• Energy Research
• basic medicine close
• US close
• DE close
• ES close
• EU close

Mouse hepatocytes respond to osmotic stress with adaptive changes in transmembrane potential, Vm, such that hypotonic stress hyperpolarizes cells and hypertonic stress depolarizes them. These changes in Vm provide electromotive force for redistribution of ions such as CI−, and this comprises part of the mechanism of hepatocyte volume regulation. We conducted the present study to determine whether ethanol administered in vitro to mouse liver slices increases hepatocyte water volume, and whether this swelling triggers adaptive changes in the Vm. Cells in mouse liver slices were loaded with tetramethylammonium ion (TMA). Changes in hepatocyte water volume were computed from measurements with Ion sensitive micro‐electrodes of changes in intracellular activity of TMA (a1TMA) that resulted from water fluxes. Ethanol (70 mM) increased hepatocyte water volume Immediately, and this peaked at 17% by 7 to 8 min, by which time a plateau was reached. Liver slices also were obtained from mice treated 12 hr prior with 4‐methylpyrazole (4 mM). The effect of ethanol on their hepatocyte water volume was identical to that from untreated mice, except that the onset and peak were delayed 2 min. Hepatocyte Vm showed no differences between control or ethanol‐treated cells during the course of volume changes. In contrast, hyposmotic stress, created by dropping external osmolality 50 mosm, increased Vm from –30 mV to –46 mV. Ethanol did not inhibit this osmotic stress‐induced hyperpolarization, except partially at high concentrations of 257 mM or greater. We infer that ethanol‐induced swelling of hepatocytes differs from that resulting from hyposmotic stress. Cellular events associated with increased activity of intracellular water most likely trigger the hyperpolarization of Vm that accompanies the latter. We conclude, therefore, that ethanol‐induced swelling occurs without change in cell water activity. This may result from the retention of macromolecules by ethanol in cells that constitutively secrete protein.

Chronic consumption of ethanol has a dramatic effect on the clinical outcome of patients with hepatitis C virus (HCV) infection, but the mechanism linking these two pathologies is unknown. Presently, in vitro systems are limited in their ability to study the interaction between a productive wild-type HCV infection and chronic ethanol exposure. Mouse models are potentially very useful in dissecting elements of the HCV-ethanol relationship. Experiments in mice that transgenically express HCV proteins are outlined, as are experiments for the generation of mice with chimeric human livers. The latter models appear to have the most promise for accurately modeling the effects of chronic ethanol intake in HCV-infected human livers.

Addition of 3' and 5' terminal phosphates to dApdA causes a decrease in conformational flexibility. pdApdAp has much fewer conformers with energies below 2.5 kcal./mole than dApdA. THE A, B and Watson-Crick (34) helices are the most preferred forms. Other important conformations are in the trans domain of psi. Thus, flexibility in psi as well as in omega and omega, and in the sugar pucker is indicated. The transformation from the B helix to the Watson-Crick helix follows a low energy path. This is significant since Watson-Crick conformations may be important for intercalation into nucleic acid polymers (40-42) above the dimer level. The B helix is preferred over the A form in these large DNA subunits.

In mid-Atlantic soft-red winter wheat (SRWW) production, the standard timing for a fungicide application is between flag leaf emergence (Feekes growth stage [FGS] 8) and heading (FGS 10.5). However, two-pass and anthesis (FGS 10.5.1) applications are becoming common, although these programs have not been thoroughly evaluated for disease control, yield, and profitability. Experiments were conducted in the mid-Atlantic in 2015 and 2016 to evaluate fungicide programs with applications at FGS 8, FGS 10.5.1, and two-pass programs with an early application at green-up (FGS 5) followed by (FB) applications at either FGS 8 or FGS 10.5.1. Fungicide programs that included an application at FGS 10.5.1 resulted in the highest probability of no disease on the flag leaf (0.29 to 0.40). The estimated mean yield increases ([Formula: see text]) relative to the nontreated check ranged from 253.65 to 634.16 kg ha−1. Using a grain price of $0.18 kg−1 ($5 bushel−1), probabilities were similar between applications at FGS 8 (0.49 to 0.56) and FGS 10.5.1 (0.53). The probability of profitability ranged from 0.48 to 0.57 for FGS 5 FB FGS 8 applications and 0.52 to 0.59 for FGS 5 FB FGS 10.5.1 applications, indicating limited benefit to two-pass programs.

Long-term ethanol exposure has deleterious effects on both glial and neuronal function. We assessed alterations in both astrocytic and neuronal viability, and alterations in N-methyl-d-aspartate receptor (NMDAR) function, in cocultures of rat cerebellar granule cells (CGCs) and astrocytes after continuous ethanol exposure (CEE). Treatment of cells with 100 mM EtOH once every 24 h for 4 days resulted in a mean ethanol concentration of 57.3 ± 2.1 mM. Comparisons between control and post-ethanol-treated cells were made 4 days after the last ethanol treatment. CEE did not alter glial cell viability, as indicated by the absence of either changes in astrocytic morphology, actin depolymerization, or disruption of astrocytic intracellular mitochondrial distribution at any day postethanol treatment. The CGCs were healthy and viable after CEE, as indicated by phase-contrast microscopy and the trypan-blue exclusion method. Whole-cell patch-clamp experiments indicated that NMDA-induced currents (I(NMDA)) were altered by CEE treatment. Similar to previous results obtained during the withdrawal phase from chronic ethanol exposure, I(NMDA) from CEE-treated cells were significantly larger than I(NMDA) from NMDARs in control CGCs, but returned to control values by the fourth day post-CEE. However, after the last ethanol dosing and during a time when ethanol concentrations remained high, I(NMDA) were significantly smaller than control values. Identical results were observed in CGCs expressing the NR2A or NR2B subunit. In summary, both neurons and astrocytes remained healthy following exposure to CEE with no signs of neurotoxicity at the cellular level, and modulation of NMDAR function is consistent with findings from prior experiments. Thus, we conclude that the CEE paradigm in glial-neuronal cocultures readily lends itself to long-term in vitro studies of ethanol effects that include glial-neuronal interactions and the ability to study ethanol withdrawal-induced neurotoxicity.

The oxidative metabolism of ethanol by the cytochrome P450 2E1 (CYP2E1) has been recognized to contribute to the ethanol-induced deleterious effects through the induction of oxidative stress. This study compared the effect of ethanol and acetaldehyde in the induction of oxidative stress and activation of transcription factors nuclear factor-κB (NF-κB) and activating protein 1 (AP-1) in HepG2 cells, which do not express CYP2E1, and HepG2 cells transfected with CYP2E1 (E47 cells). Neither ethanol (80 mmol/L) nor acetaldehyde (25-200 μmol/L) caused oxidative stress in HepG2 cells, an effect that was independent of blocking reduced glutathione (GSH) synthesis with buthionine-l -sulfoximine (BSO). However, BSO preincubation caused an overproduction of peroxides and activation of NF-κB and AP-1 in E47 cells even in the absence of ethanol. Furthermore, the incubation of E47 cells with ethanol (80 mmol/L for up to 5 days) depleted cellular GSH stores in both cytosol and mitochondria, reflecting the induction of oxidative stress. Ethanol activated NF-κB and AP-1 in E47 cells, an effect that was prevented by 4-methylpyrazole, potentiated by cyanamide, and attenuated by trolox C. Interestingly, however, despite the inability of acetaldehyde to induce oxidative stress in HepG2, acetaldehyde activated NF-κB and AP-1; in contrast, ethanol failed to activate these transcription factors in HepG2. Thus, our findings indicate that activation of NF-κB and AP-1 by ethanol and acetaldehyde occurs through distinct mechanisms. CYP2E1 is indispensable in the induction of oxidative stress from ethanol, whereas the activation of NF-κB and AP-1 by acetaldehyde is independent of oxidative stress.

AbstractThe mutant M301A of the acetylcholinesterase B from Nippostrongylus brasiliensis (NbAChE) was produced in a high‐cell‐density fermentation of a recombinant methylotrophic yeast Pichia pastoris. Dissolved oxygen (DO) spikes were used as an indicator for feeding the carbon source. Wet cell weight (WCW) reached after 8 days a maximum value of 316 g/L and the OD600 at this time was 280. The acetylcholinesterase activity increased up to 6,600 U/mL corresponding to an expression rate of 2 g of NbAChE per liter supernatant. The specific activity of the mutant NbAChE was determined after purification as 3,300 U/mg. Active site titration with chlorpyrifos, a strong AChE inhibitor, yielded in a specific activity of 3,400 U/mg. The enzyme was secreted by Pichia pastoris. Therefore, it could be concentrated from culture broth by cross‐flow‐filtration (50 kDa cut‐off membrane). It was further purified in one‐step anion‐exchange chromatography, using a XK 50/20 column filled with 125 mL Q Sepharose HP. Mutant NbAChE was purified 1.9‐fold up to a purity of 97% and a yield of 87%. The isolated enzyme was nearly homogenous, as seen on the silver stained SDS–PAGE as well as by a single peak after gel filtration. This extraordinary high expression rate and the ease of purification is an important prerequisite for their practical application, for example in biosensors for the detection of neurotoxic insecticides. © 2005 Wiley Periodicals, Inc.