BBA - Molecular Basis of Disease (v.1762, #5)

Ovalbumin (OVA)-sensitized wildtype (WT) and endotoxin-resistant (ER) mice developed similar degrees of airways eosinophilia and serum OVA-specific IgE levels after acute aerosolized OVA challenge. WT mice demonstrated methacholine hyperreactivity, whereas ER mice showed no change in responsiveness. With chronic aerosolized OVA challenge, both WT and ER mice developed local tolerance, with resolution of airway eosinophilia but persistence of anti-OVA IgE in serum. Thus, the development of local tolerance with chronic aerosol exposure to OVA is independent of any potential effects of endotoxin in the OVA aerosol solution.
Keywords: Asthma; Endotoxin; Lipopolysaccharide; Mouse; Sensitization; Tolerance;

GAPDH as a sensor of NO stress by Makoto R. Hara; Matthew B. Cascio; Akira Sawa (502-509).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a classic glycolytic enzyme, and accumulating evidence has suggested that GAPDH is a multi-functional protein. In particular, its role as a mediator for cell death has been highlighted. For the last decade, many groups reported that a pool of GAPDH translocates to the nucleus under a variety of stressors, most of which are associated with oxidative stress. At the molecular level, sequential steps lead to nuclear translocation of GAPDH during cell death as follows: first, a catalytic cysteine in GAPDH (C150 in rat GAPDH) is S-nitrosylated by nitric oxide (NO) that is generated from inducible nitric oxide synthase (iNOS) and/or neuronal NOS (nNOS); second, the modified GAPDH becomes capable of binding with Siah1, an E3 ubiquitin ligase, and stabilizes it; third, the GAPDH-Siah protein complex translocates to the nucleus, dependent on Siah1’s nuclear localization signal, and degrades Siah1’s substrates in the nucleus, which results in cytotoxicity. A recent report suggests that GAPDH may be genetically associated with late-onset of Alzheimer's disease. (−)-deprenyl, which has originally been used as a monoamine oxidase inhibitor for Parkinson's disease, binds to GAPDH and displays neuroprotective actions, but its molecular mechanism is still unclear. The NO/GAPDH/Siah1 death cascade will contribute to the molecular understanding of a role of GAPDH in neurodegenerative disorders and help to establish novel therapeutic strategies.
Keywords: GAPDH; Nuclear translocation; Oxidative stress; Nitric oxide; Siah1; Apoptosis;

ABC A-subfamily transporters: Structure, function and disease by Wolfgang E. Kaminski; Armin Piehler; Jürgen J. Wenzel (510-524).
ABC transporters constitute a family of evolutionarily highly conserved multispan proteins that mediate the translocation of defined substrates across membrane barriers. Evidence has accumulated during the past years to suggest that a subgroup of 12 structurally related “full-size” transporters, referred to as ABC A-subfamily transporters, mediates the transport of a variety of physiologic lipid compounds. The emerging importance of ABC A-transporters in human disease is reflected by the fact that as yet four members of this protein family (ABCA1, ABCA3, ABCR/ABCA4, ABCA12) have been causatively linked to completely unrelated groups of monogenetic disorders including familial high-density lipoprotein (HDL) deficiency, neonatal surfactant deficiency, degenerative retinopathies and congenital keratinization disorders. Although the biological function of the remaining 8 ABC A-transporters currently awaits clarification, they represent promising candidate genes for a presumably equally heterogenous group of Mendelian diseases associated with perturbed cellular lipid transport. This review summarizes our current knowledge on the role of ABC A-subfamily transporters in physiology and disease and explores clinical entities which may be potentially associated with dysfunctional members of this gene subfamily.
Keywords: ABC transporter; Lipid; Atherosclerosis; Retina; Surfactant; Ichthyosis;

The role of PDE4 in pulmonary inflammation and goblet cell hyperplasia in allergic rats by Hui-Fang Tang; Ji-Qiang Chen; Qiang-Min Xie; Xu-Yang Zheng; Yi-Liang Zhu; Ian Adcock; Xiangdong Wang (525-532).
Phosphodiesterase 4 (PDE4) has been suggested to a critical factor in the pathogenesis of inflammation by metabolizing cAMP in human leukocytes, endothelium and epithelium. The present study aimed at evaluating the PDE4 activity and expression, the relationship between the inflammation and cAMP- activity in the lungs, and potential interventions of PDE inhibitors and antiinflammatory drugs in the reduction of lung inflammation and goblet cell hyperplasia in allergic rats. The total leukocyte number and eosinophil number in bronchoalveolar lavegar fluid and PDE4 activity and expression in lungs significantly increased in OVA-sensitized and challenged allergic rat. Lung histology showed an increased infiltration of inflammatory cells in the perivascular and peribronchial spaces, structure changes and goblet cell hyperplasia in the OVA-sensitized and -challenged allergic rats. A significant correlation was observed between the increases in cAMP-PDE activity and inflammation in the lung. Those OVA-induced changes were prevented by pretreatment with PDE inhibitor in a dose-related patterns and with glucocorticosteroid. We found an increase in the proportion of PDE4 and PDE4 gene expression, while a decrease in the proportion of PDE3 in the lung of allergic rats. Incubation with different PDE inhibitors down-regulated OVA-induced cAMP hydrolysis. Our data suggest that PDE4C may play an important role in the airway inflammation, remodeling and goblet cell hyperplasia after repeated challenge of sensitized rats.
Keywords: Asthma; PDE4s; Piclamilast; Dexamethasone; Indomethacin; Inflammation; Goblet cell hyperplasia; Rat;

We previously reported chromatographic evidence supporting the similarity of yellow chromophores isolated from aged human lens proteins, early brunescent cataract lens proteins and calf lens proteins ascorbylated in vitro [Cheng, R. et al. Biochimica et Biophysica Acta 1537, 14–26, 2001]. In this paper, new evidence supporting the chemical identity of the modified amino acids in these protein populations were collected by using a newly developed two-dimensional LC-MS mapping technique supported by tandem mass analysis of the major species. The pooled water-insoluble proteins from aged normal human lenses, early stage brunescent cataract lenses and calf lens proteins reacted with or without 20 mM ascorbic acid in air for 4 weeks were digested with a battery of proteolytic enzymes under argon to release the modified amino acids. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column and four major A 330 nm-absorbing peaks were collected. Peaks 1, 2 and 3, which contained most of the modified amino acids were concentrated and subjected to RP-HPLC/ESI-MS, and the mass elution maps were determined. The samples were again analyzed and those peaks with a 104–106 response factor were subjected to MS/MS analysis to identify the daughter ions of each modification. Mass spectrometric maps of peaks 1, 2 and 3 from cataract lenses showed 58, 40 and 55 mass values, respectively, ranging from 150 to 600 Da. Similar analyses of the peaks from digests of the ascorbylated calf lens proteins gave 81, 70 and 67 mass values, respectively, of which 100 were identical to the peaks in the cataract lens proteins. A total of 40 of the major species from each digest were analyzed by LC-MS/MS and 36 were shown to be identical. Calf lens proteins incubated without ascorbic acid showed several similar mass values, but the response factors were 100 to 1000-fold less for every modification. Based upon these data, we conclude that the majority of the major modified amino acids present in early stage brunescent Indian cataract lens proteins appear to arise as a result of ascorbic acid modification, and are presumably advanced glycation end-products.
Keywords: Human lens; Brunescent cataract; Ascorbic acid; Advanced glycation endproduct; Maillard reaction; Yellow chromophore; Aging;

Co-expression of different subunits of human phenylalanine hydroxylase: Evidence of negative interallelic complementation by João Leandro; Cátia Nascimento; Isabel Tavares de Almeida; Paula Leandro (544-550).
To study the interaction between two different subunits of the heteromeric human phenylalanine hydroxylase (hPAH), present in hyperphenylalaninemic (HPA) compound heterozygous patients, heteroallelic hPAH enzymes were produced. A dual vector expression system was used (PRO™ Bacterial Expression System) in which each mutant subunit was expressed from a separate compatible vector, with different epitope tags, in a single bacterial host. Experimental conditions were selected in order that each plasmid produced equivalent levels of mutant subunits. In this study, we demonstrated that both subunits were expressed and that the purified heteroallelic enzymes, were catalytically active. As expected, the produced proteins displayed enzymatic activities levels lower than the predicted catalytic activity, calculated by averaging in vitro PAH activities from both alleles, and were strongly dependent on the proteins subunit composition. The obtained data suggest that interactions between the studied hPAH subunits, namely the I65T, R261Q, R270K and V388M, and the wild-type protein occurred. As postulated, this phenomenon could be a source of phenotypic variation in genetic diseases involving multimeric proteins.
Keywords: Heteroallelic human phenylalanine hydroxylase; Dual expression system; Interallelic complementation;

Comparison of protective effects of aspirin, d-penicillamine and vitamin E against high glucose-mediated toxicity in cultured endothelial cells by Jing Zhang; Mark Slevin; Yasotha Duraisamy; John Gaffney; Christopher A Smith; Nessar Ahmed (551-557).
This study compared the protective effects of three different anti-glycation compounds, aspirin, d-penicillamine and vitamin E, against high glucose and advanced glycation endproduct (AGE) mediated toxicity in cultured bovine aortic endothelial cells using two approaches. Their proliferation was assessed in culture in different concentrations of glucose (5.5–100 mmol/l) with and without these inhibitors. A monolayer of cultured endothelial cells was wounded and recovery at the wound site was measured following exposure to different concentrations of glucose with and without inhibitors. The ability of these compounds to protect cultured endothelial cells following exposure to bovine serum albumin-derived advanced glycation endproducts (BSA-AGE) was also studied. Addition of glucose to cultured endothelial cells inhibited their proliferation in a dose dependent manner. All three compounds protected against the anti-proliferative effects of high glucose, with vitamin E being the most effective. The migration of cultured endothelial cells following wounding was inhibited by increasing concentrations of glucose but was maintained in the presence of all three anti-glycation compounds with vitamin E, again giving the greatest protection. Vitamin E was also the most effective at protecting against the anti-proliferative effects of BSA-AGE. d-penicillamine was not as effective as vitamin E whereas aspirin offered no significant protection against AGE-induced cellular toxicity. Our studies suggest that compounds, such as vitamin E, with combined antiglycation and antioxidant properties offer maximum therapeutic potential in protection against high glucose and AGE-mediated cellular toxicity.
Keywords: Glycation; Advanced glycation endproduct; Aspirin; d-penicillamine; Vitamin E; Wound healing; Endothelial cell; Diabetes; Antioxidant;

Erythrocyte and plasma protein modification in alcoholism: A possible role of acetaldehyde by Olga V. Tyulina; Valentina D. Prokopieva; Alexander A. Boldyrev; Peter Johnson (558-563).
Analysis of the oxidative modification of plasma and erythrocyte ghost proteins of chronic alcoholic subjects and healthy non-alcoholics has been performed. It was found that increased levels of protein carbonyls in both plasma and erythrocyte ghosts from alcoholic subjects occurred in comparison to the levels found in preparations from non-alcoholics. Plasma proteins from alcoholic subjects did not show evidence of cross-linking, although plasma protein concentration and composition were changed. In alcoholic subjects who displayed no evidence of abnormal erythrocyte morphology no cross-linking of erythrocyte ghost proteins was detectable, whereas the ghosts obtained from alcoholic subjects who displayed morphologically abnormal erythrocytes contained cross-linked proteins. The in vitro treatment with acetaldehyde of erythrocytes from non-alcoholics caused increased levels of protein carbonyls and cross-linking products in erythrocyte ghost preparations which were similar to those found in severe alcoholics. It is concluded that chronic alcohol consumption can cause abnormal erythrocyte morphology and increased erythrocyte fragility as a result of oxidation and cross-linking of erythrocyte ghost proteins. These effects can be ascribed, in part, to exposure of erythrocytes to circulatory acetaldehyde which is a product of ethanol metabolism.
Keywords: Acetaldehyde; Alcoholism; Erythrocyte; Plasma; Protein cross-linking; Protein oxidation;

The decrease of NAD(P)H has a prominent role in dopamine toxicity by P. Giménez-Xavier; C. Gómez-Santos; E. Castaño; R. Francisco; J. Boada; M. Unzeta; E. Sanz; S. Ambrosio (564-574).
We characterized dopamine toxicity in human neuroblastoma SH-SY5Y cells as a direct effect of dopamine on cell reductive power, measured as NADH and NADPH cell content. In cell incubations with 100 or 500 μM dopamine, the accumulation of dopamine inside the cell reached a maximum after 6 h. The decrease in cell viability was 40% and 75%, respectively, after 24 h, and was not altered by MAO inhibition with tranylcypromine. Dopamine was metabolized to DOPAC by mitochondrial MAO and, at 500 μM concentration, significantly reduced mitochondrial potential and oxygen consumption. This DA concentration caused only a slight increase in cell peroxidation in the absence of Fe(III), but a dramatic decrease in NADH and NADPH cell content and a concomitant decrease in total cell NAD(P)H/NAD(P)+ and GSH/GSSG and in mitochondrial NADH/NAD+ ratios. Dopaminechrome, a product of dopamine oxidation, was found to be a MAO-A inhibitor and a strong oxidizer of NADH and NADPH in a cell-free system. We conclude that dopamine may affect NADH and NADPH oxidation directly. When the intracellular concentrations of NAD(P)H and oxidized dopamine are similar, NAD(P)H triggers a redox cycle with dopamine that leads to its own consumption. The time-course of NADH and NADPH oxidation by dopamine was assessed in cell-free assays: NAD(P)H concentration decreased at the same time as dopamine oxidation advanced. The break in cell redox equilibrium, not excluding the involvement of free oxygen radicals, could be sufficient to explain the toxicity of dopamine in dopaminergic neurons.
Keywords: Dopamine; SH-SY5Y cell; NADH; NADPH; NAD(P)H/NAD(P)+;