healthy men were divided into two groups: slow eccentric velocity (SEV; n = 8) and fast eccentric velocity (FEV; n = 8). Both groups performed four sets of eight eccentric repetitions at an intensity of 70% of their one repetition maximum eccentric (1RMecc) test, with 2-minute rest intervals between sets. The eccentric velocity was controlled to 3 seconds per range of motion for SEV and 0.5 seconds for the FEV group. There was a significant difference (P < 0.001) in the kinetics of blood lactate removal (at 3, 6, 9, 15, and 20 min) and higher mean values for peak blood lactate (P = 0.001) for the SEV group (9.1 ± 0.5 mM) compared to the FEV group (6.1 ± 0.4 mM). Additionally, serum GH concentrations were significantly higher (P < 0.001) at 15 minutes after bench press exercise in the SEV group (1.7 ± 0.6 ng · mL⁻¹) relative to the FEV group (0.1 ± 0.0 ng · mL⁻¹). In conclusion, the velocity of eccentric muscle action influences acute responses following bench press exercises performed by resistance-trained men using a slow velocity resulting in a greater metabolic stress and hormone response.     

Cytotoxicity induced by grape seed proanthocyanidins: Role of nitric oxide

Grape seed proanthocyanidin extract (GPSE) at high doses has been shown to exhibit cytotoxicity that is associated with increased apoptotic cell death. Nitric oxide (NO), being a regulator of apoptosis, can be increased in production by the administration of GSPE. In a chick cardiomyocyte study, we demonstrated that high-dose (500 μg/ml) GSPE produces a significantly high level of NO that contributes to increased apoptotic cell death detected by propidium iodide and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. It is also associated with the depletion of intracellular glutathione (GSH), probably due to increased consumption by NO with the formation of S-nitrosoglutathione. Co-treatment with L-NAME, a NO synthase inhibitor, results in reduction of NO and apoptotic cell death. The decline in reduced GSH/oxidized GSH (GSSG) ratio is also reversed. N-Acetylcysteine, a thiol compound that reacts directly with NO, can reduce the increased NO generation and reverse the decreased GSH/GSSG ratio, thereby attenuating the cytotoxicity induced by high-dose GSPE. Taken together, these results suggest that endogenous NO synthase (NOS) activation and excessive NO production play a key role in the pathogenesis of high-dose GSPE-induced cytotoxicity.     

The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster

In a recent study, we reported that the combined average mutation rate of 10 di-, 6 tri-, and 8 tetranucleotide repeats in Drosophila melanogaster was 6.3 x 10(-6) mutations per locus per generation, a rate substantially below that of microsatellite repeat units in mammals studied to date (range = 10(-2)-10(-5) per locus per generation). To obtain a more precise estimate of mutation rate for dinucleotide repeat motifs alone, we assayed 39 new dinucleotide repeat microsatellite loci in the mutation accumulation lines from our earlier study. Our estimate of mutation rate for a total of 49 dinucleotide repeats is 9.3 x 10(-6) per locus per generation, only slightly higher than the estimate from our earlier study. We also estimated the relative difference in microsatellite mutation rate among di-, tri-, and tetranucleotide repeats in the genome of D. melanogaster using a method based on population variation, and we found that tri- and tetranucleotide repeats mutate at rates 6.4 and 8.4 times slower than that of dinucleotide repeats, respectively. The slower mutation rates of tri- and tetranucleotide repeats appear to be associated with a relatively short repeat unit length of these repeat motifs in the genome of D. melanogaster. A positive correlation between repeat unit length and allelic variation suggests that mutation rate increases as the repeat unit lengths of microsatellites increase.      

Preface

"Although few people die of pain,many people die in pain and even more live in pain"——IASP On October11,2004,International Association for the Study of Pain(IASP)