BBA - Molecular Basis of Disease (v.1502, #3)

Differential alterations in antioxidant capacity in cells from Alzheimer patients by G.E. Gibson; H. Zhang; K.-F.R. Sheu; L.C.H. Park (319-329).
Oxidative stress occurs in brains of Alzheimer’s disease (AD) patients. A major question in AD research is whether the oxidative stress is just secondary to neurodegeneration. To test whether oxidative stress is an inherent property of AD tissues, the ability of cultured fibroblasts bearing the AD Presenilin-1 246 Ala→Glu mutation to handle reactive oxygen species (ROS) was compared to controls. Although ROS in cells from AD subjects were only slightly less than cells from controls under basal conditions (−10%) or after exposure to H2O2 (−16%), treatment with antioxidants revealed clear differences. Pretreatment with DMSO, a hydroxyl radical scavenger, reduced basal and H2O2-induced ROS levels significantly more in cells from controls (−22%, −22%) than in those from AD subjects (−4%, +14%). On the other hand, pretreatment with Trolox diminished H2O2-induced ROS significantly more in cells from AD (−60%) than control subjects (−39%). In summary, cells from AD patients have greater Trolox sensitive ROS and less DMSO sensitive ROS than controls. The results demonstrate that fibroblasts bearing this PS-1 mutation have altered means of handling oxidative stress and appear useful for determining the mechanism underlying the altered redox metabolism.
Keywords: Alzheimer’s disease; Fibroblast; Oxidative stress; Reactive oxygen species; Vitamin E; Trolox; Dimethylsulfoxide;

Myocardial carnitine and carnitine palmitoyltransferase deficiencies in patients with severe heart failure by Miguel A. Martı́n; Miguel A. Gómez; Fernando Guillén; Belén Börnstein; Yolanda Campos; J.C. Rubio; Carlos S. de la Calzada; Joaquı́n Arenas (330-336).
We studied myocardial tissue from 25 cardiac transplant recipients, who had end-stage congestive heart failure (CHF), and from 21 control donor hearts. Concentrations of total carnitine (TC), free carnitine (FC), short-chain acylcarnitines, long-chain acylcarnitines (LCAC) as well as carnitine palmitoyltransferase (CPT) activities were measured in myocardial tissue homogenates and referred to the concentration of non-collagen protein. Compared to controls, the concentrations of TC and FC as well as total CPT activities were significantly lower in patients. LCAC levels and the LCAC to FC ratio values were significantly greater in patients than in controls. While the malonyl-CoA sensitive fraction of CPT, which represents CPT I activity, was similar in patients and controls, the residual CPT activity after inhibition by malonyl-CoA, representing CPT II activity, was significantly reduced in patients compared to controls. Moreover, the activity of CPT in the presence of Triton X-100, which also represents the activity of CPT II, was significantly lower in patients than in controls. Malonyl-CoA concentrations required for half-maximal inhibition of CPT activity were significantly greater in patients than in controls. There was a linear relationship between ejection fraction (EF) values and concentrations of TC, FC, or total CPT activities. Values for LCAC and the LCAC to FC ratio were inversely related to EF values. We conclude that failing heart shows decreased total CPT and CPT II activities and carnitine deficiency that may be related to ventricle function.
Keywords: Carnitine; Carnitine palmitoyltransferase; Myocardium; Heart failure;

Ovarian tumor cells express a novel multi-domain cell surface serine protease by Lowell J. Underwood; Kazushi Shigemasa; Hirotoshi Tanimoto; John B. Beard; Elizabeth Nestrud Schneider; Yinxiang Wang; Tim H. Parmley; Timothy J. O’Brien (337-350).
Serine proteases serve many functions in normal biological processes. These functions are often usurped by cancer cells to allow progression of tumors by increasing the growth and metastatic potential of the neoplasia. Here, we have used a polymerase chain reaction (PCR)-based strategy to clone Tumor Associated Differentially-expressed Gene-12 (TADG-12), a new serine protease from ovarian carcinoma. This technique also revealed a variant splicing form of TADG-12 that could lead to a truncated protein product. Semi-quantitative PCR showed that TADG-12 is overexpressed in 41 of 55 ovarian cancer specimens relative to normal expression, and the variant form, TADG-12V is found at increased levels in 8 of 22 carcinomas examined. Northern blot revealed three transcripts, the largest of which is approximately 2.4 kb. An ovarian tumor cDNA library was screened, and the entire cDNA of TADG-12 has been identified. This sequence encodes a putative protein of 454 amino acids which includes a potential transmembrane domain, an LDL receptor-like domain, a scavenger receptor cysteine-rich domain, and a serine protease domain. These features imply that TADG-12 will be at the cell surface, and it may be useful as a molecular target for therapy or a diagnostic marker.
Keywords: Serine protease; Transmembrane; Cancer;

Antioxidant properties of colchicine in acute carbon tetrachloride induced rat liver injury and its role in the resolution of established cirrhosis by Debashis Das; Philip W Pemberton; Peter C Burrows; Christopher Gordon; Alexander Smith; Raymond F.T McMahon; Thomas W Warnes (351-362).
Antioxidant and antifibrotic properties of colchicine were investigated in the carbon tetrachloride (CCl4) rat model. (1) The protective effect of colchicine pretreatment on CCl4 induced oxidant stress was examined in rats subsequently receiving a single lethal dose of CCl4. Urinary 8-isoprostane, kidney and liver malondialdehyde and kidney glutathione levels increased following CCl4 treatment, but only the rise in kidney malondialdehyde was significantly inhibited by colchicine pretreatment. Serum total antioxidant levels were significantly higher in the colchicine pretreatment group. (2) The long term effects of colchicine treatment on CCl4 induced liver damage were investigated using liver histology and biochemical markers (hydroxyproline and type III procollagen peptide). Co-administration of colchicine with sub-lethal doses of CCl4 over 10 weeks did not prevent progression to cirrhosis. However, rats made cirrhotic with repeated CCl4 challenge and subsequently treated with colchicine for 12 months, all showed histological regression of cirrhosis. (3) The antioxidant effect of colchicine in vitro was evident only at very high concentrations compared to other plasma antioxidants. In summary, colchicine has only weak antioxidant properties, but does afford some protection against oxidative stress; more importantly, long term treatment with this drug may be of value in producing regression of established cirrhosis.
Keywords: Lipid peroxidation; Rat; Carbon tetrachloride; Colchicine; Cirrhosis;

Glycogen turnover and anaplerosis in preconditioned rat hearts by Silvia Bradamante; Andrea Marchesani; Livia Barenghi; Luisa Paracchini; Robert de Jonge; Jan Willem de Jong (363-379).
Using 13C NMR, we tested the hypothesis that protection by preconditioning is associated with reduced glycogenolysis during ischemia. Preconditioned rat hearts showed improved postischemic function and reduced ischemic damage relative to ischemic controls after 30 min stop-flow ischemia and 30 min reperfusion (contractility: 30±10 vs. 2±2%; creatine kinase release: 41±4 vs. 83±15 U/g; both P<0.05). Preconditioning decreased preischemic [13C]glycogen by 24% (a 10% decrease in total glycogen), and delayed ischemic [13C]glycogen consumption by 5–10 min, reducing ischemic glycogenolysis without changing acidosis relative to controls. Upon reperfusion, glycogen synthesis resumed only after preconditioning. Glutamate 13C-isotopomer analysis showed recovery of Krebs cycle activity with higher anaplerosis than before ischemia (23±4 vs. 11±3%, P<0.05), but in controls reperfusion failed to restore flux. Compared to control, preconditioning before 20 min ischemia increased contractility (86±10 vs. 29±14%, P<0.05) and restored preischemic anaplerosis (13±3 vs. 39±9%, P<0.05). Preconditioning is associated with reduced glycogenolysis early during ischemia. However, protection does not rely on major variations in intracellular pH, as proposed earlier. Our isotopomer data suggest that preconditioning accelerates metabolic and functional recovery during reperfusion by more efficient/active replenishment of the depleted Krebs cycle.
Keywords: Glutamate; Glycogen; Myocardial ischemia/reperfusion; 13C Nuclear magnetic resonance; pH; Tricarboxylic acid cycle;

Alterations in erythrocyte membrane lipid and fatty acid composition in Chediak–Higashi Syndrome by Yolanda Chico; Manuel Lafita; Pedro Ramı́rez-Duque; Fernando Merino; Begoña Ochoa (380-390).
Chediak–Higashi syndrome (CHS) is an autosomal recessive disease characterized by the presence of abnormally large cytoplasmic organelles in all body granule producing cells. The molecular mechanism for this disease is still unknown. Functional disorders in membrane-related processes have been reported. Erythrocyte membranes from four CHS patients and 15 relatives including obligatory heterozygous were studied to examine potential alterations in the lipid and fatty acid profile of erythrocyte membranes associated with this syndrome. Plasma concentrations of cholesterol, triglycerides, phospholipids, and apolipoproteins AI and B100, and the lipid components of very low-, intermediate-, low- and high-density lipoproteins were also determined. CHS erythrocyte membranes were found to be enriched with lipids in relation to protein and to show: (1) an increase in cholesterol and choline-containing phospholipids (sphingomyelin and phosphatidylcholine) that predominate in the outer monolayer, which is higher than the increase in phosphatidylserine and phosphatidylethanolamine, that are chiefly limited to the inner monolayer in normal red blood cells; (2) a relative palmitic acid and saturated fatty acid increase and arachidonic acid and unsaturated fatty acid decrease, this resulting in a lower unsaturation index than controls. Changes in CHS erythrocyte membrane lipids seem to be unrelated to serum lipid disorders as plasma lipid and apolipoprotein concentrations were apparently in the normal range, with the exception of a modest hypertriglyceridemia in patients and relatives and a decreased concentration of HDL cholesterol in patients. These findings indicate that CHS erythrocyte membranes contain an abnormal lipid matrix with which membrane proteins are defectively associated. The anomalous CHS membrane composition can be explained on the postulated effects of the CHS1/Lyst gene.
Keywords: Chediak–Higashi syndrome; Cholesterol; Erythrocyte membrane; Fatty acid composition; Phospholipid; Plasma lipid;

We have investigated the mRNA amounts of six lysosomal proteins (β-hexosaminidase α- and β-subunit, sphingolipid activator protein precursor, GM2 activator protein, lysosomal sialidase, β-glucocerebrosidase) involved in the degradation of glycosphingolipids. We analyzed extracts from brain tissues of mouse models for lysosomal storage diseases, i.e., the GM2 gangliosidoses and the deficiency of the sphingolipid activator protein precursor (prosaposin). The mRNA levels were quantified by real-time reverse transcription–polymerase chain reaction. Although storage of the respective lysosomal proteins has been reported in human and mice, no increase of their mRNA amounts could be detected here. Our results indicate that there is no transcriptional upregulation of lysosomal proteins in the examined neuronal storage disorders.
Keywords: Sphingolipidosis; mRNA quantification; Real-time reverse transcription–polymerase chain reaction; Lysosomal hydrolase; Sphingolipid activator protein; Knock-out mouse;

In the present work, pepsin digests of tail tendons from streptozotocin-diabetic rats were found to contain material that reacted rapidly at room temperature with p-dimethylaminobenzaldehyde (Ehrlich’s reagent) to give an adduct with an absorbance spectrum characteristic of the Ehrlich chromogen of pyrrolic nature determined in ageing collagens. A significant correlation of the Ehrlich adduct with tendon mechanical strength and collagen fluorescence characteristic of advanced glycation endproducts was observed. Collagen content of the Ehrlich-positive material was found to be significantly elevated in tendons of diabetic rats compared with age-matched healthy controls. The results indicate that the p-dimethylaminobenzaldehyde-reactive pyrrole moieties may contribute to the increased cross-linking of diabetic matrix collagen. Profound inhibitory effect of aminoguanidine was observed, underlining the role of non-enzymatic mechanisms of advanced glycation in pyrrolisation and cross-linking of collagen exposed to hyperglycaemia. It is hypothesised that quantification of the p-dimethylaminobenzaldehyde-reactive material in matrix collagen may provide a tissue measure of integrated hyperglycaemia over prolonged periods of time. Further research is to assess the significance of p-dimethylaminobenzaldehyde-reactive substances in diabetic collagen tissues and to reveal their relationship to enzyme-mediated physiological pyrrolisation of ageing collagens.
Keywords: Collagen; Diabetic complications; Ehrlich’s reagent; Non-enzymatic glycation; Pyrrole cross-link; Streptozotocin;

Glial cells contribute more to iron and aluminum accumulation but are more resistant to oxidative stress than neuronal cells by Satoru Oshiro; Masahiro Kawahara; Yoichiro Kuroda; Chun Zhang; Yong Cai; Shigetaka Kitajima; Mika Shirao (405-414).
Iron (Fe) and aluminum (Al) have been implicated in the pathogenesis of Alzheimer’s disease (AD). In this study, we examined neuronal and glial cells to clarify which contributes most to metal accumulation after internalization through the transferrin-independent iron uptake (Tf-IU) systems in primary neuronal and glial predominant (NP and GP) cells from rat cerebral cortex, which affect the accumulation of transition metals in a variety of cultured cells. Al more significantly upregulated the Tf-IU activity in GP cells than in NP cells. GP cells were more resistant to Fe and Al exposure than NP cells. However, a chemiluminescence analysis specific for reactive oxygen species (ROS) showed that ROS levels in Fe- or Al-loaded NP cells were twice as high as in Fe- or Al-loaded GP cells. Northern blot analysis and gel retardation assay showed that the Al and Fe exposure taken up by the cells suppress Tf receptor mRNA expression to a greater extent in GP than NP cells, indicating that Al and Fe more markedly accumulate in glial than in neuronal cells. These results suggest that glial cells rather than neuronal cells contribute to the metal accumulation and are more resistant to oxidative stress caused by metals than neuronal cells. The present study may help to explain the pathogenesis of neurodegeneration in AD disorders caused by metal-generated oxidative stress.
Keywords: Glial cell; Neuronal cell; Oxidative stress; Alzheimer’s disease; Aluminum; Transferrin-independent iron uptake system;

Mucopolysaccharidosis type IIIB (MPS-IIB) is a lysosomal storage disorder characterised by the defective degradation of heparan sulfate due to a deficiency of α-N-acetylglucosaminidase (NAG). The clinical severity of MPS-IIIB ranges from an attenuated to severely affected Sanfilippo phenotype. This paper describes the expression and characterisation of wild-type recombinant NAG and the molecular characterisation of a previously identified R297X/F48L compound heterozygous MPS-IIIB patient with attenuated Sanfilippo syndrome. We have previously shown R297X to be the most common mutation in a cohort of Dutch and Australian patients, occurring at a frequency of approximately 12.5%. To date F48L has only been described in the proband. To determine the contribution of each mutation to the overall clinical phenotype of the patient, both mutant alleles were engineered into the wild-type NAG cDNA and expressed in Chinese hamster ovary cells. The wild-type NAG and F48L mutant alleles were also retrovirally expressed in MPS-IIIB skin fibroblasts. Residual NAG activity and the stability and maturation of immunoprecipitated NAG were determined for wild-type NAG and mutant NAG. The combined biochemical phenotypes of the two NAG mutant alleles demonstrated a good correspondence with the observed attenuated Sanfilippo phenotype of the patient.
Keywords: Lysosome; α-N-acetylglucosaminidase; Mucopolysaccharidosis; Sanfilippo;

Inhibition of leptin secretion by insulin and metformin in cultured rat adipose tissue by Gail J. Mick; Xudong Wang; Chang Ling Fu; Kenneth L. McCormick (426-432).
Leptin’s role in the regulation of food intake, energy expenditure and weight control are widely recognized, especially in rodents. Likewise, the potential regulation of leptin secretion by insulin (and vice versa) has been of particular interest insofar as these nutrient signals may have meaningful, even adverse (inter)actions, in diabetes. We used a freshly isolated rat adipose tissue culture model to examine the effect of insulin, metformin and glibenclamide on basal and steroid-stimulated leptin secretion. This model was selected because of its physiologic rates of leptin formation and preservation of potentially significant cell–cell interactions compared to isolated cells. The basal rate of leptin secretion was 3.4±1.2 ng/100 mg tissue/24 h. The addition of 100 nM dexamethasone or 400 nM hydrocortisone stimulated leptin secretion by 3–4 fold over basal (no steroid). Insulin inhibited both basal and steroid-activated leptin secretion by 35–50%. This inhibition was present with either 1 mM pyruvate or 5 mM glucose as a substrate suggesting that glycolysis was not required. Metformin inhibited basal and dexamethasone-stimulated leptin secretion in a dose dependent manner (50% inhibition occurred at 1 mM metformin) while glibenclamide was ineffective. The effect of insulin on isolated fat cells versus fat tissue was tested in parallel. After 24 h in culture, insulin inhibited leptin secretion similarly in both adipose preparations. The addition of 200 nM (−)N6-(2-phenylisopropyl)-adenosine did not alter the results.
Keywords: Leptin; Insulin; Adipose tissue; Metformin;

We have systematically investigated the molecular defects resulting in a primary lipoprotein lipase (LPL) deficiency in a Japanese male infant (proband SH) with fasting hyperchylomicronemia. Neither LPL activity nor immunoreactive LPL mass was detected in pre- or postheparin plasma from proband SH. DNA sequence analysis of the LPL gene of proband SH revealed homozygosity for a novel missense mutation of F270L (Phe270→Leu/TT T 1065→TT G ) in exon 6. The function of the mutant F270L LPL was determined by both biochemical and immunocytochemical studies. In vitro expression experiments on the mutant F270L LPL cDNA in COS-1 cells demonstrated that the mutant LPL protein was synthesized as a catalytically inactive form and its total amount was almost equal to that of the normal LPL. Moreover, the synthesized mutant LPL was non-releasable by heparin because the intracellular transport of the mutant LPL to the cell surface – by which normal LPL becomes heparin-releasable – was impaired due to the abnormal structure of the mutant LPL protein. These findings explain the failure to detect LPL activities and masses in pre- and postheparin plasma of the proband. The mutant F270L allele generated an XcmI restriction enzyme site in exon 6 of the LPL gene. The carrier status of F270L in the proband’s family members was examined by digestion with XcmI. The proband was ascertained to be homozygous for the F270L mutation and his parents and sister were all heterozygous. The LPL activities and masses of the parents and the sister (carriers) were half or less than half of the control values. Regarding the phenotype of the carriers, the mother with a sign of hyperinsulinemia manifested hypertriglyceridemia (type IV hyperlipoproteinemia), whereas the healthy father and the sister were normolipidemic. Hyperinsulinemia may be a strong determinant of hypertriglyceridemia in subjects with heterozygous LPL deficiency.
Keywords: Homozygous lipoprotein lipase deficiency; Hypertriglyceridemia; Type I hyperlipoproteinemia; Type IV hyperlipoproteinemia; Heterozygous lipoprotein lipase deficiency; Hyperinsulinemia;

Altered secretion of a TIGR/MYOC mutant lacking the olfactomedin domain by Montserrat Caballero; Laura Leigh S Rowlette; Teresa Borrás (447-460).
TIGR/MYOC, a novel 504 amino acids (aa) protein of unknown function, has recently been linked to glaucoma. The protein is both intra- and extracellular and most known mutations map to its C-terminus, an olfactomedin-like domain. To investigate the properties of a TIGR/MYOC peptide lacking this important domain, we constructed a replication-deficient adenovirus with the first 344 aa and over-expressed the truncated protein in primary human trabecular meshwork cells and perfused human anterior segment cultures. The truncated mutant contains the entire N-terminus plus 98 aa of the olfactomedin-like domain. We found that the delivered truncated mutant accumulates inside the cell, reduces secretion of endogenous TIGR/MYOC and induces an increase in outflow facility at 48 h post-infection. Based on these findings, we hypothesize that TIGR/MYOC might have a dual role in trabecular meshwork function. This dual role might be that of an intracellular modulator of vesicular transport as well as that of a secreted protein involved in extracellular matrix conformation. Both functions could have a direct effect in maintaining aqueous humor outflow facility.
Keywords: TIGR/MYOC; Recombinant adenovirus; Human trabecular meshwork; Perfused organ culture;

Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane by Cathleen Magowan; Wataru Nunomura; Karena L. Waller; Jackson Yeung; Joy Liang; Heidi Van Dort; Philip S. Low; Ross L. Coppel; Narla Mohandas (461-470).
Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5′ repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.
Keywords: Plasmodium falciparum malaria; Ankyrin; Erythrocyte cytoskeleton; Knob-associated histidine-rich protein; KAHRP; PfHRP1; Phosphorylation; Protein–protein interaction;

Acute changes of myocardial creatine kinase gene expression under β-adrenergic stimulation by Stefan Hammerschmidt; Michael Bell; Nicole Büchler; Hans Wahn; Helga Remkes; Martin J Lohse; Stefan Neubauer (471-480).
Creatine kinase (CK) plays a crucial role in myocardial energy metabolism. Alterations in CK gene expression are found in hypertrophied and failing heart, but the mechanisms behind these changes are unclear. This study tests the hypothesis that increased adrenergic stimulation, which is observed in heart failure, induces changes of myocardial CK-activity, -isoenzyme distribution and -gene expression that are characteristic of the failing and hypertrophied heart. Isolated rat hearts were perfused (constant pressure of 80 mmHg) with red cell suspensions. Following a 20-min warm-up period, perfusion for 3 h with 10−8 M (iso 3 h) or without (control 3 h) isoproterenol was started or experiments were immediately terminated (control 0 h). Left ventricular tissue was analyzed for total CK-activity, CK-isoenzyme distribution and, by use of quantitative RT–PCR, for B-CK, M-CK, mito-CK and GAPDH- (as internal standard) mRNA. After β-adrenergic stimulation (iso 3 h) but not after control perfusion (control 3 h) a roughly threefold increase in B-CK mRNA levels and a decrease in M-CK mRNA levels by 18% was found. There were no significant differences among the three groups in total CK-activity and in distribution of CK-MM, CK-BB, CK-MB and mito-CK. Thus, β-adrenergic stimulation induces a switch in CK gene expression from M-CK to B-CK, which is characteristic for the hypertrophied and failing heart. This may be interpreted as an adaptive mechanism making energy transduction via CK more efficient at times of increased metabolic demand.
Keywords: Creatine kinase; Gene expression; β-Adrenergic stimulation; Isoproterenol; Isolated heart;

Nitric oxide (NO) is important in the regulation of renal tubular function. We have investigated whether glycated proteins could impair the NO production by examining the effects of Amadori products (AP-BSA) and advanced glycation end products (AGE-BSA) on primary cultures of rabbit proximal tubular epithelial (PTE) cells. Nitric oxide synthase activity was assessed by measurement of the conversion of l-arginine to l-citrulline and by production of NO, after short-term (30 min) or long-term (1 or 3 days) incubation. Short incubations of PTE cells with either 200 μg/ml AP-BSA or 40 μg/ml AGE-BSA significantly decreased NO production. AP-BSA (3000 μg/ml) inhibited the Ca2+-dependent NOS activity even though above 50 μg/ml it increased Ca2+-independent NOS activity. In contrast, 40 μg/ml AGE-BSA inhibited both isoforms of NOS. Longer incubations with 200 μg/ml AP-BSA or 250 μg/ml AGE-BSA decreased NO release and inhibited Ca2+-dependent and -independent NOS activities. APs did not affect NO release by S-nitroso-N-acetyl-penicillamine (SNAP), while 250 μg/ml AGEs decreased it. After 3 days incubation, glycation products had no effect on the NOS cell content. Cell viability and proliferation were not modified under these experimental conditions, suggesting that the fall in NO production was not due to there being fewer cells. These data indicate that APs and AGEs directly inhibit NOS activity, and additionally that AGEs quench released NO. Thus, both types of glycated proteins alter the production of NO by PTE cells and could participate in the renal tubule dysfunction associated with aging and diabetes.
Keywords: Glycation; NO synthase activity; Epithelial cell; Kidney;

Disease causing aberrations in both tuberous sclerosis predisposing genes, TSC1 and TSC2, comprise nearly every type of alteration with a predominance of small truncating mutations distributed over both genes. We performed an RNA based screening of the entire coding regions of both TSC genes applying the protein truncation test (PTT) and identified a high proportion of unusual splicing abnormalities affecting the TSC2 gene. Two cases exhibited different splice acceptor mutations in intron 9 (IVS9−15G→A and IVS9−3C→G) both accompanied by exon 10 skipping and simultaneous usage of a cryptic splice acceptor in exon 10. Another splice acceptor mutation (IVS38−18A→G) destroyed the putative polypyrimidine structure in intron 38 and resulted in simultaneous intron retention and usage of a downstream cryptic splice acceptor in exon 39. Another patient bore a C→T transition in intron 8 (IVS8+281C→T) activating a splice donor site and resulting in the inclusion of a newly recognised exon in the mRNA followed by a premature stop. These splice variants deduced from experimental results are additionally supported by RNA secondary structure analysis based on free energy minimisation. Three of the reported splicing anomalies are due to sequence changes remote from exon/intron boundaries, described for the first time in TSC. These findings highlight the significance of investigating intronic changes and their consequences on the mRNA level as disease causing mutations in TSC.
Keywords: Tuberous sclerosis; TSC2; Splicing aberration; RNA;

Ethanol potentiates hypoxic liver injury: role of hepatocyte Na+ overload by R Carini; M.G De Cesaris; R Spendore; E Albano (508-514).
Centrilobular hypoxia has been suggested to contribute to hepatic damage caused by alcohol intoxication. However, the mechanisms involved are still poorly understood. We have investigated whether alterations of Na+ homeostasis might account for ethanol-mediated increase in hepatocyte sensitivity to hypoxia. Addition of ethanol (100 mmol/l) to isolated rat hepatocytes incubated under nitrogen atmosphere greatly stimulated cell death. An increase in intracellular Na+ levels preceded cell killing and Na+ levels in hepatocytes exposed to the combination of ethanol and hypoxia were almost twice those in hypoxic cells without ethanol. Na+ increase was also observed in hepatocytes incubated with ethanol in oxygenated buffer. Ethanol addition significantly lowered hepatocyte pH. Inhibiting ethanol and acetaldehyde oxidation with, respectively, 4-methylpyrazole and cyanamide prevented this effect. 4-methylpyrazole, cyanamide as well as hepatocyte incubation in a HCO3 -free buffer or in the presence of Na+/H+ exchanger blocker 5-(N,N-dimethyl)-amiloride also reduced Na+ influx in ethanol-treated hepatocytes. 4-methylpyrazole and cyanamide similarly prevented ethanol-stimulated Na+ accumulation and hepatocyte killing during hypoxia. Moreover, ethanol-induced Na+ influx caused cytotoxicity in hepatocytes pre-treated with Na+,K+-ATPase inhibitor ouabain. Also in this condition 4-methylpyrazole and 5-(N,N-dimethyl)-amiloride decreased cell killing. These results indicate that ethanol can promotes cytotoxicity in hypoxic hepatocytes by enhancing Na+ accumulation.
Keywords: Sodium; Acidosis; Hypoxia; Cell death; Alcohol related liver injury;

Corrigendum to ‘Metabolism of carnitine in phenylacetic acid-treated rats and in patients with phenylketonuria’ by Gabor M. Fischer; Balazs Nemeti; Viktoria Farkas; Balazs Debreceni; Aranka Laszlo; Agnes Schuler; Csilla Somogyi; Attila Sandor (515).