Enhanced Phosphorylation of Bax and Its Translocation into Mitochondria in the Brains of Individuals Affiliated with Alzheimer's Disease.

BACKGROUND: Despite increased neuronal death, senile plaques, and neurofibrillary tangles observed in patients suffering from Alzheimer's disease (AD), the detailed mechanism of cell death in AD is still poorly understood. METHOD: We hypothesized that p38 kinase activates and then phosphorylates Bax, leading to its translocation to mitochondria in AD brains compared to controls. The aim of this study was to investigate the role of p38 kinase in phosphorylation and sub-cellular localization of pro-apoptotic Bax in the frontal cortex of the brains from AD and control subjects. Increased oxidative stress in AD individuals compared to control was evaluated by measuring the levels of carbonylated proteins and oxidized peroxiredoxin, an antioxidant enzyme. The relative amounts of p38 kinase and phospho-Bax in mitochondria in AD brains and controls were determined by immunoblot analysis using the respective antibody against each protein following immunoprecipitation. RESULTS: Our results showed that the levels of oxidized peroxiredoxin-SO3 and carbonylated proteins are significantly elevated in AD brains compared to controls, demonstrating the increased oxidative stress. CONCLUSION: The amount of phospho-p38 kinase is increased in AD brains and the activated p38 kinase appears to phosphorylate Thr residue(s) of Bax, which leads to its mitochondrial translocation, contributing to apoptosis and ultimately, neurodegeneration.

ASXL2 promotes proliferation of breast cancer cells by linking ERα to histone methylation.

Estrogen receptor alpha (ERα) has a pivotal role in breast carcinogenesis by associating with various cellular factors. Selective expression of additional sex comb-like 2 (ASXL2) in ERα-positive breast cancer cells prompted us to investigate its role in chromatin modification required for ERα activation and breast carcinogenesis. Here, we observed that ASXL2 interacts with ligand E2-bound ERα and mediates ERα activation. Chromatin immunoprecipitation-sequencing analysis supports a positive role of ASXL2 at ERα target gene promoters. ASXL2 forms a complex with histone methylation modifiers including LSD1, UTX and MLL2, which all are recruited to the E2-responsive genes via ASXL2 and regulate methylations at histone H3 lysine 4, 9 and 27. The preferential binding of the PHD finger of ASXL2 to the dimethylated H3 lysine 4 may account for its requirement for ERα activation. On ASXL2 depletion, the proliferative potential of MCF7 cells and tumor size of xenograft mice decreased. Together with our finding on the higher ASXL2 expression in ERα-positive patients, we propose that ASXL2 could be a novel prognostic marker in breast cancer.

Factors related with metastasis of right retroesophageal lymph nodes in papillary thyroid cancer.

AIM: Right retroesophageal lymph nodes (RRLNs) should be involved in central lymph nodes (CLNs) dissection in patients with papillary thyroid cancer (PTC). This study assessed the incidence and factors related to RRLNs metastasis. METHODS: From January 2008 to March 2010, 129 patients who underwent total thyroidectomy with CLNs dissection including RRLNs were enrolled. The predictive value of RRLNs metastasis was assessed. RESULTS: Twenty six (20.1%) of 129 patients exhibited nodal metastasis in RRLNs. Metastasis of RRLNs was associated with large tumor size (>1 cm; P<0.01), multiplicity (P=0.03), preoperative LN enlargement (P<0.01), metastasis of non-retroesophageal lateral LN (P<0.01) and large number of CLNs metastases (P<0.01) in univariate analysis. Multivariate analysis revealed that tumor size (>1 cm) and metastasis of non-retroesophageal lateral LN were independent correlates of RRLNs metastases. CONCLUSION: RRLNs may be removed during operation for PTC, particularly in patients with tumor >1 cm and lateral LN metastases.

Effects of food intake on pharmacokinetics of mosapride in beagle dogs.

This study was performed to determine the pharmacokinetic profile of mosapride in fasting and fed states. A single 5-mg oral dose of mosapride was administered to fasted (n = 15) and fed (n = 12) beagle dogs, and the plasma concentrations of mosapride were measured by liquid chromatography-tandem mass spectrometry. The resultant data were analyzed by noncompartmental analysis (NCA). Mosapride was absorbed in fasted and fed dogs with similar Tmax . Both Cmax and AUC were significantly higher in the fasting group than in fed dogs, being four times (10.51 µg/mL vs. 2.76 µg/mL) and 3.5 times higher (38.53 h · µg/mL vs. 10.22 h · µg/mL), respectively. These findings suggest that food intake affects the pharmacokinetics of mosapride and that the dosage regimen for this drug need to be reconsidered.

Roles of alcohol and tobacco exposure in the development of hepatocellular carcinoma.

The purpose of this report is to summarize the roles of alcohol and tobacco exposure in the development of hepatocellular carcinoma (HCC). Chronic heavy alcohol exposure is a major risk factor for HCC, which is the most frequent type of liver cancer. Alcohol ingestion may initiate and or promote the development of HCC by: 1) acetaldehyde-DNA adduct formation; 2) cytochrome P4502E1-associated reactive oxygen species (ROS) generation , lipid peroxidation, p53 mutation, and conversion of pro-carcinogens to carcinogens; 3) iron accumulation that leads to ROS generation, lipid peroxidation, p53 mutation, and initiation of inflammatory cascade via nuclear factor-KappaB (NF-kB) activation; 4) glutathione depletion leading to oxidative stress; 5) s-adenosylmethionine (SAM) depletion and associated DNA hypomethylation of oncogenes ; 6) retinoic acid depletion and resultant hepatocyte proliferation via up-regulation of activator protein-1 (AP-1); 7) initiating an inflammatory cascade through increased transfer of endotoxin from intestine to liver, Kupffer cell activation via CD14/toll-like receptor-4 (TLR-4), oxidative stress, NF-kB or early growth response-1(Egr-1) activation, and generation of inflammatory cytokines and chemokines; 8) induction of liver fibrosis; and 9) decreasing the number and/or function of natural killer cells. Tobacco exposure is also a risk factor for HCC. It may contribute to the initiation and promotion of HCC due the presence of mutagenic and carcinogenic compounds as well as by causing oxidative stress due to generation of ROS and depletion of endogenous antioxidants. Simultaneous exposure to alcohol and tobacco is expected to promote the development of HCC in an additive and/or synergistic manner.

Rapid degradation of hypoxia-inducible factor-1alpha by KRH102053, a new activator of prolyl hydroxylase 2.

BACKGROUND AND PURPOSE: Hypoxia-inducible factor (HIF) is a transcription factor induced by hypoxia and degraded by ubiquitin-dependent proteasomes in normoxic conditions. Under hypoxic conditions, hydroxylation of HIF-1alpha subunit by prolyl hydroxylase (PHD) is suppressed, thus leading to increased levels of HIF. Although PHD2 plays a key role in regulating the levels of HIF, chemical activators of PHD2 are relatively unknown. The aim of this study was to identify small molecule activators of PHD2 that could be used, eventually, to suppress the level of HIF-1alpha. EXPERIMENTAL APPROACH: By using the overproduced PHD2 as a target, a molecular library consisting of more than 600 small molecules with a benzopyran structure was screened with an HPLC assay method. KEY RESULTS: We found a potent activator of PHD2, KRH102053 (2-amino-4-methylsulphanyl-butylic acid-4-methoxy-6-(4-methoxy-benzylamino)-2,2-dimethyl-chroman-3-yl ester). The effects of KRH102053 on controlling HIF were studied in human HOS osteosarcoma, rat PC12 phaeochromocytoma and human HepG2 hepatoma cells. Under our experimental conditions, KRH102053 decreased the protein level of HIF-1alpha and the mRNA levels of HIF-regulated downstream target genes, such as vascular endothelial growth factor, aldolase A, enolase 1 and monocarboxylate transporter 4. Consistent with these results, KRH102053 also inhibited the rates of HIF-related migration and invasion of HOS cells as well as the degree of tube formation in human umbilical vein endothelium cells. CONCLUSIONS AND IMPLICATIONS: These results suggest that KRH102053 and its structural analogues have the potential for use as therapeutic agents against various diseases associated with HIF.

Acetaminophen induces apoptosis of C6 glioma cells by activating the c-Jun NH(2)-terminal protein kinase-related cell death pathway.

Acetaminophen (AAP), a widely used analgesic drug, can damage various organs when taken in large doses. In this study, we investigate whether AAP causes cell damage by altering the early signaling pathways associated with cell death and survival. AAP caused time- and concentration-dependent apoptosis and DNA fragmentation of C6 glioma cells used as a model. AAP activated c-Jun N-terminal protein kinase (JNK) by 5.3-fold within 15 min. The elevated JNK activity persisted for up to 4 h before it returned to the basal level at 8 h. In contrast, activities of other mitogen-activated protein (MAP) kinases and the level of Akt phosphorylation in the cell survival pathway remained unchanged throughout the treatment. Wortmannin, an inhibitor of phosphatidylinositol-3 kinase, or SB203580, an inhibitor of p38 MAP kinase, did not reduce AAP-induced toxicity, indicating that these enzymes do not play a major role in cell toxicity. AAP-induced apoptosis was preceded by the sequential elevation of the pro-apoptotic Bax protein, cytochrome c release, and caspase-3 activity. Treatment with caspase inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK) significantly reduced AAP-induced caspase-3 activation and cytotoxicity. Transfection of cDNA for the dominant-negative mutant JNK-KR or stress-activated protein kinase kinase-1 Lys-->Arg mutant (SEK1-KR), an immediate upstream kinase of JNK, significantly reduced AAP-induced JNK activation and cell death rate. The noncytotoxic analog of AAP, 3-hydroxyacetanilide, neither increased JNK activity nor caused apoptosis. Pretreatment with YH439, an inhibitor of CYP2E1 gene transcription, markedly reduced CYP2E1 mRNA, protein content, and activity, as well as the rate of AAP-induced JNK activation and cell death. These data indicate that AAP can cause cell damage by activating the JNK-related cell death pathway, providing a new mechanism for AAP-induced cytotoxicity.

Apoptosis of PC12 cells by 4-hydroxy-2-nonenal is mediated through selective activation of the c-Jun N-terminal protein kinase pathway.

Cytotoxic lipid peroxides such as 4-hydroxy-2-nonenal (HNE) are produced when cells are exposed to toxic chemicals. However, the mechanism by which HNE induces cell death has been poorly understood. In this study, we investigated the molecular mechanism of HNE-induced apoptosis in PC12 cells by measuring the activities of the mitogen-activated protein (MAP) kinases involved in early signal transduction pathways. Within 15-30 min after HNE treatment, c-Jun N-terminal protein kinase (JNK) was maximally activated, before returning to control level after 1 h post-treatment. In contrast, activities of extracellular signal regulated kinase (ERK) and p38 MAP kinase remained unchanged from their basal levels. SEK1, an upstream kinase of JNK, was also activated (phosphorylated) within 5 min after HNE treatment and remained activated for up to 60 min. Marked activation of the JNK pathway through SEK1 was demonstrated by the transient transfection of cDNA for wild type SEK1 and JNK into COS-7 cells. Furthermore, significant reductions in JNK activation and HNE-induced cell death were observed when the dominant negative mutant of SEK1 was co-transfected with JNK. Pretreatment of PC12 cells with a survival promoting agent, 8-(4-chlorophenylthio)-cAMP, prevented both the HNE-induced JNK activation and apoptosis. Nonaldehyde, a nontoxic aldehyde, caused neither apoptosis nor JNK activation. Pretreatment of PC12 cells with SB203580, a specific inhibitor of p38 MAP kinase, had no effect on HNE-induced apoptosis. All these data suggest that the HNE-mediated apoptosis of PC12 cells is likely to be mediated through the selective activation of the SEK1-JNK pathway without activation of ERK or p38 MAP kinase.

Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase.

Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.

Selective activation of the c-Jun N-terminal protein kinase pathway during 4-hydroxynonenal-induced apoptosis of PC12 cells.

The by-product of lipid peroxidation, 4-hydroxynonenal (HNE), was shown to cause apoptosis in PC12 cells. In this study, we investigated the molecular mechanism of HNE-induced apoptosis in these cells. Specifically, we determined the effect of HNE on the activities of mitogen-activated protein (MAP) kinases involved in early signal transduction. Within 15 to 30 min after HNE treatment, c-Jun N-terminal protein kinase (JNK) was maximally activated, before it returned to control level at 1 h post-treatment. In contrast, activities of extracellular signal-regulated kinase and p38 MAP kinase remained unchanged from their baseline levels. Stress-activated protein kinase kinase (SEK1), an upstream kinase of JNK, was also activated within 5 min after HNE treatment and remained activated for up to 60 min. Marked activation of the JNK pathway through SEK1 and apoptosis signal-regulating kinase 1 (ASK1), an upstream kinase of SEK1, was demonstrated by the transient transfection of cDNA for wild-type SEK1 or ASK1 together with JNK into COS-7 cells. Furthermore, significant reductions in JNK activation and HNE-induced cell death were observed when either of the dominant negative mutant of SEK1 or ASK1 was cotransfected with JNK. Pretreatment of PC12 cells with a survival-promoting agent, 8-(4-chlorophenylthio)-cAMP, prevented both the HNE-induced JNK activation and apoptosis. Nonaldehyde, a nontoxic aldehyde, neither caused apoptosis nor JNK activation. Pretreatment of PC12 cells with SB203580, a specific inhibitor of p38 MAP kinase, had no effect on HNE-induced apoptosis. All these data suggest that the selective JNK activation by HNE is critical for the apoptosis of PC12 cells and that the HNE-mediated apoptosis is likely to be mediated through the activation of the ASK1-SEK1-JNK pathway without activation of extracellular signal-regulated kinase or p38 MAP kinase.

Corneal flap dimensions in laser in situ keratomileusis using the Innovatome automatic microkeratome.

To evaluate the thickness and size consistency of the corneal flap created with the Innovatome automatic microkeratome and to determine any correlation between preoperative variables and these corneal flap dimensions, we performed a prospective study comprising of 268 eyes of 143 patients having laser in situ keratomileusis. Either No. 170 or No. 190 blade was used, and preoperative variables including the central corneal thickness, keratometry (K) reading, spherical equivalent, and the blade type were measured. The mean central corneal flap thickness was 138.8 +/- 23.5 microns (range, 71.6-193.7 microns) in blade 170 group, and 148.3 +/- 25.4 microns (range, 80.3-211.7 microns) in blade 190 group. No relationship was found between the corneal flap thickness and the preoperative K reading or the spherical equivalent, but the corneal flap thickness increased with the preoperative central corneal thickness. The mean vertical flap diameter was 9.18 +/- 0.25 mm (range, 8.50-9.75 mm) in blade 170 group, and 9.50 +/- 0.31 mm (range, 8.75-10.00 mm) in blade 190 group. No relationship was found between the corneal flap diameter and the preoperative central corneal thickness or the spherical equivalent, but the corneal flap size increased with the preoperative K reading.

Truncated form of importin alpha identified in breast cancer cell inhibits nuclear import of p53.

Disruption of the function of tumor suppressor proteins occasionally can be dependent on their subcellular localization. In about 40% of the breast cancer tissues, p53 is found in the cytoplasm as opposed to the nucleus, where it resides in normal breast cells. This means that the regulation of subcellular location of p53 is an important mechanism in controlling its function. The transport factors required for the nuclear export of p53 and the mechanisms of their nuclear export have been extensively characterized. However, little is known about the mechanism of nuclear import of p53. p53 contains putative nuclear localization signals (NLSs) which would interact with a nuclear transport factor, importin alpha. In this report we demonstrate that importin alpha binds to NLSI in p53 and mediates the nuclear import of p53. Reverse transcriptase-polymerase chain reaction and sequencing analyses showed that a truncated importin alpha deleted the region encoding the putative NLS-binding domain of p53, suggesting that it could not bind to NLSs of p53 proteins. Binding of importin alpha to p53 was confirmed by using yeast two-hybrid assay. When expressed in CHO-K1 cells, the truncated importin alpha predominantly localized to the cytoplasm. In truncated importin alpha expressing cells, p53 preferentially localized to cytoplasmic sites as well. A significant increase in the p21(waf1/cip1) mRNA level and induction of apoptosis were also observed in importin alpha overexpressing cells. These results strongly suggest that importin alpha functions as a component of the NLS receptor for p53 and mediates nuclear import of p53.

Cytochrome P450 2E1 (CYP2E1)-dependent production of a 37-kDa acetaldehyde-protein adduct in the rat liver.

Ethanol-inducible cytochrome P450 2E1 (CYP2E1) has been shown to be involved in the metabolism of both ethanol and acetaldehyde. Acetaldehyde, produced from ethanol metabolism, is highly reactive and can form various protein adducts. In this study, we investigated the role of CYP2E1 in the production of a 37-kDa acetaldehyde-protein adduct. Rats were pairfed an isocaloric control or an alcohol liquid diet with and without cotreatment of YH439, an inhibitor of CYP2E1 gene transcription, for 4 weeks. The soluble proteins from rat livers of each group were separated on SDS-polyacrylamide gels followed by immunoblot analysis using specific antibodies against the 37-kDa protein acetaldehyde adduct. In addition, catalytic activities of the enzymes involved in alcohol and acetaldehyde metabolism were measured and compared with the adduct level. Immunoblot analysis revealed that the 37-kDa adduct, absent in the pair-fed control, was evident in alcohol-fed rats but markedly reduced by YH439 treatment. Immunohistochemical analysis also showed that the 37-kDa adduct is predominantly localized in the pericentral region of the liver where CYP2E1 protein is mainly expressed. This staining disappeared in the pericentral region after YH439 treatment. The levels of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase isozymes were unchanged after YH439 treatment. However, the level of the 37-kDa protein adduct positively correlated with the hepatic content of P4502E1. These data indicate that the 37-kDa adduct could be produced by CYP2E1-mediated ethanol metabolism in addition to the ADH-dependent formation.

Identification and functional characterization of a novel, tissue-specific NAD(+)-dependent isocitrate dehydrogenase beta subunit isoform.

To understand the interactions and functional role of each of the three mitochondrial NAD(+)-dependent isocitrate dehydrogenase (IDH) subunits (alpha, beta, and gamma), we have characterized human cDNAs encoding two beta isoforms (beta(1) and beta(2)) and the gamma subunit. Analysis of deduced amino acid sequences revealed that beta(1) and beta(2) encode 349 and 354 amino acids, respectively, and the two isoforms only differ in the most carboxyl 28 amino acids. The gamma cDNA encodes 354 amino acids and is almost identical to monkey IDHgamma. Northern analyses revealed that the smaller beta(2) transcript (1.3 kilobases) is primarily expressed in heart and skeletal muscle, whereas the larger beta(1) mRNA (1.6 kilobases) is prevalent in nonmuscle tissues. Sequence analysis of the IDHbeta gene indicates that the difference in the C-terminal 28 amino acids between beta(1) and beta(2) proteins results from alternative splicing of a single transcript. Among the various combinations of human IDH subunits co-expressed in bacteria, alphabetagamma, alphabeta, and alphagamma combinations exhibited significant amounts of IDH activity, whereas subunits produced alone and betagamma showed no detectable activity. These data suggest that the alpha is the catalytic subunit and that at least one of the other two subunits plays an essential supporting role for activity. Substitution of beta(1) with beta(2) in the co-expression system lowered the pH optimum for IDH activity from 8.0 to 7.6. This difference in optimal pH was analogous to what was observed in mouse kidney and brain (beta(1) prevalent; optimal pH 8.0) versus heart (beta(2) prevalent; pH 7.6) mitochondria. Experiments with a specially designed splicing reporter construct stably transfected into HT1080 cells indicate that acidic conditions favor a splicing pattern responsible for the muscle- and heart-specific beta(2) isoform. Taken together, these data indicate a regulatory role of IDHbeta isoforms in determining the pH optimum for IDH activity through the tissue-specific alternative splicing.

Noncompetitive, Ca(2+)-independent inhibition of pyruvate dehydrogenase phosphatase by fluphenazine.

The effects of two different classes of calmodulin antagonists on the catalytic activities of purified pyruvate dehydrogenase (PDH) phosphatase and PDH complex (PDC) were studied. In general, PDH phosphatase was more strongly inhibited than PDC by the calmodulin antagonists with the following potency order: fluphenazine > chlorpromazine > thioridazine > triflupromazine. Promazine and two sulfonamides (W-5 and W-7) did not suppress PDH phosphatase activity at 1 mM concentrations, while about 20% of PDC activity was inhibited by these antagonists. Fluphenazine-mediated inhibition of PDH phosphatase was observed with the purified PDC as well as intact mitochondria. Although Ca2+ stimulates PDH phosphatase activity, the addition of exogenous Ca2+ did not overcome the inhibition by calmodulin antagonists. These results suggest that the suppression of PDH phosphatase activity is dependent upon the structure of the individual calmodulin antagonist and appears to be Ca(2+)-independent. Kinetic analysis showed a noncompetitive inhibition of PDH phosphatase by fluphenazine, indicating that it binds to different site(s) from the catalytic site of the enzyme.

Pyruvate dehydrogenase E1 alpha isoform in rat testis: cDNA cloning, characterization, and biochemical comparison of the recombinant testis and liver enzymes.

Previous data indicated a tissue-specific regulation of mitochondrial pyruvate dehydrogenase (PDH) complex, especially in the brain and testis. The lack of biochemical data on the rat testis PDH limits comparative analysis between testis and liver enzymes. Therefore, we have isolated a cDNA clone encoding rat testis PDH E1 alpha isoform, determined its nucleotide sequence, studied the tissue-specific expression, and characterized the recombinant protein produced in bacteria, compared to the liver counterpart. Our cDNA clone (2.2 kb) contained the identical open reading frame (from nt 974 to 2149) with that previously reported (Cullingford et al., 1993 Biochim Biophys Acta 1216:149-153) but contained a long 5' untranslated region, which has little identity to the other clone. Northern blot confirmed testis-specific expression of this isoform. Genomic DNA analyses by PCR amplification suggested this clone is a gene product distinct from its X-linked somatic counterpart. Our biochemical and kinetic analyses revealed that the purified recombinant rat testis PDH E1 (containing both E1 alpha and E1 beta subunits) was enzymatically active and phosphorylated in vitro by purified PDH-kinase p48 or p45, similar to the recombinant human liver enzyme. Our current data thus indicate that the differential regulation of testis PDH observed in the animal model may result from differential modulation of PDH-kinase or -phosphatase in this tissue rather than the presence of functionally different PDH E1 subunit.

Use of 99mTc-mercaptoacetyltriglycine (MAG3)-biocytin hepatobiliary scintigraphy to study the protective effect of a synthetic enzyme inhibitor on acute hepatotoxicity in mice.

Recent data suggest that inhibitors of ethanol-inducible cytochrome P450 (CYP2E1) can protect the liver from injury caused by various substrates of CYP2E1. In this study, we measured the protective effect of isopropyl-2-(1,3-dithioetane-2-ylidene)-2[N-(4-methylthiazol -2-yl)-carbamoyl]acetate (YH439), a transcriptional inhibitor of CYP2E1, against carbon tetrachloride (CCl4)-induced hepatotoxicity by using various conventional methods and dynamic scintigraphy with 99mTc-mercaptoacetyltriglycine (MAG3)-biocytin, a recently developed scintigraphic agent. Balb/c mice were pretreated with two doses of YH439 (50 or 150 mg/kg per day) at 48 h and 24 h and one dose of CCl4 (0.25 mL/kg) at 18 h before scintigraphy. The results were compared with those of two other groups, one that received CCl4 but not YH439, and the other that received neither (control). Scintigraphic images were acquired continuously at 15-sec intervals for 30 min. Pharmacokinetic parameters, such as peak liver/heart ratio (r(max)), peak liver uptake time (t(max)), and hepatic half-clearance time (HCT), were obtained from time-activity curves derived from regions-of-interest (ROI) over the liver and the heart. Acute administration of CCl4 alone caused centrilobular necrosis and serum transaminase levels to rise more than 5 times higher than those of the control group. Pharmacokinetic parameters also changed significantly from those of the control group. Administration of YH439 prevented centrilobular necrosis and significantly improved pharmacokinetic parameters. This study demonstrates for the first time that hepatobiliary scintigraphy can be used to study in vivo biochemistry of the CYP2E1 inhibitor (YH439) against liver toxicity.

Critical role of arg433 in rat transketolase activity as probed by site-directed mutagenesis.

It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405-2410]. To identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102-->Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350-->Ala, Arg506-->Ala and Arg433-->Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350-->Ala and Arg433-->Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433-->Ala mutant protein was less stable than the wild-type and Arg350-->Ala proteins at 55 degrees C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433-->Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433-->Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity.

Molecular characterization of fetal alcohol syndrome using mRNA differential display.

The molecular pathogenesis of fetal alcohol syndrome (FAS) has not been well elucidated. The technique of mRNA differential display was used to characterize the etiology and to identify potential markers for FAS. Out of approximately 1,080 mRNA transcripts in mouse embryos that were analyzed, the levels of three mRNAs were altered by ethanol. Two of these mRNAs (one novel and one encoding heat shock protein 47) were also modulated by another teratogen, 3-methylcholanthrene. The third mRNA, encoding alpha-tropomyosin, was specifically up-regulated by ethanol. Consistent with the Northern blot data, immunoblot analysis demonstrated that the level of alpha-tropomyosin protein (31 kDa, most likely a brain specific isoform) was elevated in the embryos exposed to ethanol.