BBA - Molecular Basis of Disease (v.1637, #1)

Genetic background of cholesterol gallstone disease by Astrid Kosters; Milan Jirsa; Albert K Groen (1-19).
Cholesterol gallstone formation is a multifactorial process involving a multitude of metabolic pathways. The primary pathogenic factor is hypersecretion of free cholesterol into bile. For people living in the Western Hemisphere, this is almost a normal condition, certainly in the elderly, which explains the very high incidence of gallstone disease. It is probably because the multifactorial background genes responsible for the high incidence have not yet been identified, despite the fact that genetic factors clearly play a role. Analysis of the many pathways involved in biliary cholesterol secretion reveals many potential candidates and considering the progress in unraveling the regulatory mechanisms of the responsible genes, identification of the primary gallstone genes will be successful in the near future.
Keywords: Gallstone disease; Cholesterol; Bile salt; Epidemiology; Genetics;

The study evaluated effects of hyposmotic shock on the rate of Rb+/K+ efflux, intracellular pH and energetics in Langendorff-perfused rat hearts with the help of 87Rb- and 31P-NMR. Two models of hyposmotic shock were compared: (1) normosmotic hearts perfused with low [NaCl] (70 mM) buffer, (2) hyperosmotic hearts equilibrated with additional methyl α-d-glucopyranoside (Me-GPD, 90 or 33 mM) or urea (90 mM) perfused with normosmotic buffer. Four minutes after hyposmotic shock, Rb+ efflux rate constant transiently increased approximately two-fold, while pH transiently decreased by 0.08 and 0.06 units, in the first and the second models, respectively, without significant changes in phosphocreatine and ATP. Hyposmotic shock (second model) did not change the rate of Rb+/K+ uptake, indicating that the activity of Na+/K+ ATPase was not affected. Dimethylamiloride (DMA) (10 μM) abolished activation of the Rb+/K+ efflux in the second model; however, Na+/H+ exchanger was not involved, because intracellular acidosis induced by the hyposmotic shock was not enhanced by DMA treatment. After 12 or 20 min of global ischemia, the rate of Rb+/K+ efflux increased by 120%. Inhibitor of the ATP-sensitive potassium channels, glibenclamide (5 μM), partially (40%) decreased the rate constant; however, reperfusion with hyperosmolar buffer (90 mM Me-GPD) did not. We concluded that the shock-induced stimulation of Rb+/K+ efflux occurred, at least partially, through the DMA-sensitive cation/H+ exchanger and swelling-induced mechanisms did not considerably contribute to the ischemia–reperfusion-induced activation of Rb+/K+ efflux.
Keywords: Osmotic shock; Ischemia; Cation/H+ exchanger; Intracellular pH; Rb+/K+ flux;

Inhibition of free radical-induced peroxidation of rat liver microsomes by resveratrol and its analogues by Yu-Jun Cai; Jian-Guo Fang; Lan-Ping Ma; Li Yang; Zhong-Li Liu (31-38).
Resveratrol (3,5,4′-trans-trihydroxystilbene) is a natural phytoalexin present in grapes and red wine, which possesses a variety of biological activities including antioxidative activity. To find more efficient antioxidants by structural modification, resveratrol analogues, that is, 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4′-dihydroxy-trans-stilbene (4,4′-DHS), 4-hydroxy-trans-stilbene (4-HS) and 3,5-dihydroxy-trans-stilbene (3,5-DHS), were synthesized and their antioxidant activity studied for the free radical-induced peroxidation of rat liver microsomes in vitro. The peroxidation was initiated by either a water-soluble azo compound 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH) or Fe2+/ascorbate, and monitored by oxygen uptake and formation of thiobarbituric acid reactive substances (TBARS). It was found that all of these trans-stilbene derivatives are effective antioxidants against both AAPH- and iron-induced peroxidation of rat liver microsomes with an activity sequence of 3,4-DHS>4,4′-DHS>resveratrol>4-HS>3,5-DHS. The remarkably higher antioxidant activity of 3,4-DHS is discussed.
Keywords: Lipid peroxidation; Antioxidant; Resveratrol; Microsome; Structure/activity relationship;

Cardiovascular changes of still obscure origin are sometimes correlated with co-existing liver diseases, as cholestasis.The aim of this work was to examine and compare cardiac mitochondrial bioenergetics and calcium loading capacity from rats injected with a single dose of α-naphthylisothiocyanate (ANIT), a cholestasis-inducing compound. Forty-eight hours after ANIT administration, blood samples were collected and markers for hepatic disease were determined. Heart mitochondria from both control and ANIT-injected rats were isolated and subjected to biochemical characterization, including the susceptibility to the calcium-dependent permeability transition. The results showed that cardiac mitochondria from cholestatic animals did not have significant changes in respiratory parameters or in the basal levels of adenine nucleotide. The most impressive result from this work was that cardiac mitochondria from ANIT-injected animals had a lower calcium loading capacity. The prevention of this property by cyclosporin-A, a specific inhibitor of the mitochondrial permeability transition, showed that this phenomenon was reason for the reduced calcium loading capacity in ANIT-injected animals. The results suggest that, during the development of ANIT-induced cholestasis, heart mitochondria loose their default ability to buffer calcium. Our results may contribute to explain the occurrence of cardiomyopathies sometimes associated with cholestatic disease.
Keywords: Cardiac mitochondrion; Mitochondrial permeability transition; Hepatic disease; Cholestasis;

Characterization of hydrogen peroxide removal reaction by hemoglobin in the presence of reduced pyridine nucleotides by Noriyoshi Masuoka; Hiroyuki Kodama; Tadashi Abe; Da-Hong Wang; Taku Nakano (46-54).
Hydrogen peroxide removal rates by hemoglobin were enhanced in the presence of reduced pyridine nucleotides. The species which had the activity to oxidize pyridine nucleotides was purified from human blood and identified as hemoglobin A. Hydrogen peroxide removal rates by hemoglobin A without reduced pyridine nucleotides at 0.2 mM hydrogen peroxide were 0.87±0.11 μmol/s/g hemoglobin, and the removal rates using 0.2 mM NADH and NADPH were 2.02±0.20 and 1.96±0.31 μmol/s/g hemoglobin, respectively. We deduced that the removal reaction by hemoglobin included formations of methemoglobin and the ferryl radical and reduction of the latter with pyridine nucleotides. The hydrogen peroxide removal ability by hemoglobin was less than that by catalase but was larger than that by glutathione peroxidase-glutathione reductase system at 0.2 mM hydrogen peroxide. Under acatalasemic conditions, it was suggested that NAD(P)H were important factors to prevent the oxidative degradation of hemoglobin.
Keywords: Takahara disease; Acatalasemic erythrocyte; Hydrogen peroxide removal; Hemoglobin; Reduced pyridine nucleotide; Glutathione peroxidase;

Antibody production in early life supported by maternal lymphocyte factors by Michio Shimamura; Yi-Ying Huang; Hiroshi Goji (55-58).
To examine the influence of maternal lymphocyte factors on the immune responses in offspring in early life, antibody production in neonates born to either normal or lymphocyte-deficient mothers was analyzed. Recombination activating gene (Rag)-2+/− mouse neonates born to Rag-2+/+, Rag-2+/−or Rag-2−/−mothers were injected with goat anti-mouse IgD antiserum, and IgE and IgG1 production was evaluated. The levels of IgE and IgG1 were higher in the pups born to Rag-2+/+and Rag-2+/− dams than to lymphocyte-deficient Rag-2−/− dams. The enhanced antibody production in the former compared with the latter neonates was also found following immunization with ovalbumin or TNP-Ficoll. Thus, the presence of maternal lymphocyte factors was suggested in neonates that augmented antigen-specific antibody production in both T cell-dependent and -independent pathways. A reduction in antibody production was observed in normal neonates when they were foster-nursed by Rag-2−/− mothers. Thus, the maternal lymphocyte factors enhancing the immune responses in newborns were shown to be present in breast-milk.
Keywords: Antibody production; Neonatal life; Transplacental passage; Breast-feeding;

Inhibition of protein kinase C by resveratrol by Simon J Slater; Jodie L Seiz; Anthony C Cook; Brigid A Stagliano; Christopher J Buzas (59-69).
Evidence is emerging that resveratrol (RV), a polyphenolic phytoaxelin present in dietary sources including red wine, may protect against atherosclerosis and cardiovascular disease by enhancing the integrity of the endothelium. In this study, the possibility that such beneficial effects of RV may arise from a modulation of protein kinase C (PKC)-mediated signaling was investigated by determining the effects of RV on the in vitro activities of PKC isozymes. It was found that the Ca2+-dependent activities of membrane-associated PKCα induced by either phorbol ester or diacylglycerol were potently inhibited by RV, each with an IC50 of ∼2 μM. The inhibitory effect of RV was also observed for conventional PKCβI, whereas the activities of novel PKCε and atypical PKCζ were each unaffected. The inhibition of PKCα activity was found to be competitive with respect to phorbol ester concentration but noncompetitive with respect to Ca2+ and phosphatidylserine concentrations, suggesting that the RV may compete for phorbol ester-binding to the C1 domains. Supporting this, it was found that RV bound to a fusion peptide containing the C1A and C1B domains of PKCα. Similar to the effects of diacylglycerol and phorbol ester, the interaction of RV with the C1 domains induced the association of PKCα with membrane lipid vesicles, although this did not result in activation. Overall, the results suggest that the inhibitory effect of RV on PKC activity, and therefore on the associated signaling networks, may, in part, underlie the mechanism(s) by which this agent exerts its beneficial effects on endothelial and cardiovascular function. Furthermore, the effects of RV on these signaling networks are predicted to differ according to the cellular localization and the regulating PKC isozyme.
Keywords: Protein kinase C; Resveratrol; Atherosclerosis;

Spermine induces cataract and 43-kDa protein that binds spermine possibly participates in the cataract formation by Satoru Maekawa; Moto Kataoka; Yukitaka Uji; Hiroshige Hibasami; Kunio Nakashima (70-74).
Among polyamines (putrescine, spermidine, and spermine), spermine specifically induces cataract in an organ cultured lens. Spermine uptake nearly paralleled the cataract formation. When polyamines were added to lens soluble proteins, spermine specifically induced turbidity. When lens soluble proteins were separated by gel chromatography, heavy-molecular-weight protein (HMW, high molecular form of α-crystallin) and proteins between βH- and βL-crystallin fractions reacted with spermine and aggregated. SDS-polyacrylamide gel electrophoresis of the aggregated proteins showed that 43-kDa lens protein was commonly observed in both aggregates. Spermine-affinity chromatography of the total soluble proteins showed the binding of HMW protein to the gel and the chromatogram of the second turbidity peak in the gel chromatography showed the binding of 43-kDa protein. These results indicated that 43-kDa protein, which is present as a subunit in HMW and also in free form, binds spermine and induces turbidity of lens soluble proteins and produces cataract in a cultured lens.
Keywords: Lens; Polyamine; Spermine; Cataract; Crystallin;

High in comparison with low tidal volume ventilation aggravates oxidative stress-induced lung injury by Stefan Hammerschmidt; Torsten Sandvoß; Christian Gessner; Joachim Schauer; Hubert Wirtz (75-82).
Ventilator settings influence the development and outcome of acute lung injury. This study investigates the influence of low versus high tidal volume (V t) on oxidative stress-induced lung injury.Isolated rabbit lungs were subjected to one of three ventilation patterns (V t–positive end-expiratory pressure, PEEP): LVZP (6 ml/kg–0 cm H2O), HVZP (12 ml/kg–0 cm H2O), LV5P (6 ml/kg–5 cm H2O). These ventilation patterns allowed a comparison between low and high V t without dependence on peak inspiratory pressure (PIP). Infusion of hypochlorite (1000 nmol/min) or buffer (control) was started at t=0 min. Pulmonary artery pressure (PAP), PIP and weight were continuously recorded. Capillary filtration coefficient [K f,c (10−4 ml s−1 cm H2O−1 g−1)] was gravimetrically determined (−15/30/60/90/120 min).PIP averaged 5.8±0.6/13.9±0.6/13.9±0.4 cm H2O in the LVZP, HVZP and LV5P groups. PIP, K f,c or PAP did not change in control groups, indicating that none of the ventilation patterns caused lung injury by themselves. Hypochlorite-induced increase in K f,c but not hypochlorite-induced increase in PAP, was significantly attenuated in the LVZP-/LV5P- versus the HVZP-group (K f,c,max. 1.0±0.23/1.4±0.40 versus 3.2±1.0*). Experiments with hypochlorite were terminated due to excessive edema (>50 g) at 97±2.2/94.5±4.5 min in the LVZP-/LV5P-group versus 82±3.8* min in the HVZP-group (*: P<0.05).Low V t attenuated oxidative stress-induced increase in vascular permeability independently from PIP and PEEP.
Keywords: Artificial ventilation; Tidal volume; Acute respiratory distress syndrome (ARDS); Mechanical stress; Oxidative stress; Hypochlorous acid;

Disorders of the microcirculation and reduced resistance to infection are major complications in diabetes. Histamine enhances capillary permeability, and may also reduce cellular immunity. Here we demonstrate that streptozotocin (STZ)-induced diabetes in mice not only enhances the activity of the histamine-forming enzyme, histidine decarboxylase (HDC), but also augments the lipopolysaccharide (LPS)-induced elevation of HDC activity in various tissues, resulting in a production of histamine. The augmentation of HDC activity occurred as early as 2 days after STZ injection, but was not seen in nondiabetic mice. When given to STZ-treated mice, nicotinamide, an inhibitor of poly(ADP-ribose) synthetase, reduced both the elevation of blood glucose and the elevations of HDC activity and histamine production. These results suggest that hyperglycemia may initiate a sequence of events leading not only to an enhancement of basal HDC activity, but also to a sensitization of mice to the HDC-inducing action of LPS. We hypothesize that bacterial infections and diabetic complications may mutually exacerbate one another because both involved an induction of HDC.
Keywords: Histamine; Histidine decarboxylase; Diabetes; Hyperglycemia; Periodontitis; Infectious disease;

Effect and possible role of Zn treatment in LEC rats, an animal model of Wilson's disease by Alessandro Santon; Paola Irato; Valentina Medici; Renata D'Incà; Vincenzo Albergoni; Giacomo Carlo Sturniolo (91-97).
The effect of oral zinc (Zn) treatment was studied in the liver, kidneys and intestine of Long–Evans Cinnamon (LEC) rats in relation to metals interaction and concentration of metallothionein (MT) and glutathione (GSH). We also investigated the change in the activity of antioxidant enzymes and determined the biochemical profile in the blood and metal levels in urine. We showed that the Zn-treated group had higher levels of MT in the hepatic and intestinal cells compared to both untreated and basal groups. Tissue Zn concentrations were significantly higher in the Zn-treated group compared to those untreated and basal, whereas Cu and Fe concentrations decreased. The antioxidant enzyme activities in the Zn-treated group did not change significantly with respect to those in the basal group, except for hepatic glutathione peroxidase activity. Moreover, the biochemical data in the blood of Zn-treated group clearly ascertain no liver damage. These observations suggest an important role for Zn in relation not only to its ability to compete with other metals at the level of absorption in the gastrointestinal tract producing a decrease in the hepatic and renal Cu and Fe deposits, but also to MT induction as free radical scavenger.
Keywords: Trace element; Metallothionein; Glutathione; Antioxidant enzyme; Oxidative stress; LEC rat;

Glyceraldehyde-3-phosphate dehydrogenase activity as an independent modifier of methylglyoxal levels in diabetes by Paul J Beisswenger; Scott K Howell; Kenneth Smith; Benjamin S Szwergold (98-106).
Methylglyoxal (MG) may be an important cause of diabetic complications. Its primary source is dihydroxyacetone phosphate (DHAP) whose levels are partially controlled by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using a human red blood cell (RBC) culture, we examined the effect of modifying GAPDH activity on MG production. With the inhibitor koningic acid (KA), we showed a linear, concentration-dependent GAPDH inhibition, with 5 μM KA leading to a 79% reduction of GAPDH activity and a sixfold increase in MG. Changes in redox state produced by elevated pH also resulted in a 2.4-fold increase in MG production at pH 7.5 and a 13.4-fold increase at pH 7.8. We found substantial inter-individual variation in DHAP and MG levels and an inverse relationship between GAPDH activity and MG production (R=0.57, P=0.005) in type 2 diabetes. A similar relationship between GAPDH activity and MG was observed in vivo in type 1 diabetes (R=0.29, P=0.0018).Widely varying rates of progression of diabetic complications are seen among individuals. We postulate that modification of GAPDH by environmental factors or genetic dysregulation and the resultant differences in MG production could at least partially account for this observation.
Keywords: Methylglyoxal; Glyceraldehyde-3-phosphate dehydrogenase; Diabetes; Red blood cell; Carbohydrate metabolism;

Lipid peroxidation causes the generation of the neurotoxic aldehydes acrolein and 4-hydroxy-trans-2-nonenal (HNE). These products are elevated in neurodegenerative diseases and acute CNS trauma. Previous studies demonstrate that mitochondrial class 2 aldehyde dehydrogenase (ALDH2) is susceptible to inactivation by these alkenals. In the liver and brain another mitochondrial aldehyde dehydrogenase, succinic semialdehyde dehydrogenase (SSADH/ALDH5A1), is present. In this study, we tested the hypothesis that aldehyde products of lipid peroxidation inhibit SSADH activity using the endogenous substrate, succinic semialdehyde (SSA, 50 μM). Acrolein potently inhibited SSADH activity (IC50=15 μM) in rat brain mitochondrial preparations. This inhibition was of an irreversible and noncompetitive nature. HNE inhibited activity with an IC50 of 110 μM. Trans-2-hexenal (HEX) and crotonaldehyde (100 μM each) did not inhibit activity. These data suggest that acrolein and HNE disrupt SSA metabolism and may have subsequent effects on CNS neurochemistry.
Keywords: Lipid peroxidation; Succinic semialdehyde dehydrogenase; 4-hydroxynonenal; Acrolein; Alzheimer's disease;

Human β-hexosaminidase A (Hex A) (αβ) is composed of two subunits whose primary structures are ∼60% identical. Deficiency of either subunit results in severe neurological disease due to the storage of GM2 ganglioside; Tay–Sachs disease, α deficiency, and Sandhoff disease, β deficiency. Whereas both subunits contain active sites only the α-site can efficiently bind negatively charged 6-sulfated hexosamine substrates and GM2 ganglioside. We have recently identified the αArg424 as playing a critical role in the binding of 6-sulfate-containing substrates, and βAsp452 as actively inhibiting their binding. To determine if these same residues affect the binding of the sialic acid moiety of GM2 ganglioside, an αArg424Gln form of Hex A was expressed and its kinetics analyzed using the GM2 activator protein:[3H]-GM2 ganglioside complex as a substrate. The mutant showed a ∼3-fold increase in its K m for the complex. Next a form of Hex B (ββ) containing a double mutation, βAspLeu453AsnArg (duplicating the α-aligning sequences), was expressed. As compared to the wild type (WT), the mutant exhibited a >30-fold increase in its ability to hydrolyze a 6-sulfated substrate and was now able to hydrolyze GM2 ganglioside when the GM2 activator protein was replaced by sodium taurocholate. Thus, this α-site is critical for binding both types of negatively charge substrates.
Keywords: Tay–Sachs disease; Sandhoff disease; GM2 Gangliosidosis; Structure–function;

Mitochondrial dysfunction occurs in many neurodegenerative diseases. The α-ketoglutarate dehydrogenase complex (KGDHC) catalyzes a key and arguably rate-limiting step of the tricarboxylic acid cycle (TCA). A reduction in the activity of the KGDHC occurs in brains and cells of patients with many of these disorders and may underlie the abnormal mitochondrial function. Abnormalities in calcium homeostasis also occur in fibroblasts from Alzheimer's disease (AD) patients and in cells bearing mutations that lead to AD. Thus, the present studies test whether the reduction of KGDHC activity can lead to the alterations in mitochondrial function and calcium homeostasis. α-Keto-β-methyl-n-valeric acid (KMV) inhibits KGDHC activity in living N2a cells in a dose- and time-dependent manner. Surprisingly, concentration of KMV that inhibit in situ KGDHC by 80% does not alter the mitochondrial membrane potential (MMP). However, similar concentrations of KMV induce the release of cytochrome c from mitochondria into the cytosol, reduce basal [Ca2+]i by 23% (P<0.005), and diminish the bradykinin (BK)-induced calcium release from the endoplasmic reticulum (ER) by 46% (P<0.005). This result suggests that diminished KGDHC activities do not lead to the Ca2+ abnormalities in fibroblasts from AD patients or cells bearing PS-1 mutations. The increased release of cytochrome c with diminished KGDHC activities will be expected to activate other pathways including cell death cascades. Reductions in this key mitochondrial enzyme will likely make the cells more vulnerable to metabolic insults that promote cell death.
Keywords: Mitochondrial membrane potential; α-keto-β-methyl-n-valeric acid; Neuroblastoma; Cytochrome c; Endoplasmic reticulum; Calcium;

Mitochondrially mediated synergistic cell killing by bile acids by Anabela P Rolo; Carlos M Palmeira; Kendall B Wallace (127-132).
The accumulation of endogenous bile acids contributes to hepatocellular damage during cholestatic liver disease. To examine the controversy regarding the therapeutic use of ursodeoxycholate (UDCA) in cholestatic patients, we investigated the possible cytoprotection or synergistic effects of UDCA against chenodeoxycholate (CDCA)-induced injury to isolated rat hepatocytes. Our aim was to investigate the role of the mitochondrial permeability transition (MPT) in the mechanism of cytotoxicity caused by UDCA plus CDCA. Although not toxic by itself, UDCA potentiated the mitochondrial depolarization, ATP depletion and cell killing caused by CDCA. Fructose maintained ATP levels and prevented bile acid-induced cell killing. Cyclosporine A (CyA), a potent inhibitor of the MPT, substantially reduced mitochondrial depolarization, ATP depletion and cell killing caused by CDCA. Our results demonstrate that the synergistic cytotoxicity by UDCA plus CDCA is mediated by impairment of mitochondrial function, an event that is expressed via induction of the MPT.
Keywords: Hepatocyte; Bile acid; Ursodeoxycholate; Mitochondrial permeability transition; Cholestasis;