Effects of ethanol and acetaldehyde on rat embryos developing in vitro

Summary Rat embryos were explanted on Days 9.5 or 10 of gestation and cultured for 48 to 30h, respectively, in rat serum containing 0, 3, 6, 9 mg/ml of Ethanol (Eth); 0, 10, 20 μg/ml of Acetaldehyde (Ach); 3 mg/ml Eth + 10 μg/ml Ach. At the end of the culture period the embryos were evaluated for vitality, and scored. Some of them were also examined histologically. Embryos exposed to Eth from Day 9.5 showed a dose-related growth retardation associated with a high frequency of malformations (open neural tube, heart defects, branchial arch hypoplasia). The exposure of 9.5-day embryos to 20 μg/ml Ach resulted in 100% embryolethality, whereas 10 μg/ml induced growth retardation and teratogenic effects. When 10-day embryos were exposed to 3 mg/ml Eth or 10 μl/ml Ach no effects were observed, but the highest levels of Eth produced a moderate growth retardation and morphologic defects. Exposure to 20 μg/ml Ach induced hypoplasia of the first arch, but did not alter the score value. The histologic examination of these embryos revealed severe lesions at the level of the neuroepithelium and of the branchial mesenchyma. Similar effects were observed in embryos exposed simultaneously to 3 mg/ml Eth and 10 μg/ml Ach. These results should make us reevaluate the role of Ach in the Eth-induced embryopathies.     

The role of acetaldehyde and glycerol in the adaptation to ethanol stress of Saccharomyces cerevisiae and other yeasts

Ethanol inhibition is a commonly encountered stress condition during typical yeast fermentations and often results in reduced fermentation rates and production yields. While past studies have shown that acetaldehyde addition has a significant ameliorating effect on the growth of ethanol-stressed Saccharomyces cerevisiae , this study investigated the potential ameliorating effect of acetaldehyde on a wide range of ethanol-stressed yeasts. Acetaldehyde does not appear to be a universal ameliorating agent for yeasts exposed to ethanol stress. It is also shown that as a result of an ethanol stress, most yeasts rapidly produce glycerol as an alternative means of NAD⁺ regeneration rather than having a specific requirement for glycerol. The results strongly suggest that both ethanol and acetaldehyde exposure have a direct effect on the cellular NAD⁺/NADH ratio, which can manifest itself as modulations in glycerol production.     

The major DNA polymerase in cultured plant cells: Partial purification and correlation with cell multiplication

A DNA polymerase activity was isolated from cells of Oryza sativa L. grown in suspension culture. Molecular mass ( 180,000), optimal requirements for pH (neutral), Mg²⁺ (5–10 mM), Mn²⁺ (1 mM), template preference (activated DNA), lack of activity with native or denatured DNA, and sensitivity to N-ethylmaleimide and ionic strength are similar to those of the vertebrate -polymerase. Like DNA polymerase , the DNA polymerase described in this work is the most abundant in proliferating cells of Oryza sativa L., Parthenocissus tricuspidata (Siebold et Zucc.) Planchon, Acer pseudoplatanus L., and Medicago sativa L. and responds to changes in the rate of cell multiplication. We therefore postulate that this -like DNA polymerase is the replicating enzyme of plant cells.Abbreviations BSA bovine serum albumin - EDTA ethylendiamino-tetracetic acid - DTT dithiothreitol - PTSF p-toluenesulfonyl fluoride     

Conductance states activated by glycine and GABA in rat cultured spinal neurones

Summary The conductance properties of single Cl^– channels activated by glycine and gamma-aminobutyric acid (GABA) were examined in rat spinal cord neurones grown in cell culture. The majority (85%) of spinal neurones were sensitive to both glycine and GABA as were most (83%) outside-out patches tested. Glycine and GABA activated multiple conductance state Cl^– channels with linear current-voltage properties when the chloride activities of the solutions bathing both sides of the membrane were similar. Glycine activated six distinct conductance states with conductances of 14, 20, 30, 43, 64 and 93 pS, whereas GABA activated five states with conductances of 13, 20, 29, 39 and 71 pS. The 30 and 43 pS states and the 20 and 29 pS states were observed most frequently with glycine and GABA, respectively. As the values of the glycine- and GABA-activated conductance states form a geometric progression when arranged in ascending order, we concluded that the channels do not consist of a cluster of identical pores. Additional conductance states (50 and 100 pS) were activated by glycine occasionally. The similarity between the conductances of the states activated by the two transmitters is consistent with the proposal that they both activate the same type of Cl^– channel.     

Liver collagen of rats submitted to chronic intoxication with acetaldehyde

It was found that chronic intoxication of rats with acetaldehyde results in a distinct, progressive increase of 5-³H-proline incorporation into collagen synthesized by liver. At the same time biosynthesis of other proline-containing (noncollagenous) proteins does not change significantly. On the other hand the collagen content in the rat liver did not increase in the early stage of acetaldehyde administration, but increased when acetaldehyde feeding was continued for 6 months. About 40% increase of total collagen content was found in livers of the intoxicated animals. All the investigated collagen types (I, III, IV and V) grew in the same degree. No changes in proportional relationships between collagens of different types were found.     

The use of acetaldehyde to control carnation flower longevity

Acetaldehyde is the causal agent of ethanol-induced longevity increases in carnation cut flowers. It increases the vase life of cut carnation flowers by at least 50%. The capacity of acetaldehyde to regulate carnation flower senescence was therefore investigated. Ethylene formation was reduced or inhibited as a result of acetaldehyde application. There was, however, no prevention of ethylene action. The morphological development of the ovary was also inhibited, thus eliminating the movement of metabolites from the petals. The potential use of acetaldehyde as a post-harvest treatment is however impractical, due to the inefficiency of pulse treatments and ineffectiveness in preventing the action of exogenous ethylene.     

Kinetics of ethanol and acetaldehyde release suggest a role for acetaldehyde production in tolerance of rice seedlings to micro-aerobic conditions

BACKGROUND AND AIMS: This paper examines the basis of the greater tolerance of an indica rice cultivar FR13A to complete submergence compared with relatively intolerant japonica rice CT6241. We study whether this superior tolerance is related to its greater tolerance to O2 shortage and to an ability to run a more favourable rate of alcoholic fermentation during and after O2 deprivation. METHODS Fermentation products were analysed using sensitive laser-based photoacoustics at high time resolution to establish patterns and rates of ethanol and acetaldehyde emission by intact rice seedlings exposed to micro-aerobic (0.05-0.5 % O2) or zero O2 supply, and also during their return to air. Oxygen and CO2 emission or uptake was also quantified. KEY RESULTS: In the dark, no acetaldehyde and ethanol emission was observed until external O2 concentration in a gas phase decreased to 相似文献   

Effects of ethanol on rat schwann cell proliferation and myelination in culture

Summary It is possible to treat dissociated embryonic rat dorsal root ganglia in culture to inhibit proliferation of all nonneuronal cells except Schwann cells. Neurons have been shown to produce a mitogenic stimulus for Schwann cells under these conditions. Additionally, myelin-competent neurons induce Schwann cells to elaborate myelin sheaths. Groups of sibling cultures were exposed to various nonlethal concentrations of ethanol (0, 43, 86, or 172 mM) for 4 wk. Culture were assessed weekly by light microscopy in a blind fashion for evidence of Schwann cell proliferation and myelin formation. Ethanol adversely affected both Schwann cell proliferation and myelin formation in culture. No obvious differences in neuronal morphology were observed among the various groups of cultures by light or electron microscopy. These observations suggest that ethanol might interfere with Schwann cell proliferation and myelin formation in culture by one or both of the following means: a) inhibit neuronal production of signals for Schwann cell proliferation and myelination or b) impede Schwann cell responses to neuronal signals. Investigation of these possibilities in culture may provide insight into neuropathologic mechanisms operative in the fetal alcohol syndrome or alcohol-associated peripheral neuropathy in humans. This work was supported by the Department of Veterans Affairs, Washington, D.C.     

Isolation and characterization of aDatura innoxia cell line resistant to ethanol

A cell line ofDatura innoxia was selected in suspension culture to be resistant to 1% (vol/vol) ethanol (EtOH^R). EtOH^R cells were cross-resistant to 1% (vol/vol) methanol and 1% (vol/vol) 2-propanol but not 1% (vol/vol)n-propanol orn-butanol, whereas wild type (WT) cells were resistant only to methanol. Resistance in EtOH^R cells is probably a result of a very low level of alcohol dehydrogenase (ADH) activity which was only 9 to 10% of that in WT cells and was undetectable during much of the EtOH^R growth cycle. In the absence of ethanol, EtOH^R cells have a I₅₀ for the toxic ethanol analog allyl alcohol, which is nearly 3 times higher than that in WT cells. In the presence of ethanol, EtOH^R cells have an I₅₀ for allyl alcohol which is 12 times more than WT cells. This difference correlated well with the decrease in ADH activity found in EtOH^R cells grown on ethanol. When ethanol was removed from the suspension medium, ADH activity in EtOH^R cells gradually increased to WT levels. When re-exposed to ethanol after 200 cell generations, ADH activity quickly decreased and growth resumed after a 4- to 6-day lag period. Lipid analysis showed a 37% increase in total lipid in EtOH^R cells, mostly in polar lipids, di- and triglycerides. The fatty acid composition of these lipid classes was shifted toward the more polyunsaturated. These lipid changes were probably a reflection of the increased plastid number in the EtOH^R cells and may be a result of growth in ethanol rather than a reason for resistance. EtOH^R cells seem to be regulatory mutants able to quickly lower ADH activity in the presence of ethanol.     

DNA damaging effects of carbofuran and its main metabolites on mice by micronucleus test and single cell gel electrophoresis

The DNA damaging effects of the carbamate pesticide carbofuran and its four metabolites (carbofuranphenol, 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran) on mice were evaluated by single cell gel electrophoresis (SCGE) assay and micronucleus test. KM mice were exposed to test compounds with different doses of 0.1, 0.2 and 0.4mg/kg through in-traperitoneal injection two times with an internal of 24 h, and then killed by cervical dislocation 6 h after the second injection. In SCGE assay, isolated mice peripheral blood lymphocytes were employed to determine DNA damaging degree after a 1 h treatment by test compounds and a following electrophoresis. Carbofuran and carbofuranphenol showed negative results in both test and had no obvious toxicity. 3-hydrocarbofuran and nitrosocarbofuran were positive. 3-ketocarbofuran could not induce micronucleus formation but caused significant DNA migration in SCGE test. These tests revealed that 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran are pote     

DNA damaging effects of carbofuran and its main metabolites on mice by micronucleus test and single cell gel electrophoresis

The DNA damaging effects of the carbamate pesticide carbofuran and its four metabolites (carbofuranphenol, 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran) on mice were evaluated by single cell gel electrophoresis (SCGE) assay and micronucleus test. KM mice were exposed to test compounds with different doses of 0.1, 0.2 and 0.4mg/kg through intraperitoneal injection two times with an internal of 24 h, and then killed by cervical dislocation 6 h after the second injection. In SCGE assay, isolated mice peripheral blood lymphocytes were employed to determine DNA damaging degree after a 1 h treatment by test compounds and a following electrophoresis. Carbofuran and carbofuranphenol showed negative results in both test and had no obvious toxicity. 3-hydrocarbofuran and nitrosocarbofuran were positive.3-ketocarbofuran could not induce micronucleus formation but caused significant DNA migration in SCGE test. These tests revealed that 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran are potential mutagesis and further research is needed.     

Production of ethanol by cultured insect cells

Summary Proton Nuclear Magnetic Resonance (¹H-NMR) Analysis of insect cell culture media used for cultivating insect cell lines derived from the fleshflySarcophaga peregrina, swallowtail butterflyPapilio xuthus, and cabbage armywormMamestra brassicae revealed that ethanol appeared in the medium as the cultures aged. By incorporating [¹³C-1]-glucose into the media, we pursued¹³C-NMR spectrograms to show that the ethanol was derived from glucose. Thus, it became evident that the insect cells culturedin vitro produce ethanol from glucose as a metabolite.     

Ethylene, ethanol, acetaldehyde and carbon dioxide released by Prunus avium shoot cultures

Wild cherry ( Prunus avium L.) shoots were cultured in closed vessels on a proliferation medium and the volatile substances released during incubation at photosynthetic photon flux density of 30 μmol m^–2 S^–1 were determined. Ethylene and CO₂ started forming at the beginning of the incubation period and a linear relationship between their formation was observed even at high CO₂ concentrations. After 30 days of culture, CO₂ reached a concentration of 30%. Shoots released elhanol and acetaldehyde after several days of incubation.     

Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast

Acetaldehyde, a primary metabolite of alcohol, forms DNA adducts and disrupts the DNA replication process, causing genomic instability, a hallmark of cancer. Indeed, chronic alcohol consumption accounts for approximately 3.6% of all cancers worldwide. However, how the adducts are prevented and repaired after acetaldehyde exposure is not well understood. In this report, we used the fission yeast Schizosaccharomyces pombe as a model organism to comprehensively understand the genetic controls of DNA damage avoidance in response to acetaldehyde. We demonstrate that Atd1 functions as a major acetaldehyde detoxification enzyme that prevents accumulation of Rad52-DNA repair foci, while Atd2 and Atd3 have minor roles in acetaldehyde detoxification. We found that acetaldehyde causes DNA damage at the replication fork and activates the cell cycle checkpoint to coordinate cell cycle arrest with DNA repair. Our investigation suggests that acetaldehyde-mediated DNA adducts include interstrand-crosslinks and DNA-protein crosslinks. We also demonstrate that acetaldehyde activates multiple DNA repair pathways. Nucleotide excision repair and homologous recombination, which are both epistatically linked to the Fanconi anemia pathway, have major roles in acetaldehyde tolerance, while base excision repair and translesion synthesis also contribute to the prevention of acetaldehyde-dependent genomic instability. We also show the involvement of Wss1-related metalloproteases, Wss1 and Wss2, in acetaldehyde tolerance. These results indicate that acetaldehyde causes cellular stresses that require cells to coordinate multiple cellular processes in order to prevent genomic instability. Considering that acetaldehyde is a human carcinogen, our genetic studies serve as a guiding investigation into the mechanisms of acetaldehyde-dependent genomic instability and carcinogenesis.     

Glutamate-mediated overexpression of CD38 in astrocytes cultured with neurones

Recently, a new system of astrocyte-neurone glutamatergic signalling has been identified. It is started in astrocytes by ectocellular, CD38-catalysed conversion of NAD(+) to the calcium mobilizer cyclic ADP-ribose (cADPR). This is then pumped by CD38 itself into the cytosol where the resulting free intracellular Ca(2+) concentration [Ca(2+)](i) transients elicit an increased release of glutamate, which can induce an enhanced Ca(2+) response in neighbouring neurones. Here, we demonstrate that co-culture of either cortical or hippocampal astrocytes with neurones results in a significant overexpression of astrocyte CD38 both on the plasma membrane and intracellularly. The causal role of neurone-released glutamate in inducing overexpression of astrocyte CD38 is demonstrated by two observations: first, in the absence of neurones, induction of CD38 in pure astrocyte cultures can be obtained with glutamate and second, it can be prevented in co-cultures by glutamate receptor antagonists. The neuronal glutamate-mediated effect of neurones on astrocyte CD38 expression is paralleled by increased intracellular cADPR and [Ca(2+)](i) levels, both findings indicating functionality of overexpressed CD38. These results reveal a new neurone-to-astrocyte glutamatergic signalling based on the CD38/cADPR system, which affects the [Ca(2+)](i) in both cell types, adding further complexity to the bi-directional patterns of communication between astrocytes and neurones.     

Detection of mycoplasmas infecting cell cultures by DNA hybridization

Summary Infection of cell cultures by mycoplasmas can be detected and the mycoplasma identified by Southern blot hybridization of theEco RI-digested DNA of the suspected cell cultures with a nick-translated probe consisting of cloned ribosomal RNA genes ofMycoplasma capricolum. The probe does not hybridize with eukaryotic DNA. The hybridization pattern with mycoplasmal DNA is species specific, enabling the identification of the four most prevalent mycoplasma contaminants,Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma arginini, andAcholeplasma laidlawii. The test is also very sensitive and can detect as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10⁵ mycoplasmas. The study was supported by the U.S. Public Health Service Grant GM25286 awarded to G. G. and by a grant from the United States-Israel Binational Agricultural Research Development Fund (BARD) awarded to S. R.     

Acceleration of high gravity yeast fermentations by acetaldehyde addition

In high gravity Saccharomyces cerevisiae fermentations containing 300 g glucose l^–1, daily addition of acetaldehyde to a total of 93 mM shortened the time required to ferment the first 250 g glucose l^–1 from 790 h to 585 h. Acetaldehyde feeding had no effect on the ethanol yield but increased by 135%, 78% and 77% the final concentrations of 2,3-butanediol, 2-methylpropanol and acetate, while decreasing that of glycerol by 14%. Controlled acetaldehyde feeding has potential as a technique for accelerating high gravity fuel or industrial ethanol fermentations and may be useful in preventing incomplete fermentations.     

Identification and authentication of animal cell culture by polymerase chain reaction amplification and DNA sequencing

Polymerase chain reaction (PCR) amplification and deoxyribonucleic acid (DNA) sequence analysis were used to identify the species origin of cell lines used in a cell culture facility where various cell lines of different species are routinely propagated. The aldolase gene family was selected for PCR amplification because the DNA sequences of this gene are highly conserved over a wide range of animals and humans. A total of 36 cell lines representing 13 different species were selected for this study. The DNA from each cell line was amplified, and PCR products were analyzed by agarose gel electrophoresis. The results showed unique profiles of amplified bands on agarose gels that allowed differentiation among non-closely related species. However, DNA amplification of closely related species, including rat and mouse or human and primate, resulted in similar and indistinguishable banding patterns that could be further differentiated by DNA sequence analysis. These results suggested that aldolase gene amplification coupled with DNA sequence analysis is a useful tool for identification of cell lines and has potential application for use in identification of interspecies cross-contamination.     

Fluorometric determination of DNA in mammalian cell cultures by PicoGreen

An intercalating fluorochrome, PicoGreen, was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 91.8% of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples.