Effects of phorbol myristate acetate,phorbol dibutyrate,ethanol, dimethylsulfoxide,phenol, and seven metabolities of phenol on metabolic cooperation between chinese hamster v79 lung fibroblasts

The effect of phorbol myristate acetate, phorbol dibutyrate, ethanol, dimethylsulfoxide, phenol, and seven metabolites of phenol on metabolic cooperation were assessed as a function of mutant cell recovery from populations of cocultivated hypoxanthine-guanine phosphoribosyl transferase-deficient mutant (HGPRT–) and wild-type (HGPRT+) Chinese hamster V79 lung fibroblasts. Phorbol myristate acetate and phorbol diputyrate, two established tumor promoters, were potent inhibitors of metabolic cooperation. Ethanol and dimethylsulfoxide, solvents commonly used to prepare chemicals for testing, weakly inhibited metabolic cooperation. Phenol and phenylglucuronide had no effect on metabolic cooperation. Four oxidative metabolites (1,4-benzoquinone, catechol, hydroxyquinol and quinol) inhibited metabolic cooperation. Phenylsulfate weakly inhibited metabolic cooperation. Conversely, 2-methoxyphenol, a methylated derivative of catechol, appeared to enhance metabolic cooperation. These results generallyAbbreviations CAS Chemical Abstracts Service - DMSO dimethylsulfoxide - ETOH ethanol - HGPRT hypoxanthine-guanine phosphoribosyl transferase - HGPRT+ HGPRT-competent - HGPRT– HGPRT-te]deficient - MC metabolic cooperation - MC+ metabolic cooperation-competent - MC– metabolic cooperation-deficient - MEM minimum essential medium - PDBu phorbol dibutyrate - PMA phorbol myristate acetate - 6TG 6-thioguanine - 6TG^r 6-thioguanine-resistant - 6TG^s 6-thioguanine-sensitive - V79/MC assay Chinese hamster V79 lung fibroblast assay for metabolic cooperation     

Ethanol-sensitive mutants of Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants unable to grow at ethanol concentrations at which the wild type strain S288C does grow, have been isolated. Some of them show additional phenotypic alterations in colony size, temperature sensitivity and viability in ethanol, which cosegregate with the growth sensitivity in ethanol. 21 selected monogenic ethanol-sensitive mutants define 20 complementation groups, denominated ETA1 to ETA20, which indicates that there is a high number of genes involved in the ethanol tolerance/sensitivity mechanism.Out of 21 selected monogenic mutants, 20 are not altered in the glycolytic pathway since, when maintained in glucosesupplemented medium, they can produce as much ethanol as the wild type and at about the same velocity. Nor do any of the mutants seem to be altered in the lipid biosynthetic pathway since, whether grown in the absence or in the presence of ethanol, their concentration of fatty acids and ergosterol is similar to that of the wild type under the same conditions. Therefore growth sensitivity to ethanol does not seem necessarily to be related to carbohydrate or lipid metabolism.Non-common abbreviations YP yeast extract peptone medium - YPD yeast extract peptone dextrose agar or medium - YPG yeast extract peptone glycerol agar - YPDE yeast extract peptone dextrose ethanol agar or medium - SD yeast nitrogen base dextrose agar - SPO yeast extract potassium acetate glucose agar - PD parental ditype - NPD non-parental ditype - TT tetratype     

Adaptive changes in lipid composition of rat liver plasma membrane during postnatal development following maternal ethanol ingestion

The fatty acid composition of constituent phospholipids and the cholesterol content of rat liver plasma membranes were determined subsequent to maternal alcohol ingestion during pregnancy and lactation. The alcoholic group was given a liquid Metrecal diet containing 37% ethanol-derived calories. The control group was pair-fed an isocaloric sucrose/Metrecal diet. Litters were killed for lipid analyses at days 5, 15 and 25 after birth. These studies revealed that the total phospholipid phosphorus was similar and increased significantly with age in both groups. Cholesterol also increased significantly with age in both groups but was greater in the alcoholic pups, resulting in a higher cholesterol/phospholipid molar ratio. While the phosphatidylethanolamine (PE) content increased with age in both groups, that of sphingomyelin decreased. Phosphatidylserine + phosphatidylinositol (PS + PI) was significantly higher in the control group at all ages studied. A consistent increase of C22:6 in phosphatidylcholine (PC), sphingomyelin, PS + PI and in the total phospholipid fraction from alcoholic pups was observed. Although other fatty acid changes were found in PC, PS + PI and sphingomyelin, PE was not affected. These results suggest that specific adaptive changes were induced in the liver plasma membrane lipids of the progeny from alcoholic rats.     

Acute and Chronic Effects of Ethanol on Transbilayer Membrane Domains

Alcohols, including ethanol, have a specific effect on transbilayer and lateral membrane domains. Recent evidence has shown that alcohols in vitro have a greater effect on fluidity of one leaflet as compared to the other. The present study examined effects of chronic ethanol consumption on fluidity of synaptic plasma membrane (SPM) exofacial and cytofacial leaflets using trinitrobenzenesulfonic acid (TNBS) labeling and differential polarized fluorometry of 1,6-diphenyl-1,3,5-hexatriene (DPH). Mice were administered ethanol or a control liquid diet for 3 weeks. Animals were killed and SPM prepared. The exofacial leaflet of SPM was significantly more fluid than the cytofacial leaflet in both groups, as indicated by limiting anisotropy of DPH. However, differences between the two leaflets were much smaller in the ethanol-treated group. Ethanol at concentrations seen clinically had a greater effect in vitro on the more fluid exofacial leaflet. This asymmetric effect of ethanol was significantly diminished in the exofacial leaflet of the ethanol-treated mice. Chronic ethanol consumption has a specific effect on membranes. Membrane functions that may be regulated by asymmetry of fluidity and lipid distribution may be altered by chronic ethanol consumption.     

Solvent Effects in the Spin Trapping Of Lipid Oxyl Radicals

Studies documenting spin trapping of lipid radicals in defined model systems have shown some surprising solvent effects with the spin trap DMPO. In aqueous reactions comparing the reduction of H₂O₂ and methyl linoleate hydroperoxide (MLOOH) by Fe^z+, hydroxyl (HO·) and lipid alkoxyl (LO·) radicals produce identical four-line spectra with line intensities 1:2:2:1. Both types of radicals react with commonly-used HO· scavengers, e.g. with ethanol to produce ·C(CH₃)HOH and with dirnethylsulfoxide (DMSO)togive ·CH₃. However, DMSO radicals (either ·CH₃or ·OOCH₃) react further with lipids, and when radicals are trapped in these MLOOH systems, multiple adducts are evident. When acetonitrile is added to the aqueous reaction systems in increasing concentrations, ·CH₂CN radicals resulting from HO· attack on acetonitrile are evident, even with trace quantities of that solvent. In contrast, little, if any, reaction of LO· with acetonitrile occurs, even in 100% acetonitrile. A single four-line signal persists in the lipid systems as long as any water is present, although the relative intensity of the two center lines decreases as solvent-induced changes gradually dissociate the nitrogen and β-hydrogen splitting constants. Extraction of the aqueous-phase adducts into ethyl acetate shows clearly that the identical four-line spectra in the H₂0₂ and MLOOH systems arise from different radical species in this study, but the lack of stability of the adducts to phase transfer may limit the use of this technique for routine adduct identification in more complex systems. These results indicate that the four-line 1:2:2:1. a_N = a_H = 14.9G spectrum from DMPO cannot automatically be assigned to the HO· adduct in reaction systems where lipid is present, even when the expected spin adducts from ethanol or DMSO appear confirmatory for HO-. Conclusive distinction between HO· and LO· ultimately will require use of ¹³C-labelled DMPO or HPLC-MS separation and specific identification of adducts when DMPO is used as the spin trap.     

Erk1/2 signaling mediates the HGF-induced protection against ethanol and acetaldehyde-induced toxicity in the pancreatic RINm5F cell line

Alcohol-induced pancreas damage remains as one of the main risk factors for pancreatitis development. This disorder is poorly understood, particularly the effect of acetaldehyde, the primary alcohol metabolite, in the endocrine pancreas. Hepatocyte growth factor (HGF) is a protective protein in many tissues, displaying antioxidant, antiapoptotic, and proliferative responses. In the present work, we were focused on characterizing the response induced by HGF and its protective mechanism in the RINm5F pancreatic cell line treated with ethanol and acetaldehyde. RINm5F cells were treated with ethanol or acetaldehyde for 12 h in the presence or not of HGF (50 ng/ml). Cells under HGF treatment decreased the content of reactive oxygen species and lipid peroxidation induced by both toxics, improving cell viability. This effect was correlated to an improvement in insulin expression impaired by ethanol and acetaldehyde. Using a specific inhibitor of Erk1/2 abrogated the effects elicited by the growth factor. In conclusion, the work provides mechanistic evidence of the HGF-induced-protective response to the alcohol-induced damage in the main cellular component of the endocrine pancreas.     

[125I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrhenius analysis

Binding of [¹²⁵I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca²⁺-dependent membrane binding of [¹²⁵I]calmodulin was found to have a B_max of 284 pmol/mg protein and an apparent affinity with a K_d of 131 nM. Kinetic analysis indicates that at 37°C, the dissociation of [¹²⁵I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7×10^–1 min^–1 and a half-time (t_1/2) of 1.8 min for the fast component, and a k of 4.8×10^–2 min^–1 and a t_1/2 of 14.5 min for the slow component. At 0°C, substantial dissociation still occurred, with a k of 4.5×10^–2 min^–1 and a t_1/2 of 15.3 min for the fast component, and a k of 5.5×10^–3 min^–1 and a t_1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associated and dissociate calmodulin. In Arrhenius analysis of temperature effects, [¹²⁵I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (T_d) estimated to be 23.8°C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [¹²⁵I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10^–7–10^–6 M), a steroid hormone, and decreased by ethanol (50–200 mM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.Abbreviations used CaM calmodulin - SPM synaptic plasma membrane - ATPase adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - SDS sodium dodecyl sulfate - TFP trifluoperazine - K_d dissociation constant - B_max maximum binding - k first-order rate constant - t_1/2 half-time - T_d transition temperature     

The 59-kDa polypeptide constituent of 8–10-nm cytoplasmic filaments in Neurospora crassa is a pyruvate decarboxylase

The fungus Neurospora crassa harbors large amounts of cytoplasmic filaments which are homopolymers of a 59-kDa polypeptide (P59Nc). We have used molecular cloning, sequencing and enzyme activity measurement strategies to demonstrate that these filaments are made of pyruvate decarboxylase (PDC, EC 4.1.1.1), which is the key enzyme in the glycolytic-fermentative pathway of ethanol production in fungi, and in certain plants and bacteria. Immunofluorescence analyses of 8–10-nm filaments, as well as quantitative Northern blot studies of P59Nc mRNA and measurements of PDC activity, showed that the presence and abundance of PDC filaments depends on the metabolic growth conditions of the cells. These findings may be of relevance to the biology of ethanol production by fungi, and may shed light on the nature and variable presence of filament bundles described in fungal cells.     

Flow injection assays for NADH and ethanol using photosensitized rose bengal and luminol–copper (II) chemiluminescence system

The online photoreaction of the rose bengal photosensitized luminol–copper (II) chemiluminescence (CL) system was used for the determination of β-nicotinamide adenine dinucleotide (NADH) and ethanol (EtOH) in pharmaceutical formulations combined with a flow injection technique. NADH can significantly enhance the CL emission of the reaction. For EtOH, alcohol dehydrogenase in soluble form was utilized in the presence of nicotinamide adenine dinucleotide resulting in NADH production. The limit of detection (3σ blank, 𝑛 = 3) of 4.0 × 10⁻⁸ and 2.17 × 10⁻⁵ M, and linear range 1.3 × 10⁻⁷ to 2.5 × 10⁻⁵ M (R² = 0.9998, n = 6) and 0.11–2.17 × 10⁻³ M (R² = 0.9996, n = 6) were obtained for NADH and EtOH respectively. The injection rate was 100 h⁻¹ with a relative standard deviation (n = 3) of 1.5–4.8% in the range studied for both analytes. The procedure was satisfactorily applied to pharmaceutical formulations with recoveries in the range 91.6 ± 3.0% to 110 ± 2.0% for NADH and 88 ± 3.0% to 95.4 ± 4.0% for EtOH. The results obtained were very consistent and did not differ considerably from the reported approaches at a 95% confidence limit. The possible mechanism of the CL reaction is also explained briefly.     

Soy-based medium for ethanol production byEscherichia coli KO11

An optimized soy-based medium was developed for ethanol production byEscherichia coli KO11. The medium consists of mineral salts, vitamins, crude enzymatic hydrolysate of soy and fermentable sugar. Ethanol produced after 24 h was used as an endpoint in bioassays to optimize hydrolysate preparation. Although longer fermentation times were required with soy medium than with LB medium, similar final ethanol concentrations were achieved (44–45 g ethanol L^–1 from 100 g glucose L^–1). The cost of materials for soy medium (excluding sugar) was estimated to be $0.003 L^–1 broth, $0.006 L^–1 ethanol.