Ethanol-free Medium Research Articles

Kluyveromyces marxianus developing ethanol tolerance during adaptive evolution with significant improvements of multiple pathways

BackgroundKluyveromyces marxianus, the known fastest-growing eukaryote on the earth, has remarkable thermotolerance and capacity to utilize various agricultural residues to produce low-cost bioethanol, and hence is industrially important to resolve the imminent energy shortage crisis. Currently, the poor ethanol tolerance hinders its operable application in the industry, and it is necessary to improve K. marxianus’ ethanol resistance and unravel the underlying systematical mechanisms. However, this has been seldom reported to date.ResultsWe carried out a wild-type haploid K. marxianus FIM1 in adaptive evolution in 6% (v/v) ethanol. After 100-day evolution, the KM-100d population was obtained; its ethanol tolerance increased up to 10% (v/v). Interestingly, DNA analysis and RNA-seq analysis showed that KM-100d yeasts’ ethanol tolerance improvement was not due to ploidy change or meaningful mutations, but founded on transcriptional reprogramming in a genome-wide range. Even growth in an ethanol-free medium, many genes in KM-100d maintained their up-regulation. Especially, pathways of ethanol consumption, membrane lipid biosynthesis, anti-osmotic pressure, anti-oxidative stress, and protein folding were generally up-regulated in KM-100d to resist ethanol. Notably, enhancement of the secretory pathway may be the new strategy KM-100d developed to anti-osmotic pressure, instead of the traditional glycerol production way in S. cerevisiae. Inferred from the transcriptome data, besides ethanol tolerance, KM-100d may also develop the ability to resist osmotic, oxidative, and thermic stresses, and this was further confirmed by the cell viability test. Furthermore, under such environmental stresses, KM-100d greatly improved ethanol production than the original strain. In addition, we found that K. marxianus may adopt distinct routes to resist different ethanol concentrations. Trehalose biosynthesis was required for low ethanol, while sterol biosynthesis and the whole secretory pathway were activated for high ethanol.ConclusionsThis study reveals that ethanol-driven laboratory evolution could improve K. marxianus’ ethanol tolerance via significant up-regulation of multiple pathways including anti-osmotic, anti-oxidative, and anti-thermic processes, and indeed consequently raised ethanol yield in industrial high-temperature and high-ethanol circumstance. Our findings give genetic clues for further rational optimization of K. marxianus’ ethanol production, and also partly confirm the positively correlated relationship between yeast’s ethanol tolerance and production.

Ethanol exposed maturing rat cerebellar granule cells show impaired energy metabolism and increased cell death after oxygen-glucose deprivation.

Alcohol, a widely abused drug, has deleterious effects on the immature nervous system. This study investigates the effect of chronic in vitro ethanol exposure on the metabolism of immature rat cerebellar granular cells (CGCs) and on their response to oxygen-glucose deprivation (OGD). Primary CGC cultures were exposed to ethanol (100 mM in culture medium) or to control ethanol-free medium starting day one in vitro (DIV1). At DIV8, the expression of ATP synthase gene ATP5g3 was quantified using real-time PCR, then cultures were exposed to 3 hours of OGD or normoxic conditions. Subsequently, cellular metabolism was assessed by a resazurin assay and by ATP level measurement. ATP5g3 expression was reduced by 12-fold (P = 0.03) and resazurin metabolism and ATP level were decreased to 74.4 ± 4.6% and 55.5 ± 6.9%, respectively after chronic ethanol treatment compared to control values (P < 0.01). Additionally, after OGD exposure of ethanol-treated cultures, resazurin metabolism and ATP level were decreased to 12.7 ± 1.0% and 9.0 ± 2.0% from control values (P < 0.01). These results suggest that chronic ethanol exposure reduces the cellular ATP level, possibly through a gene expression down-regulation mechanism, and increases the vulnerability to oxygen-glucose deprivation. Thus, interventions which improve metabolic function and sustain ATP-levels could attenuate ethanol-induced neuronal dysfunction and should be addressed in future studies.

Altered drug release from Orfiril® long following ethanol consumption - extent and consequences.

Orfiril® long is a widely used antiepileptic drug preparation despite being characterized by features associated with susceptibility to ethanol induced changes to drug release. In vitro dissolution studies revealed that 30 % ethanol was required in order to cause dose dumping of valproic acid within two hours at pH 1. However, after exposure to only 5 % of ethanol for 45 minutes at pH 1, the subsequent release of sodium valproate increased by ~ 10 % for the first two hours in ethanol-free media of pH 6.8. The drug solubility increases with pH, likely to also increase the vulnerability to ethanol. This indicates that Orfiril® long is affected by exposure to low concentrations of ethanol as well, only that this effect was exclusively displayed at an elevated pH value not part of the standard regulatory recommendations. Even so, simplified pharmacokinetic simulations revealed no risk of a lower therapeutic effect. Postponing the drug intake in case of moderate drinking, likely to increase the risk of omission altogether, is therefore not necessary. However, for slim patients with a small volume of distribution and low tolerability, a longer duration of mild side effects such as drowsiness and nausea might be experienced.

Acetobacter pasteurianus metabolic change induced by initial acetic acid to adapt to acetic acid fermentation conditions.

Initial acetic acid can improve the ethanol oxidation rate of acetic acid bacteria for acetic acid fermentation. In this work, Acetobacter pasteurianus was cultured in ethanol-free medium, and energy production was found to increase by 150% through glucose consumption induced by initial acetic acid. However, oxidation of ethanol, instead of glucose, became the main energy production pathway when upon culturing ethanol containing medium. Proteome assay was used to analyze the metabolism change induced by initial acetic acid, which provided insight into carbon metabolic and energy regulation of A. pasteurianus to adapt to acetic acid fermentation conditions. Results were further confirmed by quantitative real-time PCR. In summary, decreased intracellular ATP as a result of initial acetic acid inhibition improved the energy metabolism to produce more energy and thus adapt to the acetic acid fermentation conditions. A. pasteurianus upregulated the expression of enzymes related to TCA and ethanol oxidation to improve the energy metabolism pathway upon the addition of initial acetic acid. However, enzymes involved in the pentose phosphate pathway, the main pathway of glucose metabolism, were downregulated to induce a change in carbon metabolism. Additionally, the enhancement of alcohol dehydrogenase expression promoted ethanol oxidation and strengthened the acetification rate, thereby producing a strong proton motive force that was necessary for energy production and cell tolerance to acetic acid.

Reduced SP1-mediated transcriptional activation decreases expression of intestinal folate transporters in response to ethanol exposure.

The study was designed to identify the regulatory mechanisms underlying the effects of ethanol exposure on intestinal folate transport and to investigate the reversibility of such effects. Caco-2 cells were grown in control and ethanol containing medium for 96 h. Thereafter, one subgroup of cells was shifted on ethanol free medium and grown for next 72 h. For in vivo studies, rats were given 1g ethanol/kg body weight/day either for 3 or 5 months and after 3 months of ethanol treatment, one group of rats received no ethanol for 2 months. A significant decrease in folic acid transport as well as expression of folate transporters was observed on ethanol treatment and the effects were reversible upon removal of ethanol. Ethanol exposure had no impact on CpG island methylation of the folate transporters however, an increase in their mRNA half-life was observed that seems to be a homeostatic mechanism. Chromatin immunoprecipitation assay revealed a decrease in binding of SP1 transcription factor to the promoter regions of folate transporters. Reduced binding of SP1 to the promoter region of folate transporters may be a part of the regulatory mechanism resulting in decreased expression of folate transporters on ethanol exposure.

The relationship between lysine 4 on histone H3 methylation levels of alcohol tolerance genes and changes of ethanol tolerance in Saccharomyces cerevisiae.

We evaluated whether epigenetic changes contributed to improve ethanol tolerance in mutant populations of Saccharomyces cerevisiae (S. cerevisiae). Two ethanol-tolerant variants of S. cerevisiae were used to evaluate the genetic stability in the process of stress-free passage cultures. We found that acquired ethanol tolerance was lost and transcription level of some genes (HSP104, PRO1, TPS1, and SOD1) closely related to ethanol tolerance decreased significantly after the 10th passage in ethanol-free medium. Tri-methylation of lysine 4 on histone H3 (H3K4) enhanced at the promoter of HSP104, PRO1, TPS1 and SOD1 in ethanol-tolerant variants of S. cerevisiae was also diminished after tenth passage in stress-free cultures. The ethanol tolerance was reacquired when exogenous SOD1 transferred in some tolerance-lost strains. This showed that H3K4 methylation is involved in phenotypic variation with regard to ethanol tolerance with respect to classic breeding methods used in yeast.

Effects of ethanol on red blood cell rheological behavior

Consumption of red wine is associated with a decreased risk of several cardiovascular diseases (e.g., coronary artery disease, stroke), but unfortunately literature reports regarding ethanol's effects on hemorheological parameters are not concordant. In the present study, red blood cell (RBC) deformability was tested via laser ektacytometry (LORCA, 0.3-30 Pa) using two approaches: 1) addition of ethanol to whole blood at 0.25%-2% followed by incubation and testing in ethanol-free LORCA medium; 2) addition of ethanol to the LORCA medium at 0.25%-6% then testing untreated native RBC in these media. The effects of ethanol on deformability for oxidatively stressed RBC were investigated as were changes of RBC aggregation (Myrenne Aggregometer) for cells in autologous plasma or 3% 70 kDa dextran. Significant dose-related increases of RBC deformability were observed at 0.25% (p < 0.05) and higher concentrations only if ethanol was in the LORCA medium; no changes occurred for cells previously incubated with ethanol then tested in ethanol-free medium. The impaired deformability of cells pre-exposed to oxidative stress was improved only if ethanol was in the LORCA medium. RBC aggregation decreased with concentration at 0.25% and higher for cells in both autologous plasma and dextran 70. Our results indicate that ethanol reversibly improves erythrocyte deformability and irreversibly decreases erythrocyte aggregation; the relevance of these results to the health benefits of moderate wine consumption require further investigation.

Ethanol Increases the Paracellular Permeability of Monolayers of CAPAN-1 Pancreatic Duct Cells

When grown on permeable supports, pancreatic duct adenocarcinoma CAPAN-1 cells establish very high values of transepithelial resistance (TER). The addition of ethanol produced a dose-related, reversible drop in the TER of these cells, ranging from 15% (with 1% ethanol) to 65% (with 10% ethanol). The ethanol effect was rapid and reversible. The resistance decrease was associated with an increase in monolayer permeability to mannitol. No significant decrease in cell ATP was detected for ethanol concentrations lower than 7%. Confocal vertical sections of calcein-loaded monolayers of CAPAN-1 cells, grown on plasticware, showed a progressive deflation of domes detectable after 5 min of treatment with 2% ethanol. Incubation in an ethanol-free medium caused a progressive dome restoration. Immunocytochemical analysis of ethanol-treated cells indicated that ZO-1 and occludin exhibited clear cut distribution changes while the perijunctional actin pattern was slightly modified. Electron microscopy showed that a discrete intercellular space was detectable between adjacent ethanol-treated cells but not between control cells. These data indicate that ethanol is a tight junction barrier opener in pancreatic duct cells.

Fetal Alcohol Exposure Alters Neurosteroid Modulation of Hippocampal N-Methyl-d-aspartate Receptors

The actions of ethanol on brain ligand-gated ion channels have important roles in the pathophysiology of alcohol-related neurodevelopmental disorders and fetal alcohol syndrome. Studies have shown that N-methyl-d-aspartate (NMDA) receptors are among the ligand-gated ion channels affected by prenatal ethanol exposure. We exposed pregnant dams to an ethanol-containing liquid diet that results in blood ethanol levels near the legal intoxication limit in most states (0.08%). Primary cultures of hippocampal neurons were prepared from the neonatal offspring of these dams, and NMDA receptor function was assessed by patch clamp electrophysiological techniques after 6-7 days in culture in ethanol-free media. Unexpectedly, we did not detect any changes in hippocampal NMDA receptor function at either the whole-cell or single-channel levels. However, we determined that fetal alcohol exposure alters the actions of the neurosteroids pregnenolone sulfate and pregnenolone hemisuccinate, which potentiate NMDA receptor function. Western immunoblot analyses demonstrated that this alteration is not due to a change in the expression levels of NMDA receptor subunits. Importantly, in utero ethanol exposure did not affect the actions of neurosteroids that inhibit NMDA receptor function. Moreover, the actions of pregnenolone sulfate on type A gamma-aminobutyric acid and non-NMDA receptor function were unaltered by ethanol exposure in utero, which suggests that the alteration is specific to NMDA receptors. These findings are significant because they provide, at least in part, a plausible mechanistic explanation for the alterations in the behavioral responses to neurosteroids found in neonatal rats prenatally exposed to ethanol and to other forms of maternal stress (Zimmerberg, B., and McDonald, B. C. (1996) Pharmacol. Biochem. Behav. 55, 541-547).

Conditioning to ethanol in the fruit fly—a study using an inhibitor of ADH

To identify processes involved in the choice of ethanol by adult Drosophila, flies homozygous Adh F, reared in the absence of alcohol were placed in contact with: a) an ethanol-free medium, b) a medium containing ethanol, c) a medium supplemented with 4-methylpyrazole (4-MP, an inhibitor of the ADH pathway), d) a medium containing ethanol and 4-MP. The choice of ethanol over a medium without ethanol was evaluated by measuring the duration of extension of the proboscis of the flies in each of the media. A slight preference for the ethanol-supplemented medium was observed in the naive flies, which was enhanced by previous exposure to ethanol. Exposure to ethanol and 4-MP, however, led to an avoidance of ethanol. There was a reduction in ADH activity on treatment of the flies with 4-MP, and signs of malaise (reduced locomotor activity, loss of balance) were observed in the flies who ingested both ethanol and inhibitor. We concluded that the preference for ethanol stems from an associative learning related to ethanol utilization. Inhibition of enzymes of ADH pathway led to a conditioned aversion due to disturbance of ethanol metabolism giving rise to malaise.

A METHOD FOR MAINTAINING CONSTANT ETHANOL CONCENTRATIONS IN CELL CULTURE MEDIA

The present study reports on the development of a model for maintaining constant ethanol concentrations over time in cell culture media. When neuroblastoma x glioma cells (NG 108-15) were grown in ethanol containing media under standard cultivation conditions in the incubator at 37 degrees C, a 90% evaporation was observed after 24 hr. To counteract evaporation, the cell culture dishes were placed inside polystyrene boxes together with an open dish containing an appropriate amount of ethanol. By using such procedure, the decrease in ethanol concentration in the culture media was completely avoided. Cultivating cells in ethanol-free media inside sealed plastic boxes did not change their viability, growth rate, protein and phospholipid composition of the cells or the pH of the media, compared to cultures grown outside the boxes.

Long-term ethanol-exposure markedly changes the cellular composition of cerebral glial cultures

The vulnerablity of morphologically distinct glial subpopulations to ethanol toxicity was surveyed in tissue culture. Secondary cultures of rat glia were examined at intervals during 56 days of ethanol treatment for changes in growth and cellular characteristics. Beginning at 6 days in vitro (DIV), the experimental cultures were treated with either 0.2% or 0.5% (w/v) ethanol in the medium; control cultures received ethanol-free medium. Relative to control cultures, the ethanol-treated cultures exhibited a consistent and dose-dependent suppression in cell number. The development of these cultures was documented with sequential phase-contrast photomicrography. Prior to treatment day 5 (11 DIV), the preponderance of cells were epithelioid in configuration; the astrocytic character of these cells was verified by the immunocytochemical localization of glial fibrillary acidic protein. In control cultures, a subpopulation of process-bearing cells was acquired gradually during the first 3 weeks in culture. The majority of these process-bearing cells were considered to be oligodendrocytes due to their position above the astrocytic carpet and by the immunocytochemical localization of galactocerebroside. Exposure to 0.5% ethanol markedly suppressed the acquisition of process-bearing cells. This ethanol-related suppression of process-bearing cells was apparent in the photomicrographic records of culture development and was confirmed by differential cell counts after 50 days of treatment. These results suggest a possible differential sensitivity of oligodendrocytes or their precursors to ethanol toxicity at elevated (0.5% w/v) concentrations.

HERITABLE VARIATION IN ETHANOL TOLERANCE AND ITS ASSOCIATION WITH BIOCHEMICAL TRAITS IN DROSOPHILA MELANOGASTER.

To help elucidate mechanisms of larval ethanol tolerance seven isochromosomal lines of Drosophila melanogaster with different second chromosomes were fed a growth-limiting concentration of ethanol (4.5% v/v) and examined for associations between growth traits and biochemical characteristics that had previously been implicated in the determination of tolerance variation. Repeated measures of survival and development time over four generations verified the inherited nature of these traits. Significant variation among the lines were evident for flux from ethanol into lipid, for activity levels of alcohol dehydrogenase and glycerol-3-phosphate oxidase (GPO), and for levels of long chain and unsaturated fatty acids. A high degree of positive association occurred among the variables. A partial correlation analysis controlling for performance of the lines on ethanol-free medium revealed a strong association between the degree of long chain fatty acid content and line survival when ethanol was fed. The correlation between GPO activity and survival in an ethanol environment appeared to depend on the association of GPO activity with long chain fatty acid content. The positive correlations of flux from ethanol into lipid with many of the other variables suggested that the ADH pathway influenced the level of ethanol tolerance. These associations are all consistent with the hypothesis that the lipid content of body tissues, especially the levels of long chain and unsaturated fatty acids in cell membranes, may have an important influence on both spatial and interspecific variation in the ethanol tolerance of larvae.

Opposing Effects of Ethanol on Pig Ovarian Adenylyl Cyclase Desensitized by Human Choriogonadotropin or Isoproterenol*

Pig ovarian follicular membranes contain a gonadotropin-responsive adenylyl cyclase, which becomes partially desensitized (approximately 40%) upon a 40-min incubation with a saturating concentration of human (h) CG. This in vitro desensitization is time and hormone dependent and also requires the presence of micromolar concentrations of GTP. In this report we show that 10% ethanol present during the desensitization phase of the incubation increases the extent of hCG-induced desensitization of adenylyl cyclase by 2-fold. Ethanol shortened the time necessary to reach maximal hCG-induced desensitization from 20 to 10 min, but had no effect on the dose dependency for GTP. In addition, ethanol had no effect on the affinity of the LH/hCG receptor for 125I-hCG but did cause an increase in the ED50 of hCG for inducing desensitization from 0.25 to 0.75 nM. Interestingly, ethanol decreased the apparent number of LH/hCG-receptor sites by 55%, yet the control hCG-sensitive adenylyl cyclase activity was not reduced. The "hyperdesensitized" state achieved in the presence of ethanol could not be reversed by washing the membranes and incubating them in ethanol-free medium. NaF-sensitive adenylyl cyclase was also not impaired in hCG-desensitized membranes from control or ethanol-treated samples. Thus, hCG-induced desensitization was not due to a defect in the functioning of the stimulatory guanine nucleotide-binding regulatory protein (G8) or catalytic subunits, but rather was caused by an impairment of the coupling of the lutropin (LH)/hCG receptor with G8, which was exacerbated further by ethanol. In spite of the effect of ethanol on hCG-induced desensitization, this agent had an inhibitory effect on isoproterenol-induced desensitization of isoproterenol-responsive luteal adenylyl cyclase. These results indicate that membrane fluidity is important in modulating the structure and functional interaction of the LH/hCG receptor with G8 because ethanol is a well known lipid-fluidizing agent. The resistance to ethanol potentiation of desensitization of the isoproterenol-sensitive adenylyl cyclase suggests that there are differences between the LH/hCG- and beta-adrenergic receptors in factors controlling their structures and or interactions with G proteins, and that there is a fundamental difference in their mechanisms of desensitization.

Tolerance to ethanol in the rat vas deferens. Effect of a calcium channel antagonist

Previous research from this laboratory indicated that ethanol dose-dependently depressed vas deferens contractions. The experiments described here examine the role calcium plays in the inhibitory action of ethanol in this tissue. Rat vas deferens tissues obtained from control (dextrin maltose) or chronically ethanol-treated animals were stimulated in the absence and then in the presence of 181 mM ethanol and/or a calcium channel blocker. Increasing the extracellular calcium concentration from 2.1 to 5.0 mM decreased ethanol's in vitro inhibitory effect on contractions induced by norepinephrine, KCl or electrical stimulation. Following chronic in vivo ethanol administration, increasing the calcium concentration to 5 mM blocked this inhibition. Nifedipine, a calcium channel antagonist inhibited vas deferens contractions. In the presence of nifedipine, in vitro ethanol further depressed vas deferens contractions stimulated by norepinephrine, K + and by electrical stimulation. In vivo ethanol treatment attenuated ethanol's inhibition in vitro, and reduced the blocking effect of the calcium antagonist on mechanical responses of the vas in ethanol-free medium. These data suggest that changes in calcium mobilization are involved in both the acute action of ethanol and the development of tolerance.

Ethanol inhibits human and hamster sperm penetration of eggs.

The effect of alcohol on the fertilizing ability of both human and hamster spermatozoa was examined by an in vitro fertilization assay using hamster ova. Spermatozoa were incubated in capacitating media for 3 hr (hamster sperm) and 4 hr (human sperm). Hamster ova were inseminated with preincubated sperm and were examined after 2 to 3 hr. Ethanol was added to the capacitating media at concentrations of 25, 50, 100, 200, and 400 mg%. Fertilization of zona-free hamster eggs by human spermatozoa was reduced from 49.6% in no alcohol to 16.7% in 400 mg% ethanol. Fertilization of hamster eggs by hamster sperm revealed a reduction from 63.6% to 33.7% in cumulus-intact eggs and from 65.8% to 10.8% in cumulus-free eggs in the presence of ethanol at 400 mg%. Hamster sperm acrosome reaction was reduced from 47% to 12%. When these hamster sperm with reduced acrosome reaction were placed with zona-free hamster eggs, the 100% fertilization rate was not reduced; however, the fertilization index, which reflects the number of swelling sperm heads per egg, was reduced from 8.5 to 1.8. This suggests that as little as 12% of the sperm with an acrosome reaction is sufficient to fertilize 100% of the zona-free eggs. If ethanol was added to the insemination media only, there was no inhibition of fertilization by human sperm or hamster sperm that had been previously capacitated in an ethanol-free media. Removal of the ethanol from the preincubated sperm produced fertilization at control levels; thus the inhibitory effect is reversible. These results indicate that ethanol may affect fertilization by an inhibition of the capacitation and/or acrosome reaction process.

Exposure to ethanol during capacitation impairs the fertilizing ability of human spermatozoa in vitro

To validate earlier findings, mainly in laboratory animals, the effect of ethanol on the fertilizing ability of human spermatozoa has been investigated. Ethanol added to the capacitation medium reduced the penetration of zona-free hamster eggs by human spermatozoa in a dose-dependent manner at concentrations from 50 to 500 mg % (0.05-0.5%). Fertilizing capacity was at least partially restored by washing in ethanol-free medium. Ethanol exposure before capacitation had a slight stimulatory effect on the penetration of spermatozoa into zona-free hamster ova. The motility of spermatozoa was not altered significantly, either quantitatively or qualitatively, by the presence of ethanol in the capacitation medium. These results suggest that the decrease in fertilizing ability of spermatozoa induced by ethanol during capacitation is due to a specific action on the capacitation process.

Effect of chronic ethanol exposure on the electric membrane properties of DRG neurons in cell culture.

Neural cell cultures of adult mouse dorsal root ganglia were utilized to investigate the effects of chronic ethanol exposure on neuronal electric membrane properties (EMP). After 12 days of exposure to various ethanol concentrations, the EMP of the neurons were determined in ethanol-free medium. Significant changes in a number of EMP were observed. Of particular physiological significance were decreased specific membrane resistance, increased specific membrane capacitance, relatively little change in membrane time constant, and increased electrical excitability. Various features of the action potential were also affected, e.g., reduced overshoot, afterhyperpolarization, and rate of rise. In preliminary experiments, EMP were determined at varying periods after the cultures had been withdrawn from ethanol medium and maintained in ethanol-free medium. These results indicated that the altered EMP persisted as long as one (Cm) to two (Rm) weeks after ethanol withdrawal. A possible mechanism for these ethanol-induced changes in EMP was suggested, utilizing the membrane expansion theory of anesthesia. Because of few previous reports demonstrating significant electrophysiological effects of ethanol at pharmacological concentrations, the neural cell culture system provides a useful new experimental model for studying the action of chronic ethanol exposure on neuronal EMP and the physical basis of the tolerance and withdrawal phenomena found in alcoholism and addiction in general. After being maintained for 12 days in culture media containing various concentrations of ethanol, non-neuronal cell survival was observed to have decreased in an approximately linear manner with increasing ethanol levels. By contrast, neuron survival was not affected until ethanol concentrations greater than 0.34 g % were used. This decreased cell survival due to chronic exposure to physiological levels of ethanol has not been reported previously. Neural cell cultures may therefore be useful for investigating the cellular pathology of chronic alcoholism and fetal alcohol syndrome.

Probable indentity of substances in citrus that repress asexual embryogenesis.

The embryogenetic responseof culturedDaucus carota L. ‘Queen Anne's Lace’ callus was employed to attempt fractionation and identification of a repressive factor produced byCitrus medica L. ovules. The factor was evidently synthesized and released into the medium continuously, inasmuch as citron ovules that had been autoclaved with the medium were completely infeffective. The inhibition could be attributed to volatile and nonvolatile components. A substantial part of the inhibition was prevented by continuously refereshing the atmosphere within the cultures with filtered air. Monitoring of the gases produced by citron ovule sections under conditions simulating bioassays disclosed significant evolution of carbon dioxide, ethylene and ethanol. Repression of embryogenesis was not averted by trapping the liberated ethylene. On the other hand, ethanol in concentrations equivalent to those released by citron ovules suppressed asexual embryogenesis dramatically. The adverse effect of ethanol was reversed immeditaley upon transfer to ethanol-free medium. Another investigation had disclosed anti-embryogenetic effects of auxin, abscisic acid and gibberellin. Analysis ofCitrus ovules excised from young fuits disclosed those of monoembryonic citron to contain concentrations of IAA, ABA and GA3 several times higher than those of polyembryonic Ponkan mandrain. The nonvolatile protion might be identified with these hormonal substances.