BBA - Molecular Basis of Disease (v.1535, #2)

Phenotype determination of anti-GM3 positive cells in atherosclerotic lesions of the human aorta by Yuri V. Bobryshev; Reginald S.A. Lord; Natalia K. Golovanova; Elena V. Gracheva; Natalia D. Zvezdina; Nina V. Prokazova (87-99).
Earlier we reported that atherosclerotic plaques contain cells which were specifically and very intensively stained with anti-GM3 antibodies although no GM3 positive cells were detected in the normal non-diseased arterial intima. Because of their lipid inclusions, GM3 positive cells in atherosclerotic lesions seemed to be foam cells but their origin needed clarification. Using an immunohistochemical technique in the present work, we showed that some of these foam cells contained CD68 antigen. However, the most intense accumulation of GM3 occurred in the areas composed of foam cells which did not stain with any cell type-specific antibodies, including antibodies to macrophages (anti-CD68) and smooth muscle cells (anti-smooth muscle α-actin), perhaps, because the cell type-specific antigens were lost during the transformation of intimal cells into foam cells. Ultrastructural analysis of the areas where foam cells overexpressed GM3 demonstrated that some foam cells lacked both a basal membrane and myofilaments but contained a large number of secondary lysosomes and phagolysosomes, morphological features which might indicate their macrophage origin. Other foam cells contained a few myofilaments and fragments of basal membrane around their plasmalemmal membrane, suggesting a smooth muscle cell origin. These observations indicate that accumulation of excessive amounts of GM3 occurs in different cell types transforming into foam cells. We suggest that up-regulation of GM3 synthesis in intimal cells might be an essential event in foam cell formation. Shedding of a large number of membrane-bound microvesicles from the cell surface of foam cells was observed in areas of atherosclerotic lesions corresponding to extracellular GM3 accumulation. We speculate that extracellularly localised GM3 might affect the differentiation and modification of intimal cells in atherosclerotic lesions.
Keywords: GM3; Anti-GM3 antibody; Oxidized LDL; Intima; Human artery; Atherosclerosis; Immunohistochemistry; Electron microscopy;

Cationic liposomes have been used successfully for DNA delivery to airway cells in vitro and are being tested in human clinical trials for their efficacy in cystic fibrosis transmembrane conductance regulator (CFTR) gene delivery in cystic fibrosis patients. While cationic liposomes are effective for transfection of airway cells in culture, they have not been effectively used for gene delivery to human airway cells in vivo. Several barriers in cystic fibrosis lungs, including increased amounts of mucus, phagocytic cell activity and cytokine-rich milieu caused by inflammation, may cause inhibition of gene transfection. As presented in this paper, we examined the effects of inflammatory cytokines on cationic lipid-mediated transfection of model airway cells. The results of these experiments indicate that tumor necrosis factor (TNF)-α dramatically inhibits Lipofectin-mediated transfection efficiency of H441 cells. Addition of anti-TNF-α neutralizing antibody results in recovery of efficiency. Results of temporal studies are consistent with the concept that TNF-α reduces transfection efficiency by a mechanism(s) other than or in addition to gene expression. These results are corroborated by fluorescence microscopic experiments which demonstrate that endocytosis of lipoplex is altered in the presence of TNF-α.
Keywords: Transfection; Cationic liposome; Tumor necrosis factor-α; Lung epithelial cell;

The reaction of lens proteins with sugars over time results in the formation of protein-bound advanced glycation end products (AGEs). The most damaging element of AGE formation may be the synthesis of protein-protein cross-links in long-lived proteins, such as collagen or lens crystallins. A quantitative cross-linking assay, involving the sugar-dependent incorporation of [U-14C]lysine into protein, was employed to determine the efficacy of a variety of potential cross-linking inhibitors. Reaction mixtures contained 5.0 mM l-threose, 2.5 μCi [14C]lysine (1.0 mCi/mmole), 5.0 mg/ml bovine lens proteins, 0–10 mM inhibitor and 1.0 mM DTPA in 100 mM phosphate buffer, pH 7.0. Of 17 potential inhibitors tested, 11 showed 50% inhibition or less at 10 mM. The dicarbonyl-reactive compounds 2-aminoguanidine, semicarbazide and o-phenylenediamine inhibited 50% at 2.0 mM, whereas 10 mM dimethylguanidine had no effect. Several amino acids failed to compete effectively with [14C]lysine in the cross-linking assay; however, cysteine inhibited 50% at 1.0 mM. This was likely due to the sulfhydryl group of cysteine, because 3-mercaptopropionic acid and reduced glutathione exhibited similar activity. Sodium metabisulfite had the highest activity, inhibiting 50% at only 0.1–0.2 mM. Protein dimer formation, as determined by SDS-PAGE, was inhibited in a quantitatively similar manner. The dicarbonyl-reactive inhibitors and the sulfur-containing compounds produced similar inhibition curves for [14C]lysine incorporation over a 3 week assay with 250 mM glucose. A much lesser effect was observed on either the incorporation of [14C]glucose, or on fluorophore formation (360/420 nm), suggesting that non-cross-link fluorophores were also formed. The inhibitor data were consistent with cross-linking by a dicarbonyl intermediate. This was supported by the fact that the inhibitors were uniformly less effective when the 5.0 mM threose was replaced by either 3.0 mM 3-deoxythreosone or 3.0 mM threosone.
Keywords: Glycation; Protein cross-linking; l-Threose; Advanced glycation end product; Cataract; Reducing agent; 2-Aminoguanidine; Metabisulfite;

In mouse mammary epithelial C127 cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR), chloride efflux, measured with the Cl-sensitive dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), was stimulated by activation of protein kinase A with cyclic AMP elevating agents forskolin plus 3-isobutyl-1-methyl-xanthine (IBMX) and, to a less extent, by activation of protein kinase C with the phorbol 12-myristate 13-acetate (PMA). Conversely, bicarbonate influx, determined by intracellular alkalinization of cells incubated with the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluoresceintetraacetoxymethyl ester (BCECF-AM), was stimulated by cyclic AMP elevation, but not by PMA. Patch clamp analysis revealed that PMA activated a Cl current with the typical biophysical characteristics of swelling-activated current and not of CFTR.
Keywords: Cystic fibrosis; CFTR; HCO3 influx; Phorbol ester (PMA); Swelling-activated chloride current; Patch clamp;

Oligomerization of Escherichia coli enterotoxin b through its C-terminal hydrophobic α-helix by Vincent Labrie; Josée Harel; J.Daniel Dubreuil (128-133).
Using a chemical cross-linker and gel electrophoresis or a dot blot overlay assay, we studied protein–protein interaction of STb toxin, a 48-residue amphiphilic polypeptide causing intestinal disorders. For the first time, we report on the oligomerization property of STb. This enterotoxin forms hexamers and heptamers in a temperature-independent fashion in presence or absence of its receptor (sulfatide) anchored in a 50-nm liposome or as a free molecule. Full STb structure integrity is necessary for its oligomerization as this process is not observed under reducing conditions in the presence of β-mercaptoethanol. STb treatment with tetramethylurea (TMU) and different detergents prevented oligomerization. Site-directed mutagenesis decreasing overall STb hydrophobicity in the hydrophobic α-helix resulted in the incapacity to form oligomers. Taken together, these data suggest that the C-terminal hydrophobic α-helix corresponds to the domain of STb–STb inter-binding where hydrophobic interaction is involved.
Keywords: Heat-stable toxin b; Oligomerization; Hydrophobic α-helix; (Escherichia coli);

Apolipoprotein E acts to increase nitric oxide production in macrophages by stimulating arginine transport by C.A. Colton; M. Czapiga; J. Snell-Callanan; O.N. Chernyshev; M.P. Vitek (134-144).
Previous studies have shown that apolipoprotein E (apoE) plays a role in immune function by modulating tissue redox balance. Using a mouse macrophage cell line (RAW 264.7), we have examined the mechanism by which apoE regulates nitric oxide (NO) production in macrophages. ApoE potentiates NO production in immune activated RAW cells in combination with lipopolysaccharide or polyinosinic:polycytidylic acid (PIC), agents known to induce expression of inducible nitric oxide synthase mRNA and protein. The effect is not observed with apolipoprotein B or heat-inactivated apoE. The combination of PIC plus apoE produced more NO than the level expected from an additive effect of PIC and apoE alone. Furthermore, this increase was observed at submaximal extracellular arginine concentrations, suggesting that apoE altered arginine (substrate) availability. Examination of [3H]arginine uptake across the cell membrane demonstrated that arginine uptake was increased by PIC but further increased by PIC plus apoE. Treatment of RAW cells with apoE was associated with an increased apparent V max and decreased affinity for arginine as well as a switch in the induction of mRNA for subtypes of cationic amino acid transporters (CAT). Treatment of RAW cells with PIC plus apoE resulted in the loss of detectable CAT1 mRNA and expression of CAT2 mRNA. Regulation of arginine availability is a novel action of apoE on the regulation of macrophage function and the immune response.
Keywords: Arginine transport; Apolipoprotein E; Macrophage; Nitric oxide; Lipid; Alzheimer’s disease;

Acrolein inhibits respiration in isolated brain mitochondria by Matthew J Picklo; Thomas J Montine (145-152).
Lipid peroxidation is elevated in diseased regions of brain in several neurodegenerative diseases. Acrolein (2-propenal) is a major cytotoxic product of lipid peroxidation and its adduction to neuronal proteins has been demonstrated in diseased brain regions from patients with Alzheimer’s disease. Mitochondrial abnormalities are implicated in several neurodegenerative disorders, and mitochondria are targets of alkenal adduction in vivo. We examined the effects of acrolein upon multiple endpoints associated with the mitochondrial involvement in neurodegenerative disease. Acrolein inhibited state 3 respiration with an IC50 of approx. 0.4 μmol/mg protein; however, there was no reduction in activity of complexes I–V. This inhibition was prevented by glutathione and N-acetylcysteine. Acrolein did not alter mitochondrial calcium transporter activity or induce cytochrome c release. These studies indicate that acrolein is a potent inhibitor of brain mitochondrial respiration.
Keywords: Acrolein; Lipid peroxidation; Mitochondria; Neurodegeneration; Alzheimer’s disease; Respiration;

The effect of Pb2+ on the structure and hydroxyapatite binding properties of osteocalcin by T.L. Dowd; J.F. Rosen; L. Mints; C.M. Gundberg (153-163).
Lead toxicity is a major environmental health problem in the United States. Bone is the major reservoir for body lead. Although lead has been shown to impair bone metabolism in animals and at the cellular level, the effect of Pb2+ at the molecular level is largely unknown. We have used circular dichroism (CD), and a hydroxyapatite binding assay to investigate the effect of Pb2+ on the structure and mineral binding properties of osteocalcin, a noncollagenous bone protein. The CD data indicate Pb2+ induces a similar structure in osteocalcin as Ca2+ but at 2 orders of magnitude lower concentration. These results were explained by the more than 4 orders of magnitude tighter binding of Pb2+ to osteocalcin (K d=0.085 μM) than Ca2+ (K d=1.25 mM). The hydroxyapatite binding assays show that Pb2+ causes an increased adsorption to hydroxyapatite, similar to Ca2+, but at 2–3 orders of magnitude lower concentration. Low Pb2+ levels (1 μM) in addition to physiological Ca2+ levels (1 mM) caused a significant (40%) increase in the amount of mineral bound osteocalcin as compared to 1 mM Ca2+ alone. These results suggest a molecular mechanism of Pb2+ toxicity where low Pb2+ levels can inappropriately perturb Ca2+ regulated processes. In-vivo, the increased mineral bound osteocalcin could play a role in the observed low bone formation rates and decreased bone density observed in Pb2+-intoxicated animals.
Keywords: Osteocalcin; Lead; Calcium; Circular dichroism; Hydroxyapatite;

Transmissible spongiform encephalopathies are believed to be caused by an infectious form of the prion protein, designated PrPSc. The concentration of PrPSc is often poorly correlated to the level of infectivity. Infectivity can be measured in two ways, namely endpoint titration and the incubation time assay, but patterns of infectivity vary depending on which method is used. These discrepancies can be explained by variation in the aggregation state of PrPSc. Both methods of measuring infectivity are modelled mathematically, and the theoretical results are in agreement with published data. It was found to be theoretically impossible to characterise prion infectivity by a multiple of a single quantity representing ‘one prion’, no matter how it is measured. Infectivity is instead characterised by both the number and sizes of the PrPSc aggregates. Apparent discrepancies arise when these complexities are reduced to a single number.
Keywords: Prion disease; Mathematical model; One-hit model; Incubation time assay; Protein aggregation; Amyloid;

It is well-established that high levels of cAMP or glucose can produce insulin resistance. The aim of this study was to characterize the interaction between these agents and insulin with respect to adipose tissue/muscle glucose transporter isoform (glucose transporter 4, GLUT4) gene regulation in cultured 3T3-F442A adipocytes and to further elucidate the GLUT4-related mechanisms in insulin resistance. Insulin (104 μU/ml) treatment for 16 h clearly increased GLUT4 mRNA level in cells cultured in medium containing 5.6 mM glucose but not in cells cultured in medium with high glucose (25 mM). 8-Bromo-cAMP (1 or 4 mM) or N 6-monobutyryl cAMP, a hydrolyzable and a non-hydrolyzable cAMP analog, respectively, markedly decreased the GLUT4 mRNA level irrespective of glucose concentrations. In addition, these cAMP analogs also inhibited the upregulating effect of insulin on GLUT4 mRNA level. Interestingly, the tyrosine phosphatase inhibitor vanadate (1–50 μM) clearly increased GLUT4 mRNA level in a time- and concentration-dependent manner. Furthermore, cAMP-induced inhibition of the insulin effect was also prevented by vanadate. In parallel to the effects on GLUT4 gene expression, both insulin, vanadate and cAMP produced similar changes in cellular GLUT4 protein content and cAMP impaired the effect of insulin to stimulate 14C-deoxyglucose uptake. In contrast, insulin, vanadate or cAMP did not alter insulin receptor (IR) mRNA or the cellular content of IR protein. In conclusion: (1) Both insulin and vanadate elicit a stimulating effect on GLUT4 gene expression in 3T3-F442A cells, but a prerequisite is that the surrounding glucose concentration is low. (2) Cyclic AMP impairs the insulin effect on GLUT4 gene expression, but this is prevented by vanadate, probably by enhancing the tyrosine phosphorylation of signalling peptides and/or transcription factors. (3) IR gene and protein expression is not altered by insulin, vanadate or cAMP in this cell type. (4) The changes in GLUT4 gene expression produced by cAMP or vanadate are accompanied by similar alterations in GLUT4 protein expression and glucose uptake, suggesting a role of GLUT4 gene expression for the long-term regulation of cellular insulin action on glucose transport.
Keywords: Insulin resistance; Gene expression; Glucose transporter; Glucose uptake; Cyclic AMP; Vanadate; Preadipocytes; Cell culture;

Evaluation of oxidative stress during apoptosis and necrosis caused by carbon tetrachloride in rat liver by Fang Sun; Eri Hamagawa; Chihiro Tsutsui; Yoshiko Ono; Yukako Ogiri; Shosuke Kojo (186-191).
After 12, 18, and 24 h of oral administration of carbon tetrachloride (as a 1:1 mixture with mineral oil: 4 ml/kg body weight) to rats, the activity of caspase-3-like protease in the liver increased significantly compared to that in the control group that was given mineral oil (4 ml/kg). In plasma, the activity of caspase-3 was barely detectable in the control rat, but increased significantly 24 h after drug administration along with a dramatic increase in glutamate oxaloacetate transaminase. These results indicate that carbon tetrachloride causes apoptosis in the liver by activating caspase-3, which is released to plasma by secondary necrosis. After 18 and 24 h of carbon tetrachloride administration, the liver concentration of hydrophilic vitamin C was decreased significantly, while that of hydrophobic vitamin E was not affected. The plasma concentration of vitamins C and E was not influenced significantly. These results suggest that carbon tetrachloride induces oxidative stress mainly in the aqueous phase of the liver cell.
Keywords: Apoptosis; Carbon tetrachloride; Caspase-3; Necrosis; Vitamin C; Vitamin E;

Differentially expressed DNA sequences following recovery from unilateral testicular torsion in rat by Farag A. Ahmed; Anne M. Jequier; James M. Cummins; James Whelan (192-199).
The molecular response during recovery from torsion-induced stress in the testis is diverse with a variety of mechanisms. In this study, using unilateral testicular torsion in rat as a model, we used subtractive hybridisation to identify differentially expressed DNA sequences in the torsioned and control testes. Three genes were identified as being down regulated in the torsioned testis compared with controls: Control Testis genes 1, 2 and 3 (CT1, CT2 and CT3). Two genes were up regulated in the torsioned testes: Torsioned Testes genes 1 and 2 (TT1 and TT2). Differential expression was confirmed by Reverse Northern blot analysis. An homology search revealed that CT1 had 88% homology with rat metallothionein cDNA; CT2 had 81% homology with rat cell surface antigen in MHC class I, but no homology could be found for CT3. TT1 had 92% identity with rat Rieske iron–sulphur protein mRNA whereas TT2 had 73% identity with a human clone of unknown function (RP 11-252D22). These results indicate that changes in gene expression occur following torsion induced stress, and that identification of differentially expressed genes may provide insights into the mechanisms of cellular tissue damage in this model.
Keywords: Torsion; Differential gene expression; (Testis);

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln3+ binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln3+ binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln3+ binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N I), non-cooperative strong sites (N S) and non-cooperative weak sites (N W) with binding constants in decreasing order: K I>K S>K W. The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln3+ concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln3+ binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln3+ concentration. At the range of [Ln3+]/[Hb]<2, the marked increase of oxygen affinity (P 50 decrease) with the Ln3+ concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln3+ concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln3+ binding. This was indicated by the changes in secondary structure characterized by the decrease of α-helix content.
Keywords: Lanthanide ion; Hemoglobin; Conformational change; 2,3-Diphosphoglycerate hydrolysis; Oxygen affinity;