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.

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.

Monoclonal antibody-directed radioimmunoassay detects cytochrome P-450 in human placenta and lymphocytes.

A multiplicity of cytochromes P-450 is responsible for the detoxification and activation of xenobiotics such as drugs and carcinogens. Individual differences in sensitivity to these agents may reside in the cytochrome P-450 phenotype. A monoclonal antibody-directed radioimmunoassay was developed that detects epitope-specific cytochromes P-450 in human placentas and peripheral lymphocytes. Placentas from women who smoked cigarettes contained greater amounts of cytochrome P-450 with the monoclonal antibody-specific epitope than placentas from nonsmokers. The amount of this cytochrome P-450 in human peripheral lymphocytes increased after treatment of the mitogenized lymphocytes with the cytochrome P-450 inducer benz[a]anthracene.

Short chain diol metabolism in human disease states.

Recent clinical studies have shown the presence of two short chain diols, meso-2,3-butanediol and D/L-2,3-butanediol, and in most cases 1,2-propanediol in either serum or urine collected from humans in several apparently unrelated disease states: congenital propionic and methylmalonic acidemia, premature infants, and alcoholics both in the presence and absence of ethanol. In addition 1,2-propanediol has been shown in patients during prolonged starvation, and in patients with diabetic keto-acidosis. No common defect is known to exist in these metabolic states. Understanding how these compounds are produced in clinically well-defined diseases such as methyl malonic and propionic aciduria, however, may help explain how and why these compounds are produced in alcoholics.

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.

Pregnenolone 16 alpha-carbonitrile-inducible P-450 gene family: gene conversion and differential regulation.

A cytochrome P-450 cDNA clone, designated pP450PCN2, homologous to the previously characterized pregnenolone 16 alpha-carbonitrile (PCN)-induced P-450 cDNA (pP450PCN1; F. J. Gonzalez, D. W. Nebert, J. P. Hardwick, and C. B. Kasper, J. Biol. Chem. 260:7435-7441), was isolated from a rat liver cDNA expression library by use of a polyclonal anti-P450PCN1 antibody. This P-450 cDNA contains 2,014 base pairs and yields an open reading frame of a protein consisting of 504 amino acids (Mr = 57,760). P450PCN2 cDNA and protein shared 90% nucleotide and 89% amino acid similarity with P450PCN1 cDNA and protein, respectively. The 5' untranslated, coding, and 3' untranslated regions between the two cDNAs share 94, 93, and 79% similarities, respectively. Nucleotide differences in the coding regions, however, are not evenly distributed. Complete homology exists between the two mRNAs for 425 nucleotides (positions 346 through 771). Other regions of 93 nucleotides containing only one difference and 147 nucleotides containing two differences exist toward the 3' end of the coding regions. These data suggest the possibility that a gene conversion event(s) have occurred subsequent to duplication of the ancestral P450PCN gene. Oligonucleotide probes unique for P450PCN1 and P450PCN2 cDNAs were used to examine the levels of their respective mRNAs in noninduced and PCN-induced liver cells and in male and female rats of various ages. P450PCN1 mRNA was not detectable in either male or female rats at any ages. In contrast, P450PCN2 mRNA was present at a low level in newborn rats and became elevated in both males and females at 1 week of age. Levels of p450PCN2 mRNA continued to increase in males until 12 weeks, whereas the mRNA in females reached peak levels at 2 weeks of age but declined continuously at the onset of puberty (between 4 and 12 weeks). These levels of P45PCN2 mRNA closely parallel the increases in testosterone 6 beta-hydroxylase activity and P450PCN2 protein level, as analyzed by Western blots. P450PCN1 mRNA was induced by PCN, dexamethasone, and phenobarbital in both male and female rats. P450PCN2 mRNA was not significantly induced by PCN or dexamethasone but was readily induced by phenobarbital. Testosterone 6 beta-hydroxylase activity was also induced severalfold by PCN, dexamethasone, and phenobarbital. These data demonstrate that P450PCN1 and P450PCN2 genes are differentially regulated during development and after administration of inducing compounds and furthermore suggest that both enzymes possess testosterone 6 beta-hydroxylase activity.

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.

Monoclonal antibodies to ethanol-induced rat liver cytochrome P-450 that metabolizes aniline and nitrosamines.

Hybridomas were prepared from mouse myeloma cells and spleen cells derived from female BALB/c mice that had been immunized with a partially purified ethanol-induced rat liver cytochrome P-450 (P-450et). Monoclonal antibodies (MAbs) produced by the hybridomas were screened for binding to P-450et with a radioimmunoassay. Thirty-one independent hybrid clones produced MAbs that had a high affinity for P-450et. Each clone produced MAbs of a single subclass of the mouse immunoglobulins IgG1, IgG2a, IgM, or IgA. Ten of the 31 MAbs also immunoprecipitated P-450et as determined by Ouchterlony double-immunodiffusion analyses. One of the MAbs was tested for cross-reactivity with other rabbit and rat liver cytochromes P-450 and was found not to cross-react with rat liver P-450 induced by either phenobarbital, beta-naphthoflavone, or rabbit liver P-450LM2 or P-450LM4. Nine of the MAbs were tested for cross-reactivity with rat liver clofibrate-induced P-450, rat liver pregnenolone-16-alpha-carbonitrile-induced P-450, and a human liver P-450. All the MAbs showed no cross-reactivity except for one MAb which cross-reacted with both pregnenolone-16-alpha-carbonitrile and human P-450 and three MAbs which cross-reacted with human P-450. Three antigen-precipitating MAbs and four nonprecipitating MAbs were tested for their effects on the aniline p-hydroxylase activity of liver microsomes of untreated rats and from rats treated with acetone, pyrazole, methylpyrazole, or imidazole. One of the seven MAbs tested, 1-91-3, inhibited enzyme activity of acetone-, pyrazole-, or methylpyrazole-induced microsomes by 54, 47, and 48%, respectively. This indicates that at least 50% of microsomal cytochrome P-450 aniline p-hydroxylase activity in the latter is a function of a P-450 enzyme that contained the epitope to which the MAb 1-91-3 is directed. With untreated and imidazole-induced microsomes, 32 and 21% inhibition of the enzyme activity was observed. In reconstituted systems containing phospholipid and NADPH-cytochrome P-450 reductase, MAb 1-91-3 inhibited aniline p-hydroxylase activity of purified ethanol-induced P-450et and acetone-induced P-450 by more than 90%. Nitrosodimethylamine demethylase activity of acetone-induced rat microsomes was inhibited by the various MAbs up to 77% and the activity of the purified acetone-induced P-450 was inhibited up to 92%.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Isolation and characterization of murine vav2, a member of the vav family of proto-oncogenes.

We describe the isolation and characterization of a cDNA encoding murine vav2. vav2 shares 63% and 55% identity at the nucleic acid and amino acid levels, respectively, with vav, a proto-oncogene that plays an essential role in embryonic development and hematopoietic signal transduction. The 100 kDa Vav2 protein contains the characteristic array of structural motifs found in Vav. However, unlike vav, vav2 transcripts are widely distributed in both hematopoietic and non-hematopoietic tissues. In the adult, vav2 mRNA is found at high levels in the spleen, liver, testes and placenta. Northern blot analysis reveals two vav2 mRNA species (designated alpha and beta). The alpha species is expressed throughout development while the alpha and beta species are expressed tissue-specifically in adults. Transfection of NIH3T3 cells with expression vectors containing vav2 deletions demonstrate that elimination of 183 amino terminal residues of Vav2 is sufficient to activate its oncogenic potential. Vav2-induced transformation is characterized by the appearance of foci composed of cells in which cytokinesis and karyokinesis are uncoupled. This phenotype is comparable, but not identical, to morphological changes induced by Vav and other members of the DbI family of oncoproteins. Our results suggest that Vav family members mediate functions important in the regulation of cell architecture and proliferation in most, if not all, tissues.

Stabilization of cytochrome P450j messenger ribonucleic acid in the diabetic rat.

Cytochrome P450j, an enzyme involved in nitrosamine metabolism, is expressed in hepatic, pulmonary, and renal tissues and its level is elevated in ethanol- and acetone-treated rats as well as in diabetic rats induced by either streptozotocin or alloxan. Although P450j protein is substantially elevated by all inducing regimens, only in diabetic rats is P450j mRNA increased 10-fold. Nuclear run-on transcription analysis showed that this mRNA increase is not due to transcriptional activation but is due to specific stabilization of the P450j mRNA.

Cytochrome P450IIE1 is elevated in lymphocytes from poorly controlled insulin-dependent diabetics.

Cytochrome P450IIE1, a member of the cytochrome P450 supergene family, was measured in peripheral lymphocytes of 14 patients with insulin-dependent diabetes mellitus who were in poor metabolic control, as evidenced by elevated hemoglobin A1 levels (mean, 11.9 +/- 2.8%; normal, less than 7.8). Only one major form (mol wt, 48,000 daltons) of cytochrome P450IIE1 was detected with a specific polyclonal antibody against P450IIE1. Levels of cytochrome P450IIE1 were very low to undetectable in human lymphocytes from seven normal subjects. However, levels of P450IIE1 were elevated in lymphocytes from patients with insulin-dependent diabetes mellitus. Elevated levels of cytochrome P450IIE1 determined by immunoblot analysis correlate positively with the levels of hemoglobin A1 (r = 0.8), a metabolic indicator in diabetic subjects. In one study subject in whom diabetic control was improved, the drop in hemoglobin A1C levels was accompanied by normalization of P450IIE1 levels.

Rapid changes in cytochrome P4502E1 (CYP2E1) activity and other P450 isozymes following ethanol withdrawal in rats.

This study describes the effects of chronic ethanol (ETOH) treatment and withdrawal on the rat hepatic mixed-function mono-oxygenase system. Male Sprague-Dawley rats (150-200 g, 10 per group) were administered ETOH as part of the Lieber-deCarli liquid diet for 3 weeks. Ethanol was removed, and the animals were euthanized at 0, 24, 48, 72 and 168 hr post-withdrawal. Microsomes were prepared, and ethanol-inducible cytochrome P4502E1 (CYP2E1) activity was measured using the enzyme markers N-nitrosodimethylamine demethylase (NDMAd), p-nitrophenol hydroxylase (PNPH) and aniline hydroxylase (AH). Activities were found to be induced significantly after chronic ETOH feeding using all three assays (NDMAd, 5-fold; PNPH, 3.5-fold; AH, 9-fold). Upon ETOH withdrawal, all three activities dropped markedly, with NDMAd and PNPH at control values at 24 hr and all subsequent time points. AH activity remained 3-fold higher than controls at 24, 48 and 72 hr. Western blot analyses showed that immunoreactive CYP2E1 returned to control at 24 hr, consonant with NDMAd and PNPH activities. The prolonged induction of AH activity following ETOH withdrawal indicates that it is not a specific marker of CYP2E1-catalyzed reactions. Collectively, these data are suggestive of a rapid mechanism of CYP2E1 degradation in the rat liver. Of the other parameters investigated in this study, total cytochrome P450 content was increased 2.5-fold after ETOH feeding, with levels dropping markedly 24 hr post-withdrawal. NADPH-dependent cytochrome c reductase activity was unchanged throughout the course of the study. CYP1A1, CYP2B1 and CYP3A activities were assessed by the substrate probes ethoxyresorufin O-dealkylase (EROD), pentoxyresorufin O-dealkylase (PROD) and erythromycin N-demethylase (ERNd). EROD and PROD were induced significantly by ETOH administration (2-fold) at 0 hr, with EROD remaining elevated over controls 24 hr post-withdrawal. Quantitative western blot analysis of CYP1A1 and CYP2B1 revealed a pattern of immunostaining generally consistent with but less variable than levels predicted by the respective substrate markers. Both proteins were induced significantly by chronic ethanol administration (CYP1A1, 1.9-fold; CYP2B1, 4-fold). Induction of these P450 isoforms persisted for several days following withdrawal. In contrast, immunoreactive CYP1A2 was found to decrease significantly (by 30-40%) during ethanol withdrawal (24, 48, 72, 168 hr). ERNd activity was induced significantly by chronic ETOH feeding (2.5-fold) and remained so for 24 hr into the withdrawal period (2-fold). Immunoreactive CYP3A1 was also induced significantly following ETOH administration (0 hr) and 24 hr following withdrawal.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of brain cytochrome P-450IIE1 by chronic ethanol treatment.

Cytochrome P-450 mediated metabolism is potentially involved in the expression of the pharmacological and/or toxicological effects of a wide variety of drugs and environmental chemicals upon tissues which contain this metabolic system. In the present investigation, the presence of cytochrome P-450IIE1 and associated mono-oxygenase activities in brain and the effect of chronic ethanol treatment on brain cytochrome P-450 (P-450) were studied. Aniline hydroxylase, N-nitroso-dimethylamine N-demethylase and p-nitrophenol hydroxylase activities (known to be mediated by P-450IIE1) were detectable in brain microsomes from untreated rats and were about 5%, 125% and 8.3%, respectively, of the corresponding hepatic levels. Chronic ethanol treatment resulted in induction of the above enzyme activities in brain microsomes by 243%, 496% and 155%, respectively. Intake of ethanol for a prolonged period also resulted in the induction of total P-450 in the brain (150% of the control). Addition of the antisera raised against rat liver cytochrome P-450IIE1 markedly inhibited brain microsomal p-nitrophenol hydroxylase activity. Immunoblot analysis of rat brain microsomes using the above antisera also revealed the induction of brain cytochrome P-450IIE1 following chronic ethanol administration. Immunocytochemical localization of cytochrome P-450IIE1 using the above antisera, revealed the preferential localization of the enzyme in the neuronal cell bodies in the cortex, hippocampus, basal ganglia, hypothalamic nuclei and reticular nuclei in the brainstem of rats treated chronically with ethanol. Based upon these studies, it is conceivable that chronic alcohol ingestion could enhance the sensitivity of certain regions of the brain to environmental chemicals that are metabolized to more toxic derivatives by the P-450 system.

Translational activation of ethanol-inducible cytochrome P450 (CYP2E1) by isoniazid.

The molecular mechanism of ethanol-inducible cytochrome P450(CYP2E1) induction by isoniazid was studied and compared to that of pyridine, an inducer of CYP2E1. Aniline hydroxylase and immunoreactive CYP2E1 protein were significantly induced by isoniazid without or with only slight activation of other cytochromes P450. In contrast, pyridine increased the activities of a broad range of P450s. The effects of two structural analogs of isoniazid, isonicotinamide and isonicotinic acid were also tested and found to have a markedly decreased ability to induce CYP2E1. The induction of CYP2E1 by isoniazid was not accompanied by an increased level of CYP2E1 mRNA, and was completely blocked by pretreatment with cycloheximide or sodium fluoride, inhibitors of mRNA translation. These data thus suggest that CYP2E1 induction by isoniazid is due to activation of CYP2E1 mRNA translation and that the hydrazide group on the pyridine ring of isoniazid is important both in the selective induction of CYP2E1 and for magnitude of effect.

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.

Vascular reactivity in alcoholic rat aortas: in vitro interactions between catecholamines and alcohol.

To understand the nature of vascular problems of hypertensive and alcoholic subjects, an in vitro interaction between catecholamine and alcohol was investigated in male Sprague-Dawley rats. The descending aortas were isolated from either the control or alcohol treated rats. The aortic strips were cooled rapidly in ice-chilled Krebs-Henseleit (K-H) solution and the aorta was cut into a series of rings of approximately 1 mm width. The rings were fixed under 1 g of resting tension between a force-displacement transducer and anchoring electrodes at 37 degrees C. The rings were equilibrated for an hour with frequent changes of the K-H solution before testing. There was norepinephrine (NE) dose-dependent contraction of the rings, which showed the maximum tension with 1 microM NE. Acetylcholine or carbachol produced slow relaxation. Pretreatment of the rings with 10 microM prazocin prevent the contraction induced by 1 microM NE. Even in the presence of prazocin, 60 mM KCI was able to generate the maximum tension. NE-induced contraction was analyzed in the control K-H solution or in the presence of 1.5, 3.0 and 5.0% of ethanol. Ethanol (1.5%) slowed the rate of rise of the aortic tension without a remarkable compromise of the maximum tension. But, there was a significant reduction in contraction with 3% ethanol. A complete inhibition of contraction was noted with 5% ethanol. However, the effect of ethanol was fully reversible upon washings the aortic rings with K-H solution. Aortic rings prepared from the rats that were fed with alcohol for 30 days were not able to generate the maximum tension with 1 microM NE.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.