Pb(II) Induces Scramblase Activation and Ceramide-Domain Generation in Red Blood Cells.

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling.

Lipids and Oxidative Stress Associated with Ethanol-Induced Neurological Damage.

The excessive intake of alcohol is a serious public health problem, especially given the severe damage provoked by chronic or prenatal exposure to alcohol that affects many physiological processes, such as memory, motor function, and cognitive abilities. This damage is related to the ethanol oxidation in the brain. The metabolism of ethanol to acetaldehyde and then to acetate is associated with the production of reactive oxygen species that accentuate the oxidative state of cells. This metabolism of ethanol can induce the oxidation of the fatty acids in phospholipids, and the bioactive aldehydes produced are known to be associated with neurotoxicity and neurodegeneration. As such, here we will review the role of lipids in the neuronal damage induced by ethanol-related oxidative stress and the role that lipids play in the related compensatory or defense mechanisms.

Effect of vitamin E and C supplementation on oxidative damage and total antioxidant capacity in lead-exposed workers.

The molecular response of the antioxidant system and the effects of antioxidant supplementation against oxidative insult in lead-exposed workers has not been sufficiently studied. In this work, antioxidants (vitamin E 400 IU+vitamin C 1g/daily) were supplemented for one year to 15 workers exposed to lead (73 µg of lead/dl of blood) and the results were compared with those on 19 non-lead exposed workers (6.7 µg of lead/dl). Lead intoxication was accompanied by a high oxidative damage and an increment in the erythrocyte antioxidant response due to increased activity of catalase and superoxide dismutase. Antioxidant supplementations decreased significantly the oxidative damage as well as the total antioxidant capacity induced by lead intoxication with reduction of the antioxidant enzyme activities. We conclude that antioxidant supplementation is effective in reducing oxidative damage and induces modifications in the physiopathological status of the antioxidant response in lead-exposed workers.

Effects of chronic and acute lead treatments on the biophysical properties of erythrocyte membranes, and a comparison with model membranes.

Rat erythrocytes, or erythrocyte membrane ghosts, have been subjected to either chronic (drinking water containing 15 mM lead acetate for 3 months) or acute (10(-9)-10(-2 )M lead acetate for 1 h) Pb(2+) treatments and subsequent changes in membrane properties have been measured. Pb(2+) concentration in chronically treated rat plasma was 1.8 µM, which is one order of magnitude above normal values. Membrane permeability, or hemolysis, was increased in both cases. A comparative study using liposomes, in the form of large unilamellar vesicles, also indicated an increase in membrane permeability. Membrane microviscosity, or acyl chain molecular order, measured as DPH fluorescence polarization, showed an increased order in the acute treatments, at least below 700 µM Pb(2+), and a similar increase in chronically treated rats. The correlation between acute and chronic treatments, and between cell and model membranes, suggests that the present observations may be relevant in the pathogenesis of lead intoxication in humans.

Oxidative damage in young alcohol drinkers: A preliminary study.

BACKGROUND: Oxidative damage (OD) biomarkers have been used to evaluate metabolic stress undergone by alcoholic individuals. In alcoholic patients, these biomarkers are usually measured at late stages, i.e., when the alcoholic patients are showing clear signs of impaired hepatic function. OD biomarkers are sensitive indicators of impaired metabolic function, and might be useful in early stages of alcohol consumption to identify individuals who are at greater risk of damage in later stages of alcohol consumption. The aim of the present work was to evaluate some OD biomarkers in young people at early stages of alcohol consumption. METHODS: The study was carried out in a group of young people (18-23 years old) who drank alcohol, Youngsters Exposed to Alcohol (YEA) with an average intake of 118 g of ethanol/week, and a control group (CG) of non-drinkers. Blood counts, alcohol dehydrogenase (ADH) activity, glutathione peroxidase (GSH-Px) activity, oxidative damage to DNA, and lipid peroxidation were determined in both groups. RESULTS: The anthropometric and blood parameters of both groups were similar and no clinical symptoms of hepatic damage were observed. Nevertheless, ADH activity, lipid peroxidation, and percentage of damaged DNA cells were higher in the YEA group than in the control group. In contrast, GSH-Px activity was lower in the YEA group than in the control group. CONCLUSION: Alteration in OD biomarkers can be found in individuals with 4-5 years of alcohol drinking history. To our knowledge, this is the first study giving evidence of OD in individuals at early stages of alcohol abuse.

A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a ß-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins.

The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that might be inadvertently affected due to promiscuous scaffolds in proteins.