A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli.

BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

A novel mechanism underlying alcohol dehydrogenase expression: hsa-miR-148a-3p promotes ADH4 expression via an AGO1-dependent manner in control and ethanol-exposed hepatic cells.

The alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs) play critical roles in alcoholism development and alcohol toxicology; however, few studies have focused on the miRNA-mediated mechanisms underlying the expressions of alcohol-metabolizing enzymes. In the present study, we showed the expression changes of each alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the liver samples of alcoholic hepatitis (AH) patients, and predicted the miRNAs targeting the dysregulated alcohol-metabolizing genes by a systematic in silico analysis. 13 miRNAs were predicted to regulate the expressions of ADH1A, ADH4, and ALDH2, respectively, with hsa-miR-148a-3p (miR-148a) showing the most significant down-regulation in AH patients. Following experimental evidence using HepG2 cells proved that miR-148a promoted ADH4 expression by directly binding to the coding sequence of ADH4 and increasing the mRNA stability via an AGO1-dependent manner. Additional assays showed that secondary structure of ADH4 transcript affected the target accessibility and binding of miR-148a-3p. In sum, our results suggest that the expressions of key alcohol-metabolizing enzymes are repressed in AH patients, and the non-canonical positive regulation of miR-148a on ADH4 reveals a new regulationary mechanism for ADH genes.

Associações entre polimorfismos genéticos da álcool: desidrogenase e o transtorno por uso de álcool / Associations between genetic polymorphisms of alcohol: dehydrogenase and alcohol use disorder

O transtorno por uso de álcool (TUA) é influenciado pela genética, principalmente na metabolização do etanol. Os genes da álcool desidrogenase (ADH1B/ADH1C), enzima que transforma o etanol, apresentam SNPs (single nucleotide polymorphisms) que resultam em isoenzimas com diferentes taxas catalíticas. Estudos demonstraram que os SNPs Arg48His, Arg370Cys, Arg272Gln e Ile350Val contribuem para o TUA. Este artigo revisou os estudos que investigaram SNPs em ADH1B (Arg48His/Arg370Cys) e ADH1C (Arg272Gln/Ile350Val), bem como avaliou as variações nas frequências alélicas desses genes e a influência no TUA nas diferentes populações no mundo. As frequências alélicas dos polimorfismos foram comparadas pelos testes qui-quadrado de Pearson e exato de Fisher (p < 0,05). O SNP Arg48His confere proteção para o TUA em euroamericanos, latino-americanos, europeus, brasileiros, asiáticos e australianos. O SNP Arg370Cys confere proteção para o TUA em afrodescendentes. Os SNPs Arg272Gln e Ile350Val predispõem o TUA principalmente em europeus. Os SNPs Arg48His, Arg370Cys e Arg272Gln/Ile350Val foram mais frequentes em amostras de leste-asiáticos (69,7%), africanos (19,1%) e europeus (40,5%), respectivamente (p < 0,01). Os diferentes alelos dos genes ADH1B/ADH1C devido a SNPs têm uma importante contribuição no TUA. As frequências desses alelos variam conforme a população, resultando em diferentes efeitos no TUA. (AU)

Alcohol use disorder (AUD) is influenced by genetics, especially in the metabolism of ethanol. The ethanol dehydrogenase genes (ADH1B/ADH1C), which convert ethanol, have single nucleotide polymorphisms (SNPs) that result in isoenzymes with different catalytic rates. Studies have shown that the Arg48His, Arg370Cys, Arg272Gln, and Ile350Val SNPs contribute to AUD. This article reviewed the studies that investigated SNPs in ADH1B (Arg48His/Arg370Cys) and ADH1C (Arg272Gln/Ile350Val) and evaluated variations in the allele frequencies of these genes and their influence on AUD in different populations worldwide. The allele frequencies of the polymorphisms were compared by Pearson's chi-square and Fisher's exact tests (p < 0.05). The Arg48His SNP provides protection against AUD in Euro-Americans, Latin Americans, Europeans, Brazilians, Asians, and Australians. The Arg370Cys SNP provides protection against AUD in Afro-descendants. The Arg272Gln and Ile350Val SNPs predispose to AUD mainly in Europeans. The Arg48His, Arg370Cys, and Arg272Gln/Ile350Val SNPs were more frequent in East Asians (69.7%), Africans (19.1%), and Europeans (40.5%), respectively (p < 0.01). The different alleles of the ADH1B/ADH1C genes due to SNPs make an important contribution to AUD. The frequencies of these alleles vary among different populations, resulting in different effects on AUD..(AU)

Down-regulation of the expression of alcohol dehydrogenase 4 and CYP2E1 by the combination of α-endosulfan and dioxin in HepaRG human cells.

Pesticides and other persistent organic pollutants are considered as risk factors for liver diseases. We treated the human hepatic cell line HepaRG with both 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and the organochlorine pesticide, α-endosulfan, to evaluate their combined impact on the expression of hepatic genes involved in alcohol metabolism. We show that the combination of the two pollutants (25nM TCDD and 10µM α-endosulfan) led to marked decreases in the amounts of both the mRNA (up to 90%) and protein (up to 60%) of ADH4 and CYP2E1. Similar results were obtained following 24h or 8days of treatment with lower concentrations of these pollutants. Experiments with siRNA and AHR agonists and antagonist demonstrated that the genomic AHR/ARNT pathway is necessary for the dioxin effect. The PXR, CAR and estrogen receptor alpha transcription factors were not modulators of the effects of α-endosulfan, as assessed by siRNA transfection. In another human hepatic cell line, HepG2, TCDD decreased the expression of ADH4 and CYP2E1 mRNAs whereas α-endosulfan had no effect on these genes. Our results demonstrate that exposure to a mixture of pollutants may deregulate hepatic metabolism.

Editor's Highlight: Collaborative Cross Mouse Population Enables Refinements to Characterization of the Variability in Toxicokinetics of Trichloroethylene and Provides Genetic Evidence for the Role of PPAR Pathway in Its Oxidative Metabolism.

Background: Trichloroethylene (TCE) is a known carcinogen in humans and rodents. Previous studies of inter-strain variability in TCE metabolism were conducted in multi-strain panels of classical inbred mice with limited genetic diversity to identify gene-environment interactions associated with chemical exposure. Objectives: To evaluate inter-strain variability in TCE metabolism and identify genetic determinants that are associated with TCE metabolism and effects using Collaborative Cross (CC), a large panel of genetically diverse strains of mice. Methods: We administered a single oral dose of 0, 24, 80, 240, or 800 mg/kg of TCE to mice from 50 CC strains, and collected organs 24 h post-dosing. Levels of trichloroacetic acid (TCA), a major oxidative metabolite of TCE were measured in multiple tissues. Protein expression and activity levels of TCE-metabolizing enzymes were evaluated in the liver. Liver transcript levels of known genes perturbed by TCE exposure were also quantified. Genetic association mapping was performed on the acquired phenotypes. Results: TCA levels varied in a dose- and strain-dependent manner in liver, kidney, and serum. The variability in TCA levels among strains did not correlate with expression or activity of a number of enzymes known to be involved in TCE oxidation. Peroxisome proliferator-activated receptor alpha (PPARα)-responsive genes were found to be associated with strain-specific differences in TCE metabolism. Conclusions: This study shows that CC mouse population is a valuable tool to quantitatively evaluate inter-individual variability in chemical metabolism and to identify genes and pathways that may underpin population differences.

[Abnormal expression of genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer].

Retinoids are signaling molecules that control a wide variety of cellular processes and possess antitumor activity. This work presents a comprehensive description of changes in the expression of 23 genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer tumors compared to adjacent normal tissues obtained using RT-PCR. Even at early stages of malignant transformation, a significant decrease in ADH1B, ADH3, RDHL, and RALDH1 mRNA levels was observed in 82, 79, 73, and 64% of tumor specimens, respectively, and a considerable increase in AKR1B10 mRNA content was observed in 80% of tumors. Dramatic changes in the levels of these mRNAs can impair the synthesis of all-trans retinoic acid, a key natural regulatory retinoid. Apart from that, it was found that mRNA levels of nuclear retinoid receptor genes RXRγ, RARα, RXRα, and gene RDH11 were significantly decreased in 80, 67, 57, and 66% of tumor specimens, respectively. Thus, neoplastic transformation of lung tissue cells is accompanied with deregulated expression of key genes of retinoid metabolism and function.

Alcohol dehydrogenase and cytochrome P450 2E1 can be induced by long-term exposure to ethanol in cultured liver HEP-G2 cells.

It has been shown in previous studies that liver HEP-G2 cells (human hepatocellular carcinoma) lose their ability to express active alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Although both are ethanol-inducible enzymes, short-term exposure to ethanol does not cause any changes in expression or activity in cultured HEP-G2 cells. Therefore, we tested the effect of long-term exposure to ethanol on the expression and activity of both ADH and CYP2E1 in these cells. The expression of ADH and CYP2E1 was assessed at the mRNA and/or protein level using real-time PCR and Western blot analysis. Specific colorimetric assays were used for the measurement of ADH and CYP2E1 enzymatic activities. Caco-2 cells (active CYP2E1 and inactive ADH) were used as control cells. Significantly increased protein expression of ADH (about 2.5-fold) as well as CYP2E1 (about 1.6-fold) was found in HEP-G2 cells after long-term (12 mo) exposure to ethanol. The activity of ADH and CYP2E1 was also significantly increased from 12 ± 3 and 6 ± 1 nmol/h/mg of total protein to 191 ± 9 and 57 ± 9 nmol/h/mg of total protein, respectively. We suggest that the loss of activity of ethanol-metabolizing enzymes in cultured HEP-G2 cells is reversible and can be induced by prolonged exposure to ethanol. We are therefore able to reactivate HEP-G2 cells metabolic functions concerning ethanol oxidation just by modification of in vitro culture conditions without necessity of transfection with its side effect - enzyme overexpression.

Inhibition of CYP2E1 leads to decreased malondialdehyde-acetaldehyde adduct formation in VL-17A cells under chronic alcohol exposure.

AIM: Ethanol metabolism leads to the formation of acetaldehyde and malondialdehyde. Acetaldehyde and malondialdehyde can together form malondialdehyde-acetaldehyde (MAA) adducts. The role of alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1) in the formation of MAA-adducts in liver cells has been investigated. MAIN METHODS: Chronic ethanol treated VL-17A cells over-expressing ADH and CYP2E1 were pretreated with the specific CYP2E1 inhibitor - diallyl sulfide or ADH inhibitor - pyrazole or ADH and CYP2E1 inhibitor - 4-methyl pyrazole. Malondialdehyde, acetaldehyde or MAA-adduct formation was measured along with assays for viability, oxidative stress and apoptosis. KEY FINDINGS: Inhibition of CYP2E1 with 10 µM diallyl sulfide or ADH with 2mM pyrazole or ADH and CYP2E1 with 5mM 4-methyl pyrazole led to decreased oxidative stress and toxicity in chronic ethanol (100 mM) treated VL-17A cells. In vitro incubation of VL-17A cell lysates with acetaldehyde and malondialdehyde generated through ethanol led to increased acetaldehyde (AA)-, malondialdehyde (MDA)-, and MAA-adduct formation. Specific inhibition of CYP2E1 or ADH and the combined inhibition of ADH and CYP2E1 greatly decreased the formation of the protein aldehyde adducts. Specific inhibition of CYP2E1 led to the greatest decrease in oxidative stress, toxicity and protein aldehyde adduct formation, implicating that CYP2E1 accelerates the formation of protein aldehyde adducts which can be an important mechanism for alcohol mediated liver injury. SIGNIFICANCE: CYP2E1-mediated metabolism of ethanol leads to increased AA-, MDA-, and MAA-adduct formation in liver cells which may aggravate liver injury.

Ethanol impairs differentiation of human adipocyte stromal cells in culture.

BACKGROUND: Bioinformatic resources suggest that adipose tissue expresses mRNAs for alcohol dehydrogenases (ADHs) and ALDH2, and epidemiological studies indicate that heavy alcohol use reduces adipose tissue mass. We therefore characterized the expression of alcohol metabolizing enzymes in human, rat and mouse adipose tissue, preadipocytes, and adipocytes, the ability of adipocytes to metabolize ethanol, and the effects of ethanol on differentiation of human adipose stromal cells (hASCs). METHODS: Adipose tissue, preadipocytes, and adipocytes were collected from rodents or from humans undergoing bariatric surgery. hASCs were differentiated in vitro using standard methods. Gene expression and cellular differentiation were analyzed by Western blotting, RT-PCR, and microscopy. RESULTS: Class I ADH was expressed in human > mouse > rat adipose tissue, whereas ALDH2 was high in all samples. ADH, catalase, and ALDH2 were induced during differentiation of hASCs. The presence of 50 mM ethanol markedly reduced the differentiation of hASCs; this effect was associated with inhibition of expression of transcription factors required for differentiation, but did not depend on the ability of the cells to metabolize ethanol. CONCLUSIONS: Human adipose tissue expresses alcohol oxidizing enzymes. The presence of ethanol at physiologically relevant concentrations inhibits differentiation of hASCs. Ethanol could alter adipose tissue biology, inducing a form of acquired lipodystrophy, which is consistent with epidemiological studies.

ADH1 expression inversely correlates with CDR1 and CDR2 in Candida albicans from chronic oral candidosis in APECED (APS-I) patients.

Expression of the alcohol dehydrogenase gene ADH1, which converts ethanol into carcinogenic acetaldehyde, significantly inversely correlated with the expression of CDR1 and CDR2, genes linked to azole resistance in Candida albicans isolated from chronic oral candidosis in autoimmune polyendocrinopathy-candidosis-ectodermal dystrophy (APECED, APS-I) patients. This is a novel link between candidal two-carbon metabolism genes and azole resistance.

[Effect of colchicine and Triton X-100 on expression of the enzyme-encoding genes in nongerminating seeds of sugarbeet (Beta vulgaris L.)].

The expression of the enzyme-coding genes, controlling glucose-phosphate isomerase (GPI), malate dehydrogenase (MDH), and alcohol dehydrogenase (ADH), was examined in nongerminating seeds of sugarbeet after Triton X-100 (TX-100) and colchicine treatment. Two types of changes revealed included modification of the enzymatic loci expression (change of the isozyme electrophoretic mobility) and inactivation of standard profiles. In the MDH and GPI systems, these processes were found to be associated. Complete isozyme modification was accompanied with the disappearance of standard profiles. In the ADH system, the treatment with TX-100 and colchicine gave rise to two independent processes, including silencing of the Adh1 locus and the appearance of the ADH isozymes with abnormal electrophoretic mobility, which were probably the products of the Adh2 locus. It was suggested that the effect of TX-100 and colchicine on the expression of the enzyme-encoding genes examined depended on the intracellular localization of the encoded enzymes.

Synthesis of pyruvate: ferredoxin oxidoreductase and alcohol dehydrogenase E enzymes during Giardia intestinalis excystation.

INTRODUCTION: Giardia intestinalis is a unicellular parasite of worldwide distribution. It causes an intestinal illness known as giardiasis, and it is probably the earliest diverging eukaryotic microorganism. Previously, changes have been reported in the expression of mRNAs at several stages of the life cycle; however specific enzymatic activity changes have not been explored. OBJECTIVE: The expression of pyruvate ferredoxin oxidoreductase (PFOR) and alcohol dehydrogenase E (ADHE) enzymes was measured in cyst and trophozoite stages, and during the excystation process. MATERIALS AND METHODS: Recombinant proteins were generated for PFOR and ADHE to be used as antigens in the production of polyclonal antibodies for the detection of native proteins by Western Blot. The enzymatic activity of ADHE and glutamate dehydrogenase (GDH) was evaluated by spectrophotometric assays. RESULTS: PFOR (139 kDa) and ADHE (97 kDa) proteins were detected in trophozoites, but not in cysts. During excystation, ADHE protein was detected after the first phase of induction, but the PFOR protein appeared only after the second phase. This indicated that both proteins were synthesized during excystation, although at different times. ADHE enzymatic activity was present only in trophozoites and not in cysts whereas GDH activity was detected in both stages. CONCLUSION: These results conclusively showed that PFOR and ADHE enzymes were translated during the excystation process and is strong evidence that active protein synthesis was occurring during excystation.

The human lung cell line A549 does not develop adaptive protection against the DNA-damaging action of formaldehyde.

The alkaline comet assay was used to further characterize the induction of DNA-protein crosslinks (DPX) by formaldehyde (FA) and their removal in the human lung cell line A549. DPX were indirectly measured as the reduction of gamma ray-induced DNA migration. Repeated treatments of A549 cells with low FA concentrations (up to 100 microM) did not lead to significant differences in the induction of DPX in comparison with a single treatment. Pretreatment with higher FA-concentrations (200 microM and above) enhanced the crosslinking effect. There was no indication for an adaptive protection against the induction of DPX by FA. These findings are in agreement with RT-PCR measurements of the expression of genes that encode the main enzymes involved in FA detoxification. A549 cells exposed to FA (50-300 microM) for 1, 4, or 24 hr did not reveal altered expression of the GSH-dependent formaldehyde dehydrogenase (FDH, which is identical to alcohol dehydrogenase 3; ADH3), the cytosolic aldehyde dehydrogenase 1 (ALDH1A1) and the mitochondrial ALDH2. Pretreatment of A549 cells with a low FA concentration (50 microM) also did not enhance the removal of DPX induced by higher FA concentrations. Taken together, these results suggest that A549 cells do not develop adaptive protection against the genotoxic action of FA. Neither metabolic inactivation of FA nor the repair of FA-induced DPX seems to be enhanced in cells pretreated with FA.

Cardiac overexpression of alcohol dehydrogenase exacerbates chronic ethanol ingestion-induced myocardial dysfunction and hypertrophy: role of insulin signaling and ER stress.

Chronic alcohol intake leads to alcoholic cardiomyopathy characterized by cardiac hypertrophy and contractile dysfunction possibly related to the toxicity of the ethanol metabolite acetaldehyde. This study examined the impact of augmented acetaldehyde exposure on myocardial function, geometry, and insulin signaling via cardiac-specific overexpression of alcohol dehydrogenase (ADH). ADH transgenic and wild-type FVB mice were placed on a 4% alcohol diet for 12 weeks. Echocardiographic, glucose tolerance, glucose uptake, insulin signaling, and ER stress indices were evaluated. Mice consuming alcohol exhibited glucose intolerance, dampened cardiac glucose uptake, cardiac hypertrophy and contractile dysfunction, all of which with the exception of whole body glucose tolerance were exaggerated by the ADH transgene. Cardiomyocytes from ethanol-fed mice exhibited depressed insulin-stimulated phosphorylation insulin receptor (tyr1146) and IRS-1 (tyrosine) as well as enhanced serine phosphorylation of IRS-1. ADH-augmented alcohol-induced effect of IRS-1 phosphorylation (tyrosine/serine). Neither alcohol nor adh affected expression of insulin receptor and IRS-1. Alcohol reduced phosphorylation of Akt and GSK-3beta as well as GSK-3beta expression and the effect was exaggerated by ADH. The transcriptional factors GATA4, c-jun and c-jun phosphorylation were upregulated by alcohol, which was amplified by ADH. The ratios of phospho-c-Jun/c-Jun and phospho-GATA4/GATA4 remained unchanged. Chronic alcohol intake upregulated expression of the endoplasmic reticulum stress markers eIF2alpha, IRE-1alpha, GRP78 and gadd153, the effect of which was exaggerated by ADH. These data suggest that elevated cardiac acetaldehyde exposure via ADH may exacerbate alcohol-induced myocardial dysfunction, hypertrophy, insulin insensitivity and ER stress, indicating a key role of ADH gene in alcohol-induced cardiac dysfunction and insulin resistance.

Expression partitioning between genes duplicated by polyploidy under abiotic stress and during organ development.

Allopolyploidy has been a prominent mode of speciation and a recurrent process during plant evolution and has contributed greatly to the large number of duplicated genes in plant genomes [1-4]. Polyploidy often leads to changes in genome organization and gene expression [5-9]. The expression of genes that are duplicated by polyploidy (termed homeologs) can be partitioned between the duplicates so that one copy is expressed and functions only in some organs and the other copy is expressed only in other organs, indicative of subfunctionalization [10]. To determine how homeologous-gene expression patterns change during organ development and in response to abiotic stress conditions, we have examined expression of the alcohol dehydrogenase gene AdhA in allopolyploid cotton (Gossypium hirsutum). Expression ratios of the two homeologs vary considerably during the development of organs from seedlings and fruits. Abiotic stress treatments, including cold, dark, and water submersion, altered homeologous-gene expression. Most notably, only one copy is expressed in hypocotyls during a water-submersion treatment, and only the other copy is expressed during cold stress. These results imply that subfunctionalization of genes duplicated by polyploidy has occurred in response to abiotic stress conditions. Partitioning of duplicate gene expression in response to environmental stress may lead to duplicate gene retention during subsequent evolution.

Metabolic basis of ethanol-induced cytotoxicity in recombinant HepG2 cells: role of nonoxidative metabolism.

Chronic alcohol abuse, a major health problem, causes liver and pancreatic diseases and is known to impair hepatic alcohol dehydrogenase (ADH). Hepatic ADH-catalyzed oxidation of ethanol is a major pathway for the ethanol disposition in the body. Hepatic microsomal cytochrome P450 (CYP2E1), induced in chronic alcohol abuse, is also reported to oxidize ethanol. However, impaired hepatic ADH activity in a rat model is known to facilitate a nonoxidative metabolism resulting in formation of nonoxidative metabolites of ethanol such as fatty acid ethyl esters (FAEEs) via a nonoxidative pathway catalyzed by FAEE synthase. Therefore, the metabolic basis of ethanol-induced cytotoxicity was determined in HepG2 cells and recombinant HepG2 cells transfected with ADH (VA-13), CYP2E1 (E47) or ADH + CYP2E1 (VL-17A). Western blot analysis shows ADH deficiency in HepG2 and E47 cells, compared to ADH-overexpressed VA-13 and VL-17A cells. Attached HepG2 cells and the recombinant cells were incubated with ethanol, and nonoxidative metabolism of ethanol was determined by measuring the formation of FAEEs. Significantly higher levels of FAEEs were synthesized in HepG2 and E47 cells than in VA-13 and VL-17A cells at all concentrations of ethanol (100-800 mg%) incubated for 6 h (optimal time for the synthesis of FAEEs) in cell culture. These results suggest that ADH-catalyzed oxidative metabolism of ethanol is the major mechanism of its disposition, regardless of CYP2E1 overexpression. On the other hand, diminished ADH activity facilitates nonoxidative metabolism of ethanol to FAEEs as found in E47 cells, regardless of CYP2E1 overexpression. Therefore, CYP2E1-mediated oxidation of ethanol could be a minor mechanism of ethanol disposition. Further studies conducted only in HepG2 and VA-13 cells showed lower ethanol disposition and ATP concentration and higher accumulation of neutral lipids and cytotoxicity (apoptosis) in HepG2 cells than in VA-13 cells. The apoptosis observed in HepG2 vs. VA-13 cells incubated with ethanol appears to be mediated by release of mitochondrial cytochrome c via activation of caspase-9 and caspase-3. These results strongly support our hypothesis that diminished hepatic ADH activity facilitates nonoxidative metabolism of ethanol and the products of ethanol nonoxidative metabolism cause apoptosis in HepG2 cells via intrinsic pathway.

Recombinant Hep G2 cells that express alcohol dehydrogenase and cytochrome P450 2E1 as a model of ethanol-elicited cytotoxicity.

HepG2 cells were transfected with recombinant plasmids, one carrying the murine alcohol dehydrogenase (ADH) gene and the other containing the gene encoding human cytochrome P450 2E1 (CYP2E1). One of recombinant clones called VL-17A exhibited ADH and CYP2E1 specific activities comparable to those in isolated rat hepatocytes. VL-17A cells oxidized ethanol and generated acetaldehyde, the levels of which depended upon the initial ethanol concentration. Compared with unexposed VL-17A cells, ethanol exposure increased the cellular redox (lactate:pyruvate ratio) and caused cell toxicity, indicated by increased leakage of lactate dehydrogenase into the medium,. Exposure of VL-17A cells to 100mM ethanol significantly elevated caspase 3 activity, an indicator of apoptosis, but this ethanol concentration did not affect caspase 3 activity in parental HepG2 cells. Because ethanol consumption causes a decline in hepatic protein catabolism, we examined the influence of ethanol exposure on proteasome activity in HepG2, VL-17A, E-47 (CYP2E1(+)) and VA-13 (ADH(+)) cells. Exposure to 100mM ethanol caused a 25% decline in the chymotrypsin-like activity of the proteasome in VL-17A cells, but the enzyme was unaffected in the other cell types. This inhibitory effect on the proteasome was blocked when ethanol metabolism was blocked by 4-methyl pyrazole. We conclude that recombinant VL-17A cells, which express both ADH and CYP2E1 exhibit hepatocyte-like characteristics in response to ethanol. Furthermore, the metabolism of ethanol by these cells via ADH and CYP2E1 is sufficient to bring about an inhibition of proteasome activity that may lead to apoptotic cell death.

Depsipeptide-resistant KU812 cells show reversible P-glycoprotein expression, hyper-acetylated histones, and modulated gene expression profile.

Depsipeptide (FK228), a histone deacetylase inhibitor, is a promising new anticancer agent. The mechanism of resistance to this agent was studied using KU812 cells. Depsipeptide-resistant KU812 cells expressed P-glycoprotein (P-gp) and their resistance was abolished by co-treatment with verapamil. P-gp expression returned to the parental cell level when resistant cells were cultured in depsipeptide-free medium, while resistant cells cultured in the medium containing 16 nM depsipeptide still showed hyper-acetylation of histones. Moreover, resistant cells showed erythroid differentiation. Microarray analysis revealed that 28 genes showed increased expression and three genes showed decreased expression in resistant cells compared with parental cells. These 31 genes had various functions relating to signal transduction, cell cycle, apoptosis, and control of cell morphology and differentiation. Among the 28 genes that were upregulated, 15 genes also showed an increased expression in parental cells treated with 4 nM depsipeptide for 48 h, while the other 13 genes including P-gp were different. Among the three genes with decreased expression, HEP27 was most dramatically downregulated. These findings suggest that continuous exposure to depsipeptide reversibly induces P-gp, which contributes to the onset of resistance, but the altered gene expression profile of resistant cells may also play a role.

Genetic organization of the biosynthetic gene cluster for the antitumor angucycline oviedomycin in Streptomyces antibioticus ATCC 11891.

The oviedomycin biosynthetic gene cluster from Streptomyces antibioticus ATCC 11891 has been sequenced and characterized. It contains all the necessary genes for oviedomycin biosynthesis, together with several genes for the generation of malonyl-CoA extender units. Production of this unusual angucyclinone in its natural host occurs only in solid cultures in parallel with aerial mycelium and spore formation. A mutant that did not produce oviedomycin was generated by disruption of the beta-ketoacyl synthase gene ovmK. No other physiological process in the mutant appears to be affected; this rules out a direct relationship between oviedomycin production and cell differentiation in S. antibioticus.