BBA - Molecular Basis of Disease (v.1688, #3)

The urea cycle and related pathways in the liver of Walker-256 tumor-bearing rats by Sandra Regina Corbello Pereira; Elaine Darronqui; Jorgete Constantin; Mário Henrique da Rocha Alves da Silva; Nair Seiko Yamamoto; Adelar Bracht (187-196).
The urea cycle was evaluated in perfused livers isolated from cachectic tumor-bearing rats (Walker-256 tumor). Urea production in livers of tumor-bearing rats was decreased in the presence of the following substrates: alanine, alanine+ornithine, alanine+aspartate, ammonia, ammonia+lactate, ammonia+pyruvate and glutamine. Urea production from arginine was higher in livers of tumor-bearing rats. No difference was found with aspartate, aspartate+ammonia, citrulline, citrulline+aspartate and glutamine+aspartate. Ammonia consumption was smaller in livers from cachectic rats when the substance was infused together with lactate and pyruvate. Glucose production was smaller in the cachectic condition only when alanine was the gluconeogenic substrate. Blood urea was higher in tumor-bearing rats, suggesting higher rates of urea production. The availability of aspartate seems to be critical for urea synthesis in the liver of tumor-bearing rats, which is possibly unable to produce this amino acid in sufficient amounts from endogenous sources. The liver of tumor-bearing rats may have a different exogenous substrate supply of nitrogenous compounds. Arginine could be one of these compounds in addition to aspartate which seems to be essential for an efficient ureogenesis in tumor-bearing rats.
Keywords: Walker-256 tumor; Cachexia; Liver; Urea; Ammonia; Amino acid;

Glucocerebroside inhibits NADPH oxidase activation in cell-free system by Patryk Moskwa; Anita Palicz; Marie-Hélène Paclet; Marie-Claire Dagher; Melinda Erdős; László Maródi; Erzsébet Ligeti (197-203).
We reported earlier that monocytes and macrophages from patients with type I Gaucher disease have a decreased capacity to generate superoxide anion (O2 ) on stimulation with opsonized S. aureus or formyl-methionyl-leucyl-phenylalanine. In this study, various forms of the cell-free assay system were used to probe the hypothesis that glucocerebroside (GC) accumulating in Gaucher patients' phagocytes may interfere with the activation of NADPH oxidase. Xanthine/xanthine oxidase assay was applied to explore the possibility that GC may scavenge O2 . We found that addition of GC to the crude, semirecombinant or fully purified cell-free systems inhibited activation of NADPH oxidase in a concentration-dependent manner. The inhibitory effect of GC could be overcome by increased concentrations of p47phox and p67phox. In contrast, O2 generation was not decreased by GC added to the assembled, catalytically active enzyme complex. In the xanthine/xanthine oxidase system, GC had no effect on the generation of O2 . These data indicate that assembly of the respiratory burst oxidase of phagocytic cells may be a possible target of the pathologic actions of GC.
Keywords: NADPH oxidase; Glucocerebroside; Gaucher disease;

Renal elimination of organic anions in rats with bilateral ureteral obstruction by Silvina R Villar; Anabel Brandoni; Nora B Quaglia; Adriana M Torres (204-209).
Urinary tract obstruction is an important cause of acute renal failure. Several abnormalities in renal tubular function may occur in obstructive nephropathy. The tubular secretion of organic anions is an important function of the kidney that eliminates potentially toxic organic anions from the body, however, the mechanisms involved in organic anions renal elimination in rats with bilateral ureteral obstruction (BUO) have not been elucidated. In this study, it was evaluated the renal handling of p-aminohippurate (PAH) in adult male Wistar rats with BUO. A diminished renal clearance of PAH was observed in BUO rats as consequence of a diminution in the secreted load of this organic anion. The increase in the abundance of organic anions transporter 1 (OAT1) and the absence of modification in cortical renal blood flow, measured with fluorescence microspheres, do not explain the altered secretion of PAH. The diminished Na,K-ATPase activity in cortex from obstructed kidneys might condition OAT1 function. Additionally, it is also possible to conclude that in the presence of BUO, PAH clearance is not a good estimate of renal plasma flow.
Keywords: Organic anion; Obstructive nephropathy; Organic anion transporter 1 (OAT1); Na,K-ATPase; Acute renal failure;

The hepatic lesion produced as a result of oxidative stress is of wide occurrence. In the present study, the effect of tungsten on liver necrosis and fulminant hepatic failure (FHF) has been studied in rats treated with various compounds known to produce oxidative stress. Supplementation of animals with sodium tungstate for 7 weeks before the induction of liver injury by chemicals including thioacetamide (TAA), carbon tetrachloride (CCl4), or chloroform (CHCl3) could protect progression of hepatic injury. Various biochemical changes associated with liver damage and oxidative stress were measured. Hepatic malondialdehyde content, endogenous tripeptide, and reduced glutathione were measured as oxidative stress markers. The activity of xanthine oxidase, which generates reactive oxygen species (ROS) as a by-product, was also determined and found to be perturbed. Tungsten supplementation to rats caused a significant decrease in lipid peroxidation and lowered the levels of the biochemical markers of hepatic lesions produced by TAA, CCl4 (CCl4), or CHCl3. Tungsten could also cause an increase in the survival rate in rats receiving lethal doses of TAA, CCl4, or CHCl3. The protective effect of tungsten, however, is suggested to be limited to the conditions where the hepatic lesion is reported to be due to the generation of ROS. The progression of liver injury produced by the compounds causing oxidative stress without initiating the generation of free radicals such as bromobenzene (BB), or acetaminophen (AAP), could not be inhibited by tungsten. The possible mechanism explaining the role of oxyanionic form of tungsten in free radical-induced hepatic lesions is discussed.
Keywords: Liver necrosis; Fulminant hepatic failure; Tungsten; Hepatoprotection; Oxidative stress;

Evaluation of MT expression and detection of apoptotic cells in LEC rat kidneys by Alessandro Santon; Vincenzo Albergoni; Giacomo Carlo Sturniolo; Paola Irato (223-231).
To confirm our previous observations on the effectiveness of long term treatment with Zn on Long–Evans Cinnamon (LEC) rats, we extended these studies determining the effects of Zn on trace elements, metallothionein (MT) concentrations and immunolocalization, and on the levels of both MT-1 and MT-2 mRNAs in the LEC rat kidneys. We also localized the renal cells that had chromatin condensation and nuclear fragmentation typical of apoptosis. The results demonstrate that the amount of Zn increased in the treated rats with respect to both untreated and basal rats. In the treated rats the amount of Cu and Fe was similar to that of the basal rats. MT concentrations did not change either with or without Zn treatment, but were higher than the basal group. However, if we consider the percentage of oxidized MT (MTox), we note that Zn treatment is very effective in reducing this value. MTox is not able to bind metals, so it does not perform a “scavenger” function. Moreover, quantification of mRNA indicates that the MT-1 isoform was significantly higher than the MT-2 isoform following Zn treatment. Untreated group sections showed a confocal fluorescent signal that highlighted the irregular nuclei and small apoptotic bodies. The intensity and quantity of fluorescence decreased in the treated group sections. These findings suggest that, in LEC rats, Zn may contribute to cytoprotection through the regulation of MT expression which may provide a cellular defence strategy in response to DNA damage.
Keywords: Metallothionein; Apoptosis; LEC rat;

The human recombinant histamine releasing factor (HrHRF), also known as translationally controlled tumor protein (TCTP), p23 and fortilin, has been described to have both extra- and intracellular functions. To elucidate an extra- or intracellular role for HrHRF, we used the yeast two-hybrid system with HrHRF as the bait and a Jurkat T cell library. We isolated a partial cDNA clone of the human elongation factor-1 delta (EF-1δ) encoding for amino acids 12 to 281. This interaction was confirmed by co-immunoprecipitation experiments. Previously, both HrHRF and EF-1δ have been isolated and identified in association with malignancy in numerous studies. EF-1δ is part of the EF-1 complex responsible for kinetic proofreading in protein synthesis. Additionally, DNA microarray data classifies TCTP (HrHRF) as co-regulated with ribosomal proteins and recent structural analysis of TCTP (HrHRF) relates it to a guanine nucleotide-free chaperone. Our findings of an interaction between HrHRF and EF-1δ taken with some of the recently published information concerning the TCTP (HrHRF) mentioned above suggest a possible intracellular role for TCTP/HrHRF.
Keywords: Human recombinant histamine releasing factor; Translationally controlled tumor protein; Yeast two-hybrid system; Elongation factor-1 delta; eElongation factor-1B beta;

We have previously prepared two B7-1 transfectant clones (AC29 B7-6 and AC29 B7-7) from the AC29 murine mesothelioma (MM) cell line which displayed markedly different in vivo growth rates and susceptibility to cytotoxic T cell killing. Using suppression subtractive hybridisation (SSH), we searched for factors which may determine the biological distinction seen in these clones. We isolated 19 cDNA clones from two SSH generated libraries by screening using subtracted cDNA probes and characterised them using Northern hybridisation, sequencing, RT-PCR and real-time RT-PCR. The 19 cDNA clones comprised 16 different transcripts of which 15 were identified by homology to known genes and one was novel. Expression of a murine endogenous retroviral (mERV) transcript mERV-AC29 was found in the immunogenic AC29 B7-6 clone and parental AC29 but absent in AC29 B7-7. Real-time RT-PCR was used to confirm that galectin-1, the disintegrin/metalloproteinase MDC9 and ribonucleotide reductase M1 were overexpressed in AC29 B7-7. Our results show that SSH is a powerful method for the identification of genes expressed differentially between phenotypically different tumour cell lines or clones. Characterisation of the role of those identified here will provide useful information in understanding genes responsible for differential tumorigenicity.
Keywords: Mesothelioma; Suppression subtractive hybridization; Differential gene expression; Endogenous retrovirus; Real-time PCR;

Characterization of xanthine oxidase inhibition by anacardic acids by Noriyoshi Masuoka; Isao Kubo (245-249).
Anacardic acid, 6[8(Z), 11(Z), 14-pentadecatrienyl]salicylic acid, inhibits generation of superoxide radicals by xanthine oxidase. This inhibition does not follow a hyperbolic inhibition, depends on anacardic acid concentrations, but follows a sigmoidal inhibition. The inhibition was analyzed by using a Hill equation, and slope factor and EC50 were 4.3±0.5 and 53.6±5.1 μM, respectively. In addition, anacardic acid inhibited uric acid formation by xanthine oxidase cooperatively. Slope factor and EC50 were 1.7±0.5 and 162±10 μM, respectively. The results indicate that anacardic acid binds to allosteric sites near the xanthine-binding domain in xanthine oxidase. Salicylic acid moiety and alkenyl side chain in anacardic acid are associated with the cooperative inhibition and hydrophobic binding, respectively.
Keywords: Antioxidant activity; Anacardic acid; Salicylic acid; Xanthine oxidase; Cooperative inhibition kinetic; Hill equation;

High expression of both mutant and wild-type alleles of c-kit in gastrointestinal stromal tumors by Nathalie Théou; Séverine Tabone; Raphael Saffroy; Axel Le Cesne; Catherine Julié; Annie Cortez; Anne Lavergne-Slove; Brigitte Debuire; Antoinette Lemoine; Jean-François Emile (250-256).
Most gastrointestinal stromal tumors (GISTs) contain activating mutations of the proto-oncogene c-kit. The GNNK− isoform of c-kit has a greater oncogenic potential than the GNNK+ isoform. We studied tumors from 29 patients with GIST, 19 of whom had c-kit mutations, and compared them to normal cells and HMC-1 mast cell line. c-kit transcripts were quantified by real-time PCR. The ratios of GNNK−/+ isoforms and of wild-type/mutant alleles were determined by RT-PCR and fluorometric quantification. On average, GISTs contained 1.9 times more c-kit transcripts than the HMC-1 cell line and GISTs with c-kit mutations contained 2.8 times more c-kit transcripts than those without (P=0.003). The median GNNK−/+ isoform ratios in GISTs with and without c-kit mutations were 4.4 and 4.1, respectively, and there was no difference in the GNNK−/+ ratios between the GISTs and the control samples. Both mutant and wild-type alleles of c-kit were expressed in similar amounts in 13/15 mutant GISTs. The oncogenic effects of KIT in GISTs are not related to the higher expression level of the GNNK− isoform. The high expression level of both mutated and wild-type allele transcripts of c-kit suggests that interactions between spontaneously activated and normal c-kit receptors are important in GIST tumorigenesis.
Keywords: Proto-oncogene c-kit; Digestive system neoplasm; Mutation; RNA; Isoform;

Azidothymidine (AZT) and AZT monophosphate (AZT-MP) in concentrations as low as 10 and 50 μM, respectively, promote oxidation of chemically deacetylated 2′,7′-dichlorodihydrofluorescein (DCDHF) to 2′,7′-dichlorofluorescein (DCF) by rat peritoneal macrophages activated with latex. Cells were incubated with AZT and AZT-MP for 18 h, washed out from residual AZT or AZT-MP and activated with latex for 30 or 60 min in the presence of DCDHF. Latex-activated cells oxidize DCDHF extracellularly due to release of hydrogen peroxide and low-molecular iron complexes, which is verified using catalase, desferal and the peroxidase inhibitor sodium azide. AZT and AZT-MP increase DCDHF oxidation due to additional release of hydrogen peroxide as demonstrated by catalase inhibition of DCDHF oxidation and direct H2O2 measurement. Thymidine and thymidine phosphates did not show any effect on macrophage activation. In separate experiments we evaluated the in vitro prooxidant activity of AZT, AZT-MP, AZT triphosphate (AZT-TP), AZT glucuronide (GAZT) and 3′-amino-3′-deoxythymidine (AMT) in a cell-free system using the hydrogen peroxide-iron-mediated oxidation of DCDHF. Under these conditions, AZT and AZT phosphates exhibit a prooxidant effect in concentrations as low as 100 μM. Furthermore, GAZT is a less effective prooxidant and AMT acts like an antioxidant. Thymidine did not show any effect.
Keywords: AZT; Azidothymidine; Zidovudine; AZT phosphate; AZT glucuronide; Free radical; Oxidative stress; Chemically deacetylated 2′,7′-dichlorodihydrofluorescein;

Iron uncouples oxidative phosphorylation in brain mitochondria isolated from vitamin E-deficient rats by Govind T. Vatassery; Eugene G. DeMaster; James C.K. Lai; W.Ed Smith; Hung T. Quach (265-273).
Few, if any, studies have examined the effect of vitamin E deficiency on brain mitochondrial oxidative phosphorylation. The latter was studied using brain mitochondria isolated from control and vitamin E-deficient rats (13 months of deficiency) after exposure to iron, an inducer of oxidative stress. Mitochondria were treated with iron (2 to 50 μM) added as ferrous ammonium sulfate. Rates of state 3 and state 4 respiration, respiratory control ratios, and ADP/O ratios were not affected by vitamin E deficiency alone. However, iron uncoupled oxidative phosphorylation in vitamin E-deficient mitochondria, but not in controls. In vitamin E-deficient mitochondria, iron decreased ADP/O ratios and markedly stimulated state 4 respiration; iron had only a modest effect on these parameters in control mitochondria. Thus, vitamin E may have an important role in sustaining oxidative phosphorylation. Low concentrations of iron (2 to 5 μM) oxidized mitochondrial tocopherol that exists in two pools. The release of iron in brain may impair oxidative phosphorylation, which would be exacerbated by vitamin E deficiency. The results are important for understanding the pathogenesis of human brain disorders known to be associated with abnormalities in mitochondrial function as well as iron homeostasis (e.g., Parkinson's disease).
Keywords: Vitamin E; Iron; Tocopherol; Mitochondria; Uncoupling; Deficiency;

Impaired regulation of sterol regulatory element binding protein 2 in cholesterol gallstone-susceptible mice by Guoqiang Xu; Oliver Müller; Eduard F. Stange; Michael Fuchs (274-279).
Lipid synthesis is under tight transcriptional control involving sterol regulatory element binding proteins (SREBP1, SREBP2). Rising cellular cholesterol levels prevent SREBP2 from entering the nucleus to directly activate the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr), the key enzyme of cholesterol synthesis. The failure to down-regulate cholesterol synthesis in gallstone-susceptible C57L/J but not AKR/J mice prompted us to study the processing of SREBP2 in these mice. Male mice of each strain received a control or lithogenic diet for 28 days. Membrane and nuclear extracts of pooled livers were prepared for immunoblot analysis of SREBP. Steady-state mRNA levels of Hmgcr, Srebp1, Srebp2 and the SREBP cleavage activating protein (Scap) were measured from individual livers. There was no marked difference between the two strains with regard to processing of SREBP1 as well as steady-state mRNA levels of Srebp1, Srebp2 and Scap. However, a near-complete suppression of nuclear SREBP2 related to low Hmgcr mRNA levels was noticed only for gallstone-resistant AKR mice. Abnormal regulation of SREBP2 appears to be responsible for the failure to suppress cholesterol synthesis in genetically cholesterol gallstone-susceptible mice. This defect may contribute to cholesterol hypersecretion and gallstone formation.
Keywords: Gallstone; HMGCoA reductase; Biliary cholesterol; SREBP; SCAP; Lith gene;