BBA - Molecular Basis of Disease (v.1741, #1-2)

Different response of the knockout mice lacking b-series gangliosides against botulinum and tetanus toxins by Masaru Kitamura; Shizunobu Igimi; Keiko Furukawa; Koichi Furukawa (1-3).
We assessed the response in knockout mice lacking the b-series (GD2, GD1b, GT1b and GQ1b) gangliosides against Clostridium botulinum (types A, B and E) and tetani toxins. We found that botulinum toxins were fully toxic, while tetanus toxin was much less toxic in the knockout mice. Combining the present results with our previous finding that tetanus toxin and botulinum types A and B toxins showed essentially no toxic activity in the knockout mice lacking both the a-series and b-series gangliosides (complex gangliosides), we concluded that the b-series gangliosides is the major essential substance for tetanus toxin, while b-series gangliosides may be not the essential substance for botulinum toxins, at the initial step during the intoxication process in mouse.
Keywords: Botulinum toxin; Tetanus toxin; Toxin; Clostridium; Ganglioside; Receptor; Knockout mouse;

JNK and PI3k/Akt signaling pathways are required for establishing persistent SARS-CoV infection in Vero E6 cells by Tetsuya Mizutani; Shuetsu Fukushi; Masayuki Saijo; Ichiro Kurane; Shigeru Morikawa (4-10).
Persistence was established after most of the SARS-CoV-infected Vero E6 cells died. RNA of the defective interfering virus was not observed in the persistently infected cells by Northern blot analysis. SARS-CoV diluted to 2 PFU failed to establish persistence, suggesting that some particular viruses in the seed virus did not induce persistent infection. Interestingly, a viral receptor, angiotensin converting enzyme (ACE)-2, was down-regulated in persistently infected cells. G418-selected clones established from parent Vero E6 cells, which were transfected with a plasmid containing the neomycin resistance gene, were infected with SARS-CoV, resulting in a potential cell population capable of persistence in Vero E6 cells. Our previous studies demonstrated that signaling pathways of extracellular signal-related kinase (ERK1/2), c-Jun N-terminal protein kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3′-kinase (PI3K)/Akt were activated in SARS-CoV-infected Vero E6 cells. Previous studies also showed that the activation of p38 MAPK by viral infection-induced apoptosis, and a weak activation of Akt was not sufficient to protect from apoptosis. In the present study, we showed that the inhibitors of JNK and PI3K/Akt inhibited the establishment of persistence, but those of MAPK/ERK kinase (MEK; as an inhibitor for ERK1/2) and p38 MAPK did not. These results indicated that two signaling pathways of JNK and PI3K/Akt were important for the establishment of persistence in Vero E6 cells.
Keywords: SARS-CoV; Persistent infection; c-Jun N-terminal protein kinase; Phosphatidylinositol 3′-kinase/Akt;

The maintenance of phospholipid asymmetry in membrane bilayers is a paradigm in cell biology. However, the mechanisms and proteins involved in phospholipid translocation are still poorly understood. Members of the type 4 subfamily of P-type ATPases have been implicated in the translocation of phospholipids from the outer to the inner leaflet of membrane bilayers. In humans, several inherited disorders have been identified which are associated with loci harboring type 4 P-type ATPase genes. Up to now, one inherited disorder, Byler disease or progressive familial intrahepatic cholestasis type 1 (PFIC1), has been directly linked to mutations in a type 4 P-type ATPase gene. How the absence of an aminophospholipid translocase activity relates to this severe disease is, however, still unclear. Studies in the yeast Saccharomyces cerevisiae have recently identified important roles for type 4 P-type ATPases in intracellular membrane- and protein-trafficking events. These processes require an (amino)phospholipid translocase activity to initiate budding or fusion of membrane vesicles from or with other membranes. The studies in yeast have greatly contributed to our cell biological insight in membrane dynamics and intracellular-trafficking events; if this knowledge can be translated to mammalian cells and organs, it will help to elucidate the molecular mechanisms which underlie severe inherited human diseases such as Byler disease.
Keywords: Type 4 P-type ATPase; Aminophospholipid translocase; Phospholipid asymmetry; Byler disease; Membrane- and protein-trafficking; Yeast Saccharomyces cerevisiae;

Expression of cell survival/death genes: Bcl-2 and Bax at the rate of colon cancer prognosis by Monika Paul-Samojedny; Danuta Kokocińska; Arkadiusz Samojedny; Urszula Mazurek; Robert Partyka; Zbigniew Lorenz; Tadeusz Wilczok (25-29).
The rate of tumour growth is dependent on the balance between proliferation and apoptosis at all stages of carcinogenesis. Apoptosis inhibition, in turn, depends partly on the balance between expression of two cell death regulatory genes, Bcl-2 and Bax. Colon cancer has long been associated with disturbances in apoptosis regulation. The aim of our study was to determine the expression levels of Bcl-2 and Bax mRNAs in 1 μg sample of total RNA obtained from normal colon and colon adenocarcinoma. This study was intended to evaluate possible differences in Bcl-2 and Bax mRNA levels at particular stages of colon adenocarcinoma classified according to Duke's system. The apoptotic frequency (represented by Bax mRNA copy number) was inversely proportional to the decrease of Bcl-2 gene expression. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed to confirm apoptosis.
Keywords: Apoptosis and proliferation; Bcl-2 gene; Bax gene; Colon cancer prognosis; Quantitative mRNA analysis;

Reduced urinary excretion of sulfated polysaccharides in diabetic rats by Cilene R. de Lima; Jair A.K. Aguiar; Yara M. Michelacci (30-41).
The aim of the present study was to further understand the changes in renal filtration that occur in the early stages of diabetes mellitus. Diabetes was induced in male Wistar rats by a single injection of streptozotocin. Glycemia, body weight, 24-h urine volume and urinary excretion of creatinine, protein and glycosaminoglycans were measured 10 and 30 days after diabetes induction. All the diabetic animals used in the present study were hyperglycemic, did not gain weight, and presented proteinuria and creatinine hyperfiltration. In contrast, the glycosaminoglycan excretion decreased. Dextran sulfates of different molecular weights (6.0 to 11.5 kDa) were administered to the diabetic rats, and to age-matched, sham-treated controls. Most of the dextran sulfate was excreted during the first 24 h, and the amounts excreted in the urine were inversely proportional to the dextran sulfate molecular weight for all groups. Nevertheless, diabetic rats excreted less and accumulated more dextran sulfate in kidney and liver, as compared to controls. These differences, which were observed only for the dextran sulfates of higher molecular weights (>7 kDa), increased with the duration of diabetes. Our findings suggest differential renal processing mechanisms for proteins and sulfated polysaccharides, with the possible involvement of kidney cells.
Keywords: Diabetes mellitus; Renal filtration; Dextran sulfate; Glycosaminoglycan;

Dermal fibroblasts from pseudoxanthoma elasticum patients have raised MMP-2 degradative potential by Daniela Quaglino; Luigi Sartor; Spiridione Garbisa; Federica Boraldi; Antonietta Croce; Alberto Passi; Giancarlo De Luca; Roberta Tiozzo; Ivonne Pasquali-Ronchetti (42-47).
Cultured fibroblasts from the dermis of normal subjects and of Pseudoxanthoma elasticum (PXE) patients were analysed for enzyme activity, protein and mRNA expression of metalloproteases (MMP-2, MMP-3, MMP-9, MT1-MMP) and of their specific inhibitors (TIMP-1, TIMP-2 and TIMP-3). MMP-3, MMP-9 and TIMP-3 mRNAs and proteins failed to be detected in both the medium and the cell layer of both controls and PXE patients. MMP-2 mRNA was significantly more expressed in PXE than in control cell lines, whereas MT1-MMP, TIMP-1 and TIMP-2 mRNAs appeared unchanged. MMP-2 was significantly higher in the cell extracts from PXE fibroblasts than in control cells, whereas differences were negligible in the cell medium. Data suggest that PXE fibroblasts have an increased proteolytic potential, and that MMP-2 may actively contribute to connective tissue alterations in this genetic disorder.
Keywords: Matrix degradation; MMP-2; Pseudoxanthoma elasticum; Skin fibroblast; TIMP;

Supplementation with N-acetylcysteine and taurine failed to restore glutathione content in liver of streptozotocin-induced diabetics rats but protected from oxidative stress by Stefania Patriarca; Anna Lisa Furfaro; Cinzia Domenicotti; Patrizio Odetti; Damiano Cottalasso; Umberto M. Marinari; M. Adelaide Pronzato; Nicola Traverso (48-54).
Rats were rendered diabetic with streptozotocin and supplemented or not with N-acetylcysteine (NAC) and taurine (TAU). The liver was examined for the quantity of glutathione (GSH), both total and oxidised (GSSG), by HPLC assay. Moreover, the liver expression of gamma-glutamyl-cysteine synthetase, cysteine dioxygenase and heme oxygenase 1 was evaluated. Streptozotocin-diabetic rats showed decreased levels of liver glutathione (GSH); dietary supplementation with the antioxidants NAC and TAU failed to restore liver GSH to the level of control rats. Gamma-glutamyl-cysteine synthetase expression was not reduced in the diabetic rats, so the low hepatic GSH level in the supplemented diabetic rats cannot be ascribed to decreased expression of the biosynthetic key enzyme. Moreover, the diabetic rats showed no evidence of increased expression of cysteine dioxygenase, which could have indicated that NAC-derived cysteine was consumed in metabolic pathways different from GSH synthesis. However, NAC+TAU treatment provided partial protection from glutathione oxidation in the liver of diabetic rats; moreover, the antioxidant treatment reduced the hepatic overexpression of heme oxygenase 1 (HO-1) mRNA which was detected in the diabetic rats. In conclusion, although NAC was not able to restore liver GSH levels, the antioxidant treatment restrained GSH oxidation and HO-1 overexpression, which are markers of cellular oxidative stress: diabetic rats probably exploit NAC as an antioxidant itself rather than as a GSH precursor.
Keywords: Streptozotocin; Taurine; Gamma-glutamyl-cysteine synthetase; Cysteine dioxygenase; Heme oxygenase 1;

Astrocytes are susceptible to HIV-1 infection. We have recently demonstrated that human mannose receptor (hMR) is directly involved in CD4-independent HIV-1 infection of astrocytes. The apparent paradox between the vivid binding affinity of HIV-1 gp120 protein to hMR and the low efficiency of hMR-mediated HIV-1 infection raises the possibility that HIV-1 binding to hMR alone may negatively affect astrocyte function. In this study, we examined the relationship between HIV-1 interaction with hMR and the production of matrix metalloproteinases (MMPs) in astrocytes. We took advantage of an astroglial cell line U87.MR stably expressing hMR as an in vitro astrocyte model system and human primary astrocytes, and demonstrated that HIV-1 binding to astrocytes induced the production of MMP-2. This induction appeared to be most potent with M-tropic HIV-1 viruses. Increased MMP-2 production was not due to hMR-mediated HIV-1 entry and/or HIV-1 viral gene expression, as the transfection of HIV-1 proviral DNA did not result in MMP-2 production, and the infection of AT-2-treated HIV-1 viruses did not inhibit MMP-2 production. Direct involvement of hMR in HIV-induced MMP-2 production was confirmed by the inhibition of the yeast mannan, an hMR ligand antagonist, and an anti-hMR serum. Furthermore, HIV-induced MMP-2 production in astrocytes was shown to involve hMR-mediated intracellular signaling. Taken together, these results suggest that HIV-1 binding to astrocytes in the absence of HIV-1 viral entry is sufficient to alter astrocyte function through hMR-mediated intracellular signaling. In addition, these results provide new evidence to support the notion that hMR is capable of eliciting intracellular signaling upon ligand binding.
Keywords: HIV-1; Human mannose receptor; Intracellular signaling; Astrocyte; Matrix metalloproteinase 2 production;

Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: Implications for Parkinson's disease by Firoj Hossain Khan; Tanusree Sen; Arpan Kumar Maiti; Sirsendu Jana; Uttara Chatterjee; Sasanka Chakrabarti (65-74).
Several studies on mitochondrial functions following brief exposure (5–15 min) to dopamine (DA) in vitro have produced extremely variable results. In contrast, this study demonstrates that a prolonged exposure (up to 2 h) of disrupted or lysed mitochondria to DA (0.1–0.4 mM) causes a remarkable and dose-dependent inhibition of complex I and complex IV activities. The inhibition of complex I and complex IV activities is not prevented by the antioxidant enzyme catalase (0.05 mg/ml) or the metal-chelator diethylenetriaminepentaacetic acid (0.1 mM) or the hydroxyl radical scavengers like mannitol (20 mM) and dimethyl sulphoxide (20 mM) indicating the non-involvement of ·OH radicals and Fenton's chemistry in this process. However, reduced glutathione (5 mM), a quinone scavenger, almost completely abolishes the DA effect on mitochondrial complex I and complex IV activities, while tyrosinase (250 units/ml) which catalyses the conversion of DA to quinone products dramatically enhances the former effect. The results suggest the predominant involvement of quinone products instead of reactive oxygen radicals in long-term DA-mediated inactivation of complex I and complex IV. This is further indicated from the fact that significant amount of quinones and quinoprotein adducts (covalent adducts of reactive quinones with protein thiols) are formed during incubation of mitochondria with DA. Monoamine oxidase A (MAO-A) inhibitor clorgyline also provides variable but significant protection against DA induced inactivation of complex I and complex IV activities, presumably again through inhibition of quinoprotein formation. Mitochondrial ability to reduce tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in presence of a respiratory substrate like succinate (10 mM) is also reduced by nearly 85% following 2 h incubation with 0.4 mM DA. This effect of DA on mitochondrial function is also dose-dependent and presumably mediated by quinone products of DA oxidation. The mitochondrial dysfunction induced by dopamine during extended periods of incubation as reported here have important implications in the context of dopaminergic neuronal death in Parkinson's disease (PD).
Keywords: Dopamine; Mitochondria; Electron transport chain; Quinones; Oxygen radicals; Parkinson's disease;

The hepatitis B virus X protein up-regulates lymphotoxin α expression in hepatocytes by Sang Hun Lee; Sung Gyoo Park; Seung Oe Lim; Guhung Jung (75-84).
Hepatitis B virus X protein (HBx) is involved in intrahepatic inflammatory processes by inducing several pro-inflammatory cytokines. It has been suggested that these inflammatory processes play an important role in causing hepatocarcinogenesis. In this study, we investigated the role of HBx in the expression of lymphotoxin α (LTα) in hepatoma cells such as Huh-7 and Chang. Our experiments showed that (i) transient HBx expression in Huh-7 cells up-regulated LTα mRNA expression; (ii) this up-regulation was predominantly affected by HBx-induced nuclear factor-κB (NF-κB) activation. In addition, the HBx-induced NF-κB activation was decreased by the treatment of LTα neutralizing antibodies in a dose-dependent manner. We conclude that HBx up-regulates LTα expression at the transcriptional level through an NF-κB-dependent mechanism and, therefore, the up-regulated LTα may be secreted and involved in the HBx-induced NF-κB activation.
Keywords: Lymphotoxin α; Hepatitis B virus X protein; Nuclear factor-κB; Tumor necrosis factor α;

Cloning, characterization and DNA immunization of an Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) by Klaus D. Erttmann; André Kleensang; Erik Schneider; Sven Hammerschmidt; Dietrich W. Büttner; Michaela Gallin (85-94).
In the search for Onchocerca volvulus antigens possibly involved in protection against human onchocerciasis, partial amino acid sequence analysis of one of the O. volvulus antigens of the serologically identified proteins showed a close relationship to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein family. Subsequent adult worm cDNA library screening and cloning produced a clone of 1650 bp. An open reading frame spans over 1020 bp encoding for a protein of 340 amino acids with an apparent molecular weight of 38 000. Comparison of the complete amino acid sequence identified this protein as a member of the GAPDH protein family. The recombinantly expressed protein shows GAPDH enzymatic activity as well as plasminogen-binding capacity. DNA sequence analysis of the corresponding gene revealed the presence of two introns. Using immunohistology Ov-GAPDH was observed in microfilariae, infective larvae, and adult male and female worms. Most striking was the labelling of the musculature of the body wall. Labelling was also observed in the pseudocoeloma cavity and in a subset of cell nuclei, suggesting additional, non-glycolytic functions of the Ov-GAPDH. Gene gun immunization with the DNA-construct in cattle led to specific humoral immune responses. Thus, the protective potential of the DNA-construct of Ov-GAPDH can be evaluated in vaccination trials using animal models such as the cattle/Onchocerca ochengi model.
Keywords: Glyceraldehyde-3-phosphate dehydrogenase; Filaria; Onchocerca volvulus; Plasminogen-binding; Protective antigen; DNA immunization;

Elevated formation of pyridinoline cross-links by profibrotic cytokines is associated with enhanced lysyl hydroxylase 2b levels by Annemarie J. van der Slot; Ernst A. van Dura; Elly C. de Wit; Jeroen DeGroot; Tom W.J. Huizinga; Ruud A. Bank; Anne-Marie Zuurmond (95-102).
The hallmark of fibrosis is the excessive accumulation of collagen. The deposited collagen contains increased pyridinoline cross-link levels due to an overhydroxylation of lysine residues within the collagen telopeptides. Lysyl hydroxylase 2b (LH2b) is the only lysyl hydroxylase consistently up-regulated in several forms of fibrosis, suggesting that an enhanced LH2b level is responsible for the overhydroxylation of collagen telopeptides. The present paper reports the effect of profibrotic cytokines on the expression of collagen, lysyl hydroxylases and lysyl oxidase in normal human skin fibroblasts, as well as the effect on pyridinoline formation in the deposited matrix. All three isoforms of TGF-β induce a substantial increase in LH2b mRNA levels, also when expressed relatively to the mRNA levels of collagen type I α2 (COL1A2). The TGF-β isoforms also clearly influence the collagen cross-linking pathway, since higher levels of pyridinoline cross-links were measured. Similar stimulatory effects on LH2b/COL1A2 mRNA expression and pyridinoline formation were observed for IL-4, activin A, and TNF-α. An exception was BMP-2, which has no effect on LH2b/COL1A2 mRNA levels nor on pyridinoline formation. Our data show for the first time that two processes, i.e., up-regulation of LH2b mRNA levels and increased formation of pyridinoline cross-links, previously recognized to be inherent to fibrotic processes, are induced by various profibrotic cytokines.
Keywords: Fibrosis; Lysyl hydroxylase 2b; Pyridinoline; Cytokine; TGF-β; Collagen;

Chronic depletion of glutathione (GSH) and minimal modification of LDL in vivo: its prevention by glutathione mono ester (GME) therapy by Namakkal Soorappan Rajasekaran; Srinivasan Sathyanarayanan; Niranjali S. Devaraj; Halagowder Devaraj (103-112).
A decline in reduced glutathione (GSH) level is associated with aging and free radical mediated diseases. The objective of this study was to determine whether the chronic depletion of extra cellular GSH causes oxidative damage to the circulating macromolecules such as lipoproteins. Decreased concentrations of plasma glutathione, vitamin E and ascorbic acid were recorded in the rats treated with buthionine sulfoximine (BSO), a selective GSH inhibitor. In LDL isolated from BSO-treated animals, the concentration of malondialdehyde (MDA) and conjugated dienes were significantly increased (P<0.01), whereas the levels of vitamin E were decreased (P<0.01). The analysis of total and LDL cholesterol revealed significant changes between the control and experimental groups. Of interest, altered concentrations of lyso-phosphatidyl choline (Lyso-PC) and phosphatidyl choline (PC) were recorded from the BSO mediated minimally modified LDL. A negative correlation between LDL-BDC/MDA and its antioxidant capacity was noted. Upon in vitro oxidation with CuSO4, the electrophoretic behavior of purified LDL-apoprotein-B on agarose gel showed an increased mobility in BSO-treated rats, indicative of in vivo modification of LDL to become susceptible for in vitro oxidation. The increased mobility of LDL (after in vitro oxidation) isolated from the BSO-treated animals correlates with a decrease in its amino groups, as determined by the trinitrobenzene sulfonic acid (TNBS) reactants. However, the mobility of LDL molecule was not altered due to BSO treatment in vivo. Interestingly, the minimal modification on LDL does not lead to any vascular damage in the dorsal aorta of the rats injected with BSO. The administration of glutathione monoester (GME), at a dose of 5 mmol/kg body weight, twice a day, for 30 days, to animals treated with l-buthionine-SR-sulfoximine (BSO, 4 mmol/kg body weight, twice a day, for 30 days) normalized the antioxidant status and prevented the minimal modifications on LDL. Thus, increasing the cellular GSH levels may trigger beneficial effects against oxidative stress.
Keywords: Glutathione monoester; Buthionine sulfoximine; LDL oxidation; Atherogenesis; Antioxidant-therapy;

We evaluated MAPK (Erk 1/2 and p38) signaling mechanisms of altered T-cell-mediated immune responses in thermal injury condition. Rats were subjected to 30% body surface scald burn, and their mesenteric lymph node (MLN) and Peyer's patch (PP) T cells were purified using nylon wool method. Activation of MAPKs, Erk 1/2 and p38 was assessed in T cells by determining its phosphorylation using immunoblot analysis, intracellular immunostaining and confocal microscopy. The results showed a down-regulation of Erk 1/2 and p38 activation in anti-CD3-stimulated T cells from thermally injured animals, compared to Erk 1/2 and p38 in sham rat T cells. The down-regulation of MAPKs in T cells was reversed by treatment of T cells with calcium agonist, ionomycin. These data indicate that attenuated MAPKs (Erk 1/2, p38) activation in thermally injured animals' T cells could result from derangement of Ca2+ mobilization. This finding suggests that T cell signaling derangements with thermal injury involve an altered cross-talk between Ca2+ mobilization and MAPK signaling mechanisms.
Keywords: T cell; Immune response; Burn; Rat; MAPK; Erk 1/2; p38; Calcium; Cell signaling;

Carnosine, an endogenous histidine-containing dipeptide, protects protein from oxidation and glycation, which may contribute to a potential treatment for some conformational diseases including cataract. Glycation, the non-enzymic reaction of sugars with proteins, promotes cross-linking and further aggregation. Prolonged use of glucocorticoids is a risk factor for cataract, as is diabetes. Esterase activity in the lens is decreased in senile cataract and diabetes. Previously, we reported that glycation and a steroid inactivate esterase. Here we tested the inactivation of esterase with fructose, fructose 6-phosphate (F6P) and ribose as model glycation reactions and prednisolone-21-hemisuccinate (P-21-H) as a model steroid and investigated the ability of carnosine to protect esterase against inactivation. The activity of esterase was measured by a spectrophotometric assay using p-nitrophenyl acetate as the substrate. The modified esterase was examined electrophoretically. The esterase was progressively inactivated by F6P, fructose, ribose and P-21-H. P-21-H was more effective than the sugars. Carnosine significantly inhibited the inactivation of esterase induced by all four compounds. Carnosine decreased the extent of the cross-linking. These results provide further evidence for carnosine's role as an anti-glycation compound. It is also proposed that carnosine may be an anti-steroid agent.
Keywords: Carnosine; Esterase; Glycation; Steroid;

Bigendothelin-1 via p38-MAPK-dependent mechanism regulates adult rat ventricular myocyte contractility in sepsis by Akanksha Gupta; Nicholas S. Aberle; Ruchita Kapoor; Jun Ren; Avadhesh C. Sharma (127-139).
We tested the hypothesis that exogenous administration of the ET-1 precursor, bigET-1, would regulate adult rat ventricular myocyte (ARVM) contractility in a p38-mitogen activated protein kinase (p38-MAPK)-dependent mechanism during sepsis. Ventricular myocytes from adult rat hearts (both sham and septic) were stimulated to contract at 0.5 Hz and mechanical properties were evaluated using an IonOptix Myocam system. Immunoblot analysis was used to determine the phosphorylation of p38-MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2). ARVMs were treated with vehicle, bigET-1 and inhibitors for 24 h and then subjected to functional and biochemical estimations. Septic ARVM displayed a distorted cell membrane and irregular network within the cells along with increased cell contractility as evidenced by elevated peak shortening (PS), maximal velocity of shortening (+dL/dt) and relengthening (−dL/dt) in comparison to sham ARVM. BigET-1 treatment caused ARVM enlargement in both sham and sepsis groups. BigET-1 (100 nM) produced an increase in ARVM contractility in sham group as compared to vehicle treatment. However, septic ARVM treated with bigET-1 exhibited unaltered ARVM contractility, and upregulated ETB receptors as compared to respective sham group. BigET-1 increased the concentration of ET-1 and upregulated phosphorylation of p38-MAPK but not of ERK1/2 in sham and septic ARVM. Furthermore, inhibition of p38-MAPK by SB203580 (10 μM) increased ARVM contractility in sham but not in sepsis group. BigET-1 reversed SB203580-induced increase in PS in sham group but accentuated it in sepsis group. BigET-1 also reversed SB203580-induced inhibition of p38-MAPK phosphorylation in sham but not in septic ARVM. SB203580 pretreatment followed by bigET-1 administration significantly decreased p38-MAPK phosphorylation and downregulated ETB receptor expression as compared to bigET-1 treatment per se in sepsis group but not in sham. We concluded that a bigET-1-induced non-responsive effect on septic ARVM contractile function could be due to upregulation of p38-MAPK phosphorylation and ETB receptor expression.
Keywords: p38 -MAPK; ERK1/2; Endothelin-1; Adult rat ventricular myocyte; Peak shortening; Immunoblot analysis;

Amyloid-beta (1–42) [Aβ (1–42)] deposition in the brain is a hallmark of Alzheimer's disease (AD) and has been shown to induce apoptosis and disrupt cellular ion homeostasis. Aβ (1–42) induces membrane lipid peroxidation, and 4-hydroxynonenal (HNE) and 2-propenal (acrolein) are the two reactive products of lipid peroxidation, which structurally modify proteins by covalent interaction and inhibit enzyme function. Phosphatidylserine (PS), an aminophospholipid, is sequestered in the inner leaflet of the plasma membrane in nonstimulated cells. An early signal of synaptosomal apoptosis is the loss of phospholipid asymmetry and the appearance of phosphatidylserine in the outer leaflet of the membrane. The ATP-requiring enzyme, flippase, maintains phospholipid asymmetry of PS. Here, we have investigated the inactivation of the transmembrane enzyme aminophospholipid-translocase (or flippase) by Aβ (1–42). Flippase activity depends on a critical cysteine residue, a putative site of covalent modification by the Aβ (1–42)-induced lipid peroxidation products, HNE or acrolein. The present study is aimed to investigate the protective effects of tricyclodecan-9-xanthogenate (D609) and ferulic acid ethyl ester (FAEE) on Aβ (1–42) induced modulation in phospholipid asymmetry in the synaptosomal membranes. Pretreatment of synaptosomes with D609 and FAEE significantly protected Aβ (1–42)-induced loss of phospholipid asymmetry in synaptosomal membranes. Our results suggest that D609 and FAEE exert protective effects against Aβ (1–42) induced apoptosis. The increase in intracellular Ca2+ might not be the sole cause for the loss of flippase activity. Rather, other mechanisms that could modulate the function of flippase might be important in the modulation of phospholipid asymmetry. The results of this study are discussed with relevance to neuronal loss in the AD brain.
Keywords: Alzheimer's disease; Oxidative stress; Lipid asymmetry; Lipid peroxidation; Phosphatidylserine; Synaptosome; Amyloid beta-peptide (1–42); Flippase;

The finding of new genetic polymorphism of UCP-1 A-1766G and its effects on body fat accumulation by Kil Soo Kim; Dae-Yeon Cho; Young Joo Kim; Sun Mi Choi; Jong Yeol Kim; Seung Uoo Shin; Yoo Sik Yoon (149-155).
A-1766G polymorphism, for the first time, has been found in the sequencing of pooled and individual genomic DNA of Korean subjects at the 5′ flanking region of the UCP-1 gene. The effects of new polymorphism on body fat were elucidated among 387 Korean female subjects. It was shown that the genotypes AA, AG, and GG were consisted of 57.4%, 37.7%, and 4.9%, respectively, which was in agreement with Hardy-Weinberg equilibrium (P=0.327). The frequency of major A allele was 0.762 and that of minor G allele was 0.238. It is found that the waist–hip ratio (WHR) (P=0.008), body fat mass (P=0.023), and percent body fat (P=0.014) are significantly higher in the AG/GG type compared to the AA type. When the subjects were analyzed using computerized tomography, there were significant increases in the AG/GG type compared to the AA type in the abdominal subcutaneous fat (P=0.015) and the abdominal visceral fat (P=0.013), respectively. A-1766G is approximately 2 kb downstream from the well-known A-3826G polymorphism, and no linkage between them was found (D′=0.929, R 2=0.283). Three haplotypes (frequency >0.05) were examined from two polymorphisms and studied for their physiological effects. It was found that haplotype [GG] was significantly associated with increased body fat, while haplotype [GA] was associated with decreased body fat.
Keywords: UCP-1; A-1766G; Polymorphism; SNP; Haplotype; Fat;

A combined defect in the biosynthesis of N- and O-glycans in patients with cutis laxa and neurological involvement: the biochemical characteristics by Suzan Wopereis; Éva Morava; Stephanie Grünewald; Philippa B. Mills; Bryan G. Winchester; Peter Clayton; Paul Coucke; Karin M.L.C. Huijben; Ron A. Wevers (156-164).
Based on our preliminary observation of abnormal glycosylation in a cutis laxa patient, nine cutis laxa patients were analyzed for congenital defects of glycosylation (CDG). Isoelectric focusing of plasma transferrin and apolipoproteinC-III showed that three out of nine patients had a defect in the biosynthesis of N-glycans and core 1 mucin type O-glycans, respectively. Mass spectrometric N-glycan analyses revealed a relative increase of glycans lacking sialic acid and glycans lacking sialic acid and galactose residues. Mutation analysis of the fibulin-5 gene (FBLN5), which has been reported in cases of autosomal recessive cutis laxa, revealed no mutations in the patients' DNA. Evidence is presented that extracellular matrix (ECM) proteins of skin are likely to be highly glycosylated with N- and/or mucin type O-glycans by using algorithms for predicting glycosylation. The conclusions in this study were that the clinical phenotype of autosomal recessive cutis laxa seen in three patients is not caused by mutations in the FBLN5 gene. Our findings define a novel form of CDG with cutis laxa and neurological involvement due to a defect in the sialylation and/or galactosylation of N- and O-glycans. Improper glycosylation of ECM proteins of skin may form the pathophysiological basis for the cutis laxa phenotype.
Keywords: Cutis laxa; Congenital disorder of glycosylation; N-glycosylation; O-glycosylation; Glycan biosynthesis defect;

KIT overexpression and amplification in gastrointestinal stromal tumors (GISTs) by Séverine Tabone; Nathalie Théou; Agnieszka Wozniak; Raphael Saffroy; Laure Deville; Catherine Julié; Patrice Callard; Anne Lavergne-Slove; Maria Debiec-Rychter; Antoinette Lemoine; Jean-François Emile (165-172).
The tyrosine kinase receptor KIT plays a major role in gastrointestinal stromal tumors (GISTs) oncogenesis. Indeed, 95% of GISTs express KIT protein, and about 70% exhibit activating mutations of the KIT gene. However, little is known about KIT overexpression mechanisms in these tumors, and the correlation with KIT mutations. GISTs with mutations within exon 11 (n  = 12) or 9 (n  = 1) of KIT were compared with GISTs without KIT mutations in exons 9, 11, 13, and 17 (n  = 10), two of them had PDGFRA mutations. KIT amplification was studied by real-time PCR of KIT and beta-ACTIN genes, and by fluorescence in situ hybridization (FISH) using KIT and chromosome 4 centromere specific probes. KIT transcripts and protein expression were quantified by reverse transcription real-time PCR and Western blot respectively. Genomic analysis revealed a single mutated GIST with KIT amplification. KIT protein and RNA levels were highly variable in GISTs but closely correlated (r  = 0.82, P  < 1.10−5), and were higher in GISTs with KIT mutations (P  = 0.07 and P  = 0.03 respectively). In conclusion, contrasting with the regulation of other tyrosine kinase receptors, KIT overexpression in GISTs is rarely related to a gene amplification, which suggests a deregulation of KIT gene transcription.
Keywords: Proto-oncogene protein KIT; KIT overexpression; Digestive system neoplasm; Gastrointestinal stromal tumor; KIT mutation; Gene amplification;

Cardiac volume overload rapidly induces oxidative stress-mediated myocyte apoptosis and hypertrophy by C. Fiorillo; C. Nediani; V. Ponziani; L. Giannini; A. Celli; N. Nassi; L. Formigli; A.M. Perna; P. Nassi (173-182).
Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated the role of oxidative stress in the initial phases of cardiac remodeling induced in an animal model by volume overload. As plausible candidates for a connection between oxidative stress and cardiomyocyte apoptosis or hypertrophy, we explored the behaviour of two MAPKs, specifically JNK and ERK. At 48 h of overload, the greatest increase in oxidative stress coincided with a peak of cardiomyocyte apoptosis. This was possibly induced through the mitochondrial metabolism, as evidenced by the release of cytochrome c and a significant increase in the active forms of caspase-9 and -3, but not caspase-8. Oxidative stress markers significantly decreased at 96 h of overload, combined with a marked attenuation of apoptosis and the appearance of hypertrophy. The highest levels of JNK and the lowest levels of ERK phosphorylation were observed at 48 h of overload. Conversely, a sharp increase in ERK phosphorylation was detected at 96 h of overload coinciding with the hypertrophic response. Together these results show that oxidative stress is an early and transient event in myocardial volume overload. They suggest that oxidative stress mediates amplitude dependent apoptotic and hypertrophic responses in cardiomyocytes through the selective activation of, respectively, JNK and ERK.
Keywords: Oxidative stress; Cardiac volume overload; Apoptosis; Hypertrophy; MAPKs;

In an attempt to model the processes of free radical-mediated cataractogenesis, we investigated the oxidative modification of rat eye lens proteins by peroxyl radicals generated by thermal decomposition of 2,2′-azobis(2-amidinopropane)hydrochloride (AAPH) under aerobic conditions. When incubated with AAPH, the soluble eye lens proteins precipitated in a time-dependent manner. The insolubilisation was accompanied by the accumulation of protein free carbonyls and the diminution of sulfhydryls, yet the processes were shifted in time. The SDS-PAGE analysis of the AAPH-treated proteins revealed the presence of high molecular weight cross-links and, to a lesser extent, fragments. The aggregation and cross-linking of proteins along with the generation of free carbonyls was significantly inhibited by the chain-breaking antioxidants stobadine and Trolox. On the other hand, the AAPH-initiated sulfhydryl consumption was much less sensitive to the antioxidants studied. The results point to a complex mechanism of peroxyl-radical-mediated modification of eye lens proteins with implications for cataract development and they indicate a potentially protective role of antioxidants.
Keywords: 2,2′-Azobis(2-amidinopropane)hydrochloride; Cataract; Eye lens protein; Protein carbonyl; Peroxyl radical; Protein sulfhydryl;

Loss of ecto-5′nucleotidase from porcine endothelial cells after exposure to human blood: Implications for xenotransplantation by Zain Khalpey; Ada H. Yuen; Kameljit K. Kalsi; Zdzislaw Kochan; Joanna Karbowska; Ewa M. Slominska; Monica Forni; Massimo Macherini; Maria L. Bacci; Puspa Batten; Marialuisa Lavitrano; Magdi H. Yacoub; Ryszard T. Smolenski (191-198).
The endothelial cell surface expression of ecto-5′-nucleotidase (E5′N, CD73) is thought to be essential for the extracellular formation of cytoprotective, anti-thrombotic and immunosuppressive adenosine. Decreased E5′N activity may play a role in xenograft acute vascular rejection, preventing accommodation and tolerance mechanisms. We investigated the extent of changes in E5′N activity and other enzymes of purine metabolism in porcine hearts or endothelial cells when exposed to human blood or plasma and studied the role of humoral immunity in this context.Pig hearts, wild type (WT, n = 6) and transgenic (T, n = 5) for human decay accelerating factor (hDAF), were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were exposed for 3 h to autologous porcine plasma (PP), normal (NHP) or heat inactivated human plasma (HHP), with and without C1-inhibitor. Enzyme activities were measured in heart or endothelial cell homogenates with an HPLC based procedure.The baseline activity of E5′N in WT and T porcine hearts were 6.60 ± 0.33 nmol/min/mg protein and 8.54 ± 2.10 nmol/min/mg protein respectively (P < 0.01). Ex vivo perfusion of pig hearts with fresh human blood for 4 h resulted in a decrease in E5′N activity to 4.01 ± 0.32 and 4.52 ± 0.52 nmol/min/mg protein (P < 0.001) in WT and T hearts respectively, despite attenuation of hyperacute rejection in transgenic pigs. The initial PAEC activity of E5′N was 9.10 ± 1.40 nmol/min/mg protein. Activity decreased to 6.76 ± 0.57 and 4.58 ± 0.47 nmol/min/mg protein (P < 0.01) after 3 h exposure of HHP and NHP respectively (P < 0.05), whereas it remained unchanged at 9.62 ± 0.88 nmol/min/mg protein when incubated with PP controls. C1-inhibitor partially preserved E5′N activity, similar to the effect of HHP. Adenosine deaminase, adenosine kinase and AMP deaminase (other enzymes of purine metabolism) showed a downward trend in activity, but none were statistically significant.We demonstrate a specific decrease in E5′N activity in pig hearts following exposure to human blood which impairs adenosine production resulting in a loss of a cytoprotective phenotype, contributing to xenograft rejection. This effect is triggered by human humoral immune responses, and complement contributes but does not fully mediate E5′N depletion.
Keywords: Ecto-5′-nucleotidase; Adenosine; Xenotransplantation; Complement; Cytoprotection;

RAGE and amyloid beta interactions: Atomic force microscopy and molecular modeling by Michael O. Chaney; W. Blaine Stine; Tyler A. Kokjohn; Yu-Min Kuo; Chera Esh; Afroza Rahman; Dean C. Luehrs; Ann Marie Schmidt; David Stern; Shi Du Yan; Alex E. Roher (199-205).
In the AD brain, there are elevated amounts of soluble and insoluble Aβ peptides which enhance the expression of membrane bound and soluble receptor for advanced glycation end products (RAGE). The binding of soluble Aβ to soluble RAGE inhibits further aggregation of Aβ peptides, while membrane bound RAGE-Aβ interactions elicit activation of the NF-κB transcription factor promoting sustained chronic neuroinflammation. Atomic force microscopy observations demonstrated that the N-terminal domain of RAGE, by interacting with Aβ, is a powerful inhibitor of Aβ polymerization even at prolonged periods of incubation. Hence, the potential RAGE-Aβ structural interactions were further explored utilizing a series of computational chemistry algorithms. Our modeling suggests that a soluble dimeric RAGE assembly creates a positively charged well into which the negative charges of the N-terminal domain of dimeric Aβ dock.
Keywords: Molecular modeling; RAGE; IgG; Atomic force microscopy; Alzheimer's disease;

Reduced insulin-mediated citrate synthase activity in cultured skeletal muscle cells from patients with type 2 diabetes: Evidence for an intrinsic oxidative enzyme defect by Niels Ørtenblad; Martin Mogensen; Ingrid Petersen; Kurt Højlund; Klaus Levin; Kent Sahlin; Henning Beck-Nielsen; Michael Gaster (206-214).
In myotubes established from patients with type 2 diabetes (T2D), lipid oxidation and insulin-mediated glucose oxidation are reduced, whereas in myotubes from obese non-diabetic subjects, exposure to palmitate impairs insulin-mediated glucose oxidation. To determine the underlying mechanisms of these metabolic malfunctions, we studied mitochondrial respiration, uncoupled respiration and oxidative enzyme activities (citrate synthase (CS), 3-hydroxy-acyl-CoA-dehydrogenase activity (HAD)) before and after acute exposure to insulin and/or palmitate in myotubes established from healthy lean and obese subjects and T2D patients. Basal CS activity was lower (14%) in diabetic myotubes compared with myotubes from lean controls (P  = 0.03). Incubation with insulin (1 μM) for 4 h increased the CS activity (26–33%) in myotubes from both lean (P  = 0.02) and obese controls (P  < 0.001), but not from diabetic subjects. Co-incubation with palmitate (0.6 mM) for 4 h abolished the stimulatory effect of insulin on CS activity in non-diabetic myotubes. No differences were detected in mitochondrial respiration and HAD activity between myotubes from non-diabetic subjects and T2D patients, and none of these measures responded to high levels of insulin and/or palmitate. These results provide evidence for an intrinsic defect in CS activity, which may play a role in the pathogenesis of T2D. Moreover, the data suggest that insulin resistance at the CS level can be induced by exposure to high free fatty acid levels.
Keywords: Citrate synthase; Insulin resistance; Free fatty acid; Myotube; Obese; Type 2 diabetes; Uncoupled respiration;

Upregulation of the amino acid transporter ATB0,+ (SLC6A14) in colorectal cancer and metastasis in humans by Naren Gupta; Seiji Miyauchi; Robert G. Martindale; Anne V. Herdman; Robert Podolsky; Katsuya Miyake; Sela Mager; Puttur D. Prasad; Malliga E. Ganapathy; Vadivel Ganapathy (215-223).
ATB0,+ (SLC6A14) is a Na+/Cl-coupled arginine transporter expressed at low levels in normal colon. Arginine is an essential amino acid for tumor cells. Arginine is also the substrate for nitric oxide synthases (NOSs). Since arginine and arginine-derived nitric oxide (NO) play a critical role in cancer, we examined the expression of ATB0,+ in colorectal cancer. Paired normal and cancer tissues from colectomy specimens of 10 patients with colorectal cancer and from the liver tissue of one patient with hepatic metastasis from a colonic primary were used for the analysis of the levels of ATB0,+ mRNA, inducible NOS (iNOS) mRNA and the corresponding proteins. Tissues samples from the colon, liver, and lymph nodes of an additional patient with metastatic colon cancer were analyzed for ATB0,+ protein alone. We also examined the levels of nitrotyrosylated proteins. The ATB0,+ mRNA increased 22.9 ± 3.0-fold in colorectal cancer compared to normal tissue and the increase was evident in each of the 10 cases examined. iNOS mRNA increased 5.2 ± 1.1-fold in cancer specimens. The changes in mRNA levels were associated with an increase in ATB0,+, iNOS, and nitrotyrosylated proteins. The increased expression of ATB0,+ and iNOS was also demonstrated in liver and lymph node specimens with metastases from colonic primaries. This study strongly suggests that the upregulation of ATB0,+ may have a pathogenic role in colorectal cancer. Since ATB0,+ is a versatile transporter not only for arginine but also for several drugs including NOS inhibitors, these findings have significant clinical and therapeutic relevance.
Keywords: Colon; Cancer; Amino acid transporter ATB0,+; Nitric oxide; Nitric oxide synthase;