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PubMed2002

Ethanol vapor is an efficient inducer of the alc gene expression system in model and crop plant species.

We have demonstrated that low concentrations of ethanol vapor efficiently induce the alc gene expression system in tobacco (Nicotiana tabacum cv Samsun NN), potato (Solanum tuberosum cv Solara), and oilseed rape (Brassica napus cv Westar). For many situations, this may be the preferred method of induction because it avoids direct application of comparatively high concentrations of an ethanol solution. Although induction was seen with less than 0.4 microM ethanol vapor, maximal induction of the chloramphenicol acetyl transferase gene was achieved after 48 h in leaves of tobacco plants enclosed with 4.5 microM ethanol vapor. In the absence of ethanol, there is no detectable gene expression. Treatment of potato tubers with ethanol vapor results in uniform beta-glucoronidase (GUS) expression. Vapor treatment of a single oilseed rape leaf resulted in induction of GUS in the treated leaf only and (14)C-ethanol labeling in tobacco confirmed that the inducer was not translocated. In contrast, enclosure of the roots, aerial parts, or whole plant with ethanol vapor resulted in induction of GUS activity in leaves and roots. The data reported here broaden the utility of the alc system for research and crop biotechnology.

Brassica napus↗

PubMed2001

Is the tert-butyl chloride solvolysis the most misunderstood reaction in organic chemistry? Evidence against nucleophilic solvent participation in the tert-butyl chloride transition state and for increased hydrogen bond donation to the 1-adamantyl chloride solvolysis transition state.

Despite theoretical calculations to the contrary, it has been argued that the 1-adamantyl cation is more stable than the tert-butyl cation in media of high dielectric constant. This argument has been utilized to suggest that the higher rate of solvolysis of tert-butyl chloride in aqueous ethanol is evidence for nucleophilic solvent participation in this classic reaction. Further, in "more highly ionizing" solvents, the rate of 1-adamantyl chloride is nearly the same as that of tert-butyl chloride, which is interpreted as a manifestation of the relative stabilities of the cations. However, the evidence cited does not explain the increased sensitivity of the rate of solvolysis of 1-adamantyl chloride over tert-butyl chloride to solvents which are better able to donate hydrogen bonds. The hypothesis developed here is that 1-adamantyl chloride solvolysis is assisted by hydrogen bond donation departing chloride ion to a greater extent than that of tert-butyl chloride solvolysis, most likely due to lessened steric interactions in a developing pyramidal cation. This hypothesis is supported by multiparameter solvent effect factor analyses utilizing the KOMPH2 equation which, in addition, quantifies the important role of ground-state destabilization due to strong solvent-solvent interactions. An important result from the good correlation of free energies of transfer of the tert-butyl chloride solvolysis transition state is that there is no change in mechanism, and, in particular, no nucleophilic participation even in non-hydroxylic basic solvents. The equation is also applied to the case of dimethylsulfonium ion solvolyses where the tert-butyl salt reacts substantially faster than the 1-adamantyl salt in ethanol and the gas phase. The decreased rate of the former in hydrogen bond donating solvents relative to the gas phase is as yet unclear. Solvent N values that were generated to characterize solvent nucleophilicity are shown not to be correlated by measures of solvent basicity but rather by the negative of measures of solvent hydrogen bond donor ability.

Journal Article↗

PubMed2006

Assessing appetitive and consummatory phases of ethanol self-administration in C57BL/6J mice under operant conditions: regulation by mGlu5 receptor antagonism.

RATIONALE: The development of mouse models of ethanol consumption and ethanol-seeking behavior is of particular importance in understanding the underlying mechanisms of drug abuse because these models can enable an analysis of an effect of specific genotype on drug-seeking behavior and the interaction of potential therapeutics with genotype. However, there are some limitations with present models, notably the inability to examine appetitive and consummatory behavior separately. MATERIALS AND METHODS: In the present study, C57BL/6 mice were trained to self-administer 10% ethanol in a modified operant protocol that allowed a clear delineation of consummatory and appetitive phases. The utility of this procedure was confirmed with the use of the metabotropic glutamate 5 (mGlu5) receptor antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP). RESULTS: Limited-access consumption during the dark phase of the light-dark cycle with intermittent access (every second or third day) led to a high level of consumption by the mice. MTEP caused a dose-dependent decrease in both the consumption of ethanol and the appetitive response for ethanol. Furthermore, this effect was unrelated to any effect of MTEP on locomotor activity. CONCLUSIONS: The model provides a useful paradigm for examining both the appetitive and consummatory phases of ethanol consumption in mice; furthermore, the data indicate mGlu5 receptors are involved in both phases.

Alcohol Drinking↗

PubMed1993

Spectrophotometric quantitation of DNA on blots after ethanol-solubilization of the MTT-formazan from anti-digoxigenin-based detection of nucleic acids.

The tetrazolium salt, 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) has recently been established as a substitute for Nitro-blue tetrazolium (NBT) in stain mixtures using antibody-conjugated alkaline phosphatase for the location of proteins on Western blots (Heegaard, 1990). Experiments reported here show that MTT is as sensitive as NBT in digoxigenin-labeled probe localization (on nucleic acid blots) utilizing alkaline-phosphatase-labelled, anti-digoxigenin antibodies. Moreover, as the formazan from MTT is soluble in ethanol, it is shown that spectrophotometric quantitation can be used to estimate the amount of target DNA on dot and Southern blots. For dot blotting, pBR328 was used as the probe and pBR322 as target. For Southern blots, human rDNA was used as the probe and total genomic calf DNA as the target. Staining response was linear over at least six twofold DNA dilutions in both types of blot.

Antibodies↗

PubMed1988

Hydrogen partial pressures in a thermophilic acetate-oxidizing methanogenic coculture.

Hydrogen partial pressures were measured in a thermophilic coculture comprised of a eubacterial rod which oxidized acetate to H(2) and CO(2) and a hydrogenotrophic methanogen, Methanobacterium sp. strain THF. Zinder and Koch (S. H. Zinder and M. Koch, Arch. Microbiol. 138:263-272, 1984) originally predicted, on the basis of calculations of Gibbs free energies of reactions, that the H(2) partial pressure near the midpoint of growth of the coculture should be near 4 Pa (ca. 4 x 10 atm; ca. 0.024 muM dissolved H(2)) for both organisms to be able to conserve energy for growth. H(2) partial pressures in the coculture were measured to be between 20 and 50 Pa (0.12 to 0.30 muM) during acetate utilization, approximately one order of magnitude higher than originally predicted. However, when DeltaG(f) (free energy of formation) values were corrected for 60 degrees C by using the relationship DeltaG(f) = DeltaH(f) - TDeltaS (DeltaH(f) is the enthalpy or heat of formation, DeltaS is the entropy value, and T is the temperature in kelvins), the predicted value was near 15 Pa, in closer agreement with the experimentally determined values. The coculture also oxidized ethanol to acetate, a more thermodynamically favorable reaction than oxidation of acetate to CO(2). During ethanol oxidation, the H(2) partial pressure reached values as high as 200 Pa. Acetate was not used until after the ethanol was consumed and the H(2) partial pressure decreased to 40 to 50 Pa. After acetate utilization, H(2) partial pressures fell to approximately 10 Pa and remained there, indicating a threshold for H(2) utilization by the methanogen. Axenic cultures of the acetate-oxidizing organism were combined with pure cultures of either Methanobacterium sp. strain THF or Methanobacterium thermoautotrophicum DeltaH to form reconstituted acetate-oxidizing cocultures. The H(2) partial pressures measured in both of these reconstituted cocultures were similar to those measured in the original acetate-oxidizing rod coculture. Since M. thermoautotrophicum DeltaH did not use formate as a substrate, formate is not necessarily involved in interspecies electron transfer in this coculture.

Journal Article↗

PubMed1994

DNA strand cleavage as a sensitive assay for the production of hydroxyl radicals by microsomes: role of cytochrome P4502E1 in the increased activity after ethanol treatment.

There is increasing interest in the role of reactive oxygen radicals in the hepatotoxicity associated with ethanol consumption. Reactive oxygen intermediates interact with DNA and can cause single-strand breaks of supercoiled DNA. Experiments were carried out to evaluate the utility of this system as a sensitive assay for the detection of potent oxidants generated by rat liver microsomes isolated from pair-fed control rats and rats treated chronically with ethanol. DNA strand cleavage was assayed by monitoring the migration of the supercoiled and open circular forms in agarose. Microsomes catalysed DNA strand breakage with either NADPH or NADH as cofactors; iron was required to catalyse the reaction and various ferric complexes were effective in promoting the reaction. DNA strand cleavage was prevented by catalase, superoxide dismutase, GSH and hydroxyl-radical-scavenging agents, suggesting that a hydroxyl-radical-like species was the oxidant responsible for the breakage. This assay system proved to be much more sensitive in detecting hydroxyl radicals than are other methods, such as e.s.r. spectroscopy or oxidation of chemical scavenging agents with respect to the amount of microsomal protein and the nature and concentration of the iron catalyst required. Microsomes from ethanol-treated rats were more reactive than control microsomes in catalysing the DNA strand cleavage with either NADPH or NADH; increased catalytic activity was observed with various ferric complexes and was sensitive to the above antioxidants. Compared with preimmune IgG, anti-(cytochrome P4502E1) IgG had no effect on DNA strand cleavage by the control microsomes, but completely prevented the NADPH- and the NADH-dependent increased activity found with microsomes from the ethanol-treated rats. Inhibitors of cytochrome P4502E1, such as diethyl dithiocarbamate and tryptamine, also lowered the extent of increase of DNA strand cleavage produced by microsomes from the ethanol-treated rats. These results indicate that DNA strand cleavage is a very sensitive assay for detecting the production of hydroxyl radicals by microsomes and to demonstrate increased activity by microsomes after chronic ethanol treatment. This increased activity with NADPH and NADH is due, at least in part, to induction of cytochrome P4502E1.

Animals↗

PubMedDate not supplied

Amino-acid-dependent, differential effects of ethanol on glucose production in rabbit kidney-cortex tubules.

AIMS: The effect of ethanol on glucose synthesis in kidney-cortex tubules of control and diabetic rabbits has been investigated. METHODS: Both freshly isolated and grown in primary cultures, kidney-cortex tubules were incubated with alanine or aspartate plus lactate or glycerol plus octanoate in the absence and presence of 100 mmol/l ethanol. RESULTS: In freshly isolated renal tubules incubated in the presence of alanine plus lactate or glycerol plus octanoate, and in tubules grown in primary culture in the medium containing alanine plus lactate plus octanoate alcohol, resulted in about 30% decrease in glucose formation. A diminished glucose production in freshly isolated tubules was accompanied by: (i) a decrease in alanine utilization, (ii) an increase in lactate or glycerol consumptions and (iii) a decline in GSH:GSSG ratio. The ethanol action was not abolished by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase (ADH). In view of ethanol-induced changes in gluconeogenic intermediates it is likely that in the presence of alanine plus glycerol plus octanoate ethanol causes a decline in flux through phosphoenolpyruvate carboxykinase, probably due to either an increase in intracellular content of 2-oxoglutarate, inhibitor of this key gluconeogenic enzyme and/or an enhanced flux through pyruvate kinase, as concluded from an increased lactate formation in the presence of glycerol in the incubation medium. In renal tubules grown in primary cultures in the presence of alanine plus lactate plus octanoate a decrease in GSH:GSSG ratio was accompanied by elevated generation of reactive oxygen species (ROS). Upon replacement of alanine by aspartate ethanol affected neither glucose production, substrate uptake, ROS accumulation nor GSH:GSSG ratio. CONCLUSIONS: In the presence of alanine ethanol-induced decrease in glucose production and elevation of ROS might cause a limited NADPH generation resulting in a decrease in the intracellular GSH:GSSG ratio. On the contrary, aspartate might protect against ROS generation, so intensive gluconeogenesis supports NADPH generation and in consequence high values of the intracellular GSH:GSSG ratio are maintained.

Amino Acids↗

PubMed1966

Effects of prolonged ingestion of glucose or ethanol on fatty acid synthesis by mitochondria and cell sap of rat liver and adipose tissue.

Effects of prolonged ingestion of glucose and ethanol on the rate of fatty acid synthesis by liver and adipose tissue have been investigated in male rats. Ethanol significantly enhanced the rate of fatty acid synthesis from malonyl-2-(14)C CoA in liver cell sap; glucose feeding enhanced the rate of fatty acid synthesis from both malonyl-2-(14)C and acetyl-1-(14)C CoA. Neither dietary supplement modified the types of fatty acid synthesized in this enzyme system. Palmitic acid was the principal product synthesized from a mixture of malonyl and acetyl CoA, whereas myristic and palmitic acids were the predominant products formed from acetyl CoA alone. Neither glucose nor ethanol affected fatty acid synthesis by adipose tissue cell sap. Mitochondria derived from liver and adipose tissue of control, glucose-fed, and ethanol-fed animals all incorporated acetyl-1-(14)C CoA into lipid at about the same rate, but did not utilize malonyl CoA for lipid synthesis to any significant degree. The label appeared in fatty acids, one-half of which were contained in phospholipid. Both unsaturated and saturated fatty acids synthesized by mitochondria contained isotope, most of which was present in the carboxyl groups. Ethanol and glucose feeding stimulated the labeling of monoenoic fatty acids in liver mitochondria, but only glucose did so for adipose tissue. These findings agree with results previously obtained when lipogenesis was measured with acetate-(14)C in vivo.

Acetates↗

PubMed1999

Effective utilization of N2-ethyl-2'-deoxyguanosine triphosphate during DNA synthesis catalyzed by mammalian replicative DNA polymerases.

Acetaldehyde is produced by metabolic oxidation of ethanol after drinking alcoholic beverages. This agent reacts with nucleosides and nucleotides, resulting in the formation of N2-ethyl-guanine residues. N2-ethyl-2'-deoxyguanosine (N2-ethyl-dG) adduct has been detected in the lymphocyte DNA of alcoholic patients [Fang, J. L., and Vaca, C. E. (1997) Carcinogenesis 18, 627-632]. Thus, the nucleotide pool is also expected to be modified by acetaldehyde. N2-Ethyl-2'-deoxyguanosine triphosphate (N2-ethyl-dGTP) was chemically synthesized. The utilization of N2-ethyl-dGTP during DNA synthesis was determined by steady-state kinetic studies. N2-Ethyl-dGTP was efficiently incorporated opposite template dC in reactions catalyzed by mammalian DNA polymerase alpha and delta. When pol alpha was used, the insertion frequency of N2-ethyl-dGTP was 400 times less than that of dGTP, but 320 times higher than that of 7,8-dihydro-8-oxo-2'-deoxyguanosine triphosphate (8-oxo-dGTP), an oxidative damaged nucleotide. Using pol delta, the insertion frequency of N2-ethyl-dGTP was only 37 times less than that of dGTP. The chain extension from dC:N2-ethyl-dG pair occurred much more rapidly: the extension frequencies for pol alpha and pol delta were only 3.8 times and 6.3 times, respectively, lower than that of dC:dG pair. We also found that N2-ethyl-dG can be detected in urine samples obtained from healthy volunteers who had abstained from drinking alcohol for 1 week before urine collection. This indicates that humans are exposed constantly to acetaldehyde even without drinking alcoholic beverages. Incorporation of N2-ethyl-dG adducts into DNA may cause mutations and may be related to the development of alcohol- and acetaldehyde-induced human cancers.

Adult↗

PubMed1976

Utilization of cellulosic materials through enzymatic hydrolysis. II. Preliminary assessment of an integrated processing scheme.

An integrated processing scheme is described for the conversion of a celluosic waste (newsprint) to sugrsa by enzymatic hydrolysis and then to ethanol and yeast by fermentation. The unconverted solids are burned to produce process energy requirements and surplus electrical power. Preliminary designs and cost studies are developed to provide a rough perspective on the potential economic feasibility of this method of cellulose utilization.

Cellulose↗

PubMed2005

Astrocytes protect neurons from ethanol-induced oxidative stress and apoptotic death.

Ethanol induces oxidative stress in cultured fetal rat cortical neurons and this is followed by apoptotic death, which can be prevented by normalization of cell content of reduced glutathione (GSH). Because astrocytes can play a central role in maintenance of neuron GSH homeostasis, the following experiments utilized cocultures of neonatal rat cortical astrocytes and fetal cortical neurons to determine if astrocytes could protect neurons from ethanol-mediated apoptotic death via this mechanism. In cortical neurons cultured in the absence of astrocytes, ethanol (2.5 and 4 mg/ml; 6-, 12-, and 24-hr exposures) decreased trypan blue exclusion and the MTT viability measures by up to 45% (P < 0.05), increased levels of reactive oxygen species (ROS) by up to 81% (P < 0.05), and decreased GSH within 1 hr of treatment by 49 and 51% for 2.5 and 4 mg/ml, respectively (P < 0.05). This was followed by onset of apoptotic cell death as determined by increased Annexin V binding and DNA fragmentation by 12 hr of ethanol exposure. Coculturing neurons with astrocytes prevented GSH depletion by 2.5 mg/ml ethanol, whereas GSH content was increased over controls in neurons exposed to 4 mg/ml ethanol (by up to 341%; P < 0.05). Ethanol generated increases in neuron ROS and apoptosis; decreases in viability were also prevented by coculture. Astrocytes were largely insensitive to ethanol, using the same measures. Only exposure to 4.0 mg/ml ethanol decreased GSH content in astrocytes, concomitant with a 204% increase in GSH efflux (P < 0.05). These studies illustrate that astrocytes can protect neurons from ethanol-mediated apoptotic death and that this may be related to maintenance of neuron GSH.

Animals↗

PubMed1998

Fatty acid omega- and (omega-1)-hydroxylation in experimental alcoholic liver disease: relationship to different dietary fatty acids.

Arachidonic acid concentrations in liver are decreased in response to ethanol administration. In addition, the oxygenated products of arachidonic acid metabolites could affect the severity of alcoholic liver injury. Selective utilization of arachidonic acid by the cytochrome P-450 system could, in part, account for the decrease in arachidonic acid. To evaluate this pathway further, male Wistar rats were fed different dietary fats: medium chain triglycerides, palm oil, and corn oil or fish oil with either ethanol or isocaloric amounts of dextrose. Histopathology, cytochrome P-4502E1 (CYP2E1) and cytochrome P-4504A (CYP4A), and omega- and (omega-1)-hydroxylation products of lauric and arachidonic acids were evaluated. Ethanol induction of CYP2E1 was related to the concentration of polyunsaturated fatty acids in the diet; induction of CYP4A by ethanol was seen in all groups. The highest levels of 11-hydroxy-lauric acid and 19-hydroxyarachidonic acid (omega-1) were seen in rats fed ethanol with palm oil and corn oil. Highly significant correlations were seen between the (omega-1)-hydroxylation products and CYP2E1 activity. No correlation was seen between the omega-hydroxylation products and CYP2E1 activity. In contrast, the levels of omega-hydroxylation products correlated with CYP4A. The overall results showed a significant increase in (omega-1)-hydroxylation products in rats fed diets containing significant amounts of linoleic acid (i.e., palm oil and corn oil).

Animals↗

PubMed1989

Ethanol causes decreased partitioning into biological membranes without changes in lipid order.

One of the adaptive responses of cell membranes to chronic ethanol consumption is the acquisition of a resistance to fluidization or disordering of the lipids by ethanol in vitro and a reduced partitioning of ethanol into the membrane (membrane tolerance). The degree to which the effects on partitioning and lipid disordering share common features has not previously been explored and in addition the relevance of the value of lipid order in the absence of added ethanol (baseline lipid order) to membrane tolerance has not been established. The location in the bilayer and the nature of the modification underlying these effects is also unknown. The effect of chronic ethanol treatment was examined using 5-doxyl decane as a model hydrophobic compound. Its partitioning into the membranes was determined by utilizing its ability to quench fluorophores (1,6-diphenyl-2,3,5-hexatriene and 3- and 12-anthroyl stearates) by collisional quenching. The partition coefficient of 5-doxyl decane into the bilayer central region was reduced as a result of the chronic ethanol treatment. The effect could also be demonstrated in vesicles of phospholipids and was lost 4 days after withdrawal of the ethanol from the diet. These results closely parallel those relating to resistance to lipid disordering and suggest that both techniques detect a common modification. Lipid order was assessed using fluorescence anisotropy measurements of a range of fluorophores, including those used to determine the partitioning properties of the membrane. No effect of chronic ethanol treatment on lipid order was found, either in the intact membranes or in vesicles of extracted phospholipids. This suggests that changes in baseline order are not critical features of membrane tolerance in liver microsomes. In addition it appears that the altered partitioning of the 5-doxyl decane into the central region of the membrane is not related to lipid order changes in this region. The reduced partitioning of 5-doxyl decane may be a reflection of a redistribution in the lipid bilayer, perhaps due to modifications in other locations in the membrane, such as the lipid head group region.

Alcoholism↗

PubMed1988

Rat heart-derived endothelial and smooth muscle cell cultures: isolation, cloning and characterization.

This report describes the initiation, cloning and establishment of long-term serial cultures of rat heart-derived vascular endothelial (EC) and smooth muscle cells (SMC). Populations of these cells derived from both the macro-and microcirculation were obtained utilizing isolated heart perfusion technique. Elimination of potential mesothelial cell contamination was achieved by ethanol fixation of the pericardial surface prior to perfusion. Initial outgrowths from perfusate yielded both endothelial (rapid adhering) and smooth muscle (slow adhering) appearing cell populations. Subsequent pooling of individual EC colonies resulted in maintaining, with gradual subcultivation, a stable homogeneous population which was designated RHE-parent. Upon continual subculture late passage (greater than P10) RHE-parent cell cultures expressed a marked heterogeneity in endothelial phenotypes. Cloning experiments resulted in establishing two distinct EC populations designated RHE-clone 1A and RHE-clone 2A. All RHE cell cultures exhibited the typical cobblestone growth pattern and positive immunofluorescent staining for factor VIII related antigen. In contrast, rat heart-derived smooth muscle cell (RH-SMC) cultures displayed the typical multilayered 'hill and valley' pattern and positive fluorescence for SMC-specific actin and myosin antibodies. Additional EC preparations, obtained without prior fixation of the pericardial surface, revealed cell clusters which stained positive for cytokeratin. On the other hand, RHE parent and cloned populations stained exclusively for vimentin, further confirming the absence of mesothelial cell contamination in these cultures. Cell growth studies on early (less than P10) and late (greater than P10) passage RHE-parent population revealed markedly different cell growth responses and cell morphology. Both EC cloned populations and more notably RHE-parent (less than P10) cultures were capable of significant growth when maintained in limiting serum concentration. Growth studies using serum-free RHE-parent conditioned medium demonstrated mitogenic activity when tested on RHE-parent cultures indicating the presence of an endothelial cell-derived growth factor. These studies indicate that long-term RHE and RH-SMC derived cell cultures can serve as a useful model to study the biology of vascular cells derived from different sites. In addition the demonstration of mitogenic activity in these cultures will enable us to explore further the nature of this response and compare this phenomenon with growth factors identified in large vessel cell systems.

Animals↗

PubMed1986

Effect of fasting and acute ethanol administration on the energy state of in vivo liver as measured by 31P-NMR spectroscopy.

The effects of 48 h fasting, administration of ethanol or 2,4-dinitrophenol, on the phosphorus-containing metabolites in liver in vivo have been determined utilizing 31P nuclear magnetic resonance spectroscopy. These measurements were combined with determinations of metabolite concentrations in livers which were freeze-clamped immediately after the NMR measurements were completed. Administration of sub-lethal amounts of dinitrophenol dramatically decreased ATP and increased Pi concentrations in liver in vivo as indicated by a 2.7-fold increase in the NMR-derived [Pi]/[ATP] ratio. Ethanol administration to fed animals increased the NMR-derived [Pi]/[ATP] ratio 27%; in contrast, the same amount of ethanol administered to fasted animals decreased the NMR-derived [Pi]/[ATP] ratio 30%. The NMR visible Pi and ADP represent about 50% and 15% of the total Pi and ADP, respectively. The phosphorylation potentials calculated from the NMR visible Pi and ADP were an order of magnitude higher than those obtained from metabolite concentrations in freeze-clamped tissue. There was no apparent correlation between the phosphorylation potentials derived from either the NMR spectral analyses or from metabolite concentrations and the hepatic [NAD+]/[NADH] ratio. The chemical shift of Pi indicated that ethanol administration elicited a decrease in pH of 0.1 unit in liver in vivo. Hepatic free [Mg2+] was increased 21% in fasted animals, but was unaffected by ethanol administration.

Acetoacetates↗

PubMed2004

In vivo monitoring of amino acids by microdialysis sampling with on-line derivatization by naphthalene-2,3-dicarboxyaldehyde and rapid micellar electrokinetic capillary chromatography.

An analytical method was developed to monitor amino acids collected by in vivo microdialysis. Microdialysate was continuously derivatized on-line by mixing 6 mM naphthalene-2,3-dicarboxyaldehyde (NDA) and 10 mM potassium cyanide with the dialysate stream in a fused silica capillary to form fluorescent products. Reaction time, determined by the flow rate and volume of reaction capillary, was 3 min. Derivatized amino acids were continuously delivered into a flow-gated interface and periodically injected onto a capillary electrophoresis unit equipped with a laser-induced fluorescence detection based on a commercial microscope. Separation was performed in the micellar electrokinetic chromatography mode using 30 mM sodium dodecyl sulfate in 15 mM phosphate buffer at pH 8.0 as the separation media. An electric field of 1.3 kV/cm was applied across a 10 cm long, 10 microm internal diameter separation capillary. These conditions allowed 17 amino acid derivatives to be resolved in less than 30 s. On-line injections could be performed at 30 s intervals for in vivo samples. Detection limits were from 10 to 30 nM for the amino acids. The method was applied to monitor the acute ethanol-induced amino acid level changes in freely moving rats. The results demonstrate the utility of the method to reveal dynamics of amino acid concentration in vivo.

Amino Acids↗

PubMed2001

Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures.

Typical methods used for encapsulating antisense oligodeoxynucleotides (ODN) and plasmid DNA in lipid vesicles result in very low encapsulation efficiencies or employ cationic lipids that exhibit unfavorable pharmacokinetic and toxicity characteristics when administered intravenously. In this study, we describe and characterize a novel formulation process that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities (0.15--0.25 g ODN/g lipid) of polyanionic ODN in lipid vesicles. This process requires the presence of up to 40% ethanol (v/v) and initial formulation at acidic pH values where the DODAP is positively charged. In addition, the presence of a poly(ethylene glycol)-lipid was required during the formulation process to prevent aggregation. The 'stabilized antisense-lipid particles' (SALP) formed are stable on adjustment of the external pH to neutral pH values and the formulation process allows encapsulation efficiencies of up to 70%. ODN encapsulation was confirmed by nuclease protection assays and (31)P NMR measurements. Cryo-electron microscopy indicated that the final particles consisted of a mixed population of unilamellar and small multilamellar vesicles (80--140 nm diameter), the relative proportion of which was dependent on the initial ODN to lipid ratio. Finally, SALP exhibited significantly enhanced circulation lifetimes in mice relative to free antisense ODN, cationic lipid/ODN complexes and SALP prepared with quaternary aminolipids. Given the small particle sizes and improved encapsulation efficiency, ODN to lipid ratios, and circulation times of this formulation compared to others, we believe SALP represent a viable candidate for systemic applications involving nucleic acid therapeutics.

Animals↗

PubMed2002

Antioxidant and pro-oxidant effects of a manganese porphyrin complex against CYP2E1-dependent toxicity.

Superoxide dismutases (SOD) mimetics have been shown to be protective against cell injury caused by reactive oxygen species. The objective of this study was to investigate the effects of the manganese (III) tetrakis(N-methyl-2-pyridyl)porphyrin (MnTMPyP) on CYP2E1-dependent toxicity. The synergistic toxicity of iron and arachidonic acid has been associated with oxidative stress and lipid peroxidation in HepG2 cells that overexpress CYP2E1. Iron plus arachidonic acid caused loss of viability, increased lipid peroxidation and reactive oxygen species generation, and mitochondrial membrane injury in these cells. MnTMPyP partially protected against the decrease in cell viability, the enhanced lipid peroxidation and oxygen radical production, and the loss of mitochondrial membrane potential. The effect of MnTMPyP on arachidonic acid (absence of iron) toxicity was also evaluated. Arachidonic acid also caused toxicity, lipid peroxidation and reduction of the mitochondrial membrane potential. However, in this model, all of these alterations were actually enhanced by MnTMPyP. MnTMPyP also enhanced toxicity in CYP2E1-expressing HepG2 cells depleted of reduced glutathione (GSH). MnCl(2) had little or no effect on the toxicity by arachidonic acid, and MnTMPyP itself did not peroxidize arachidonic acid. MnTMPyP, an SOD mimetic that also scavenges hydrogen peroxide and peroxynitrite, thus showed an antioxidant and protective effect against iron plus arachidonic acid toxicity, but a pro-oxidant and cytotoxic effect against arachidonic acid toxicity in CYP2E1-expressing cells. These different actions may relate to the ability of MnTMPyP to either scavenge or produce free radicals in cells depending upon the prevailing MnTMPyP oxidation-reduction pathways. MnTMPyP and related manganese porphyrin compounds may have potential clinical utility against diseases associated with the overproduction of reactive oxygen species such as ethanol-induced liver injury but it is clear that further investigation of all the pathways of manganese porphyrin oxidation-reduction are necessary.

Animals↗