Characterization of two cDNAs for novel drought-inducible genes in the highly drought-tolerant cowpea

Ten cDNAs for drought-inducible genes were isolated using differential screening of a cDNA library prepared from 10-hr dehydrated cowpea plants,Vigna unguiculata (S. Iuchi, K. Yamaguchi-Shinozaki, T. Urao, T. Terao, K. Shinozaki; Plant Cell Physiology, 1996 in press). Two of the cDNA clones, designated CPRD12 and CPRD46, were sequenced and characterized. The CPRD12 and CPRD46 cDNAs encode putative proteins related to nonmetallo-short-chain alcohol dehydrogenase (CPRD12) and chloroplastic lipoxygenase (CPRD46). Northern blot analysis revealed that these genes are induced by high-salinity stress and exogenous abscisic acid, but not by cold stress. The CPRD46 gene is also responsive to heat stress and methyl jasmonate and salicylic acid. Genomic Southern blot analysis suggested that CPRD12 constitutes a small gene family, but that CPRD46 is a single copy gene. We discuss the possible functions of these two CPRD gene products under drought stress.     

Production of fuel alcohol from oats by fermentation

Very high gravity (>30 g dissolved solids per 100 ml) mashes were prepared from hulled and hulless oats and fermented at 20° C with active dry yeast to produce ethanol. Excessive viscosity development during mashing was prevented by hydrolyzing -glucan with crude preparations of -glucanase or Biocellulase. Both these preparations possessed endo--glucanase activity. By using these enzymes and by decreasing the water to grain ratio, very high gravity mashes with low viscosity were prepared. Unlike wheat and barley mashes, oat mashes contained sufficient amounts of assimilable nitrogen to promote a fast rate of fermentation. The free amino nitrogen (FAN) content of oat mash could be predicted by the equation, mg FAN L^–1=8.9n wheren is the number of grams of dissolved solids in 100 ml of mash supernatant fluid. Ethanol yields of 353.2±3.7 L and 317.6±1.3 L were obtained per tonne (dry weight basis) of hulless (59.8% starch) and hulled (50.8% starch) oats respectively. The efficiency of conversion of starch to ethanol was the same in normal and very high gravity mashes.     

Perturbation of gene frequencies in a natural population of Drosophila melanogaster: evidence for selection at the Adh locus

The cellar population of Drosophila melanogaster at the Chateau Tahbilk Winery (Victoria, Australia) was perturbed for alcohol dehydrogenase (Adh) gene frequencies. Phenol oxidase (Phox) frequencies were also perturbed and monitored as a control. Subsequent gene frequency changes, together with information on population structure, indicated that selection acted on the chromosome regions of both loci. Adh gene frequencies returned to preperturbation levels in a predictable manner. A model in which the relative fitness of Adh phenotypes was determined by temperature-dependent specific activities of enzymes of Adh genotypes adequately accounts for the rate of gene frequency change at this locus. Thus temperature behaves as a selective agent in modulating Adh gene frequencies in this cellar environment.     

Sequence of the gene for a NAD(P)-dependent formaldehyde dehydrogenase (class III alcohol dehydrogenase) from a marine methanotroph Methylobacter marinus A45

Abstract A fragment of Methylobacter marinus A45 DNA has been cloned and sequenced, and an open reading frame has been identified that could code for a 46-kDa polypeptide. Comparison of the deduced amino acid sequence of the polypeptide against the protein data bank has revealed strong similarity with a number of alcohol dehydrogenases, with highest similarity towards class III alcohol dehydrogenases, which recently have been shown to be identical to glutathione-dependent formaldehyde dehydrogenases. We were unable to measure appreciable levels of NAD(P)-dependent formaldehyde dehydrogenases or alcohol dehydrogenase activities using aldehydes or primary or secondary alcohols in cell-free extracts from batch cultures of M. marinus A45. However, formaldehyde dehydrogenases activity was detected on zymograms. Our data suggest that, although NAD(P)-linked formaldehyde dehydrogenase or alcohol dehydrogenase activities are undetectable in cell-free extracts of most methylotrophs employing the ribulose monophosphate pathway for formaldehyde assimilation and dissimilation, the gene encoding formaldehyde dehydrogenase is present in M. marinus A45 and may be present in more of these organisms as well.     

Osmium tetroxide footprinting of a scaffold attachment region in the maizeAdh1 promoter

Osmium tetroxide (OsO₄) reacts with the thymine residues of double-stranded DNA, but thymines that are unpaired or under torsional stress are hyperreactive. Although OsO₄ hyperreactivity has been primarily utilized to identify Z-DNA structures in supercoiled plasmids, OsO₄ will also identify other torsional perturbations of DNA. In this study, OsO₄ was used to footprint an AT-rich region (between –780 and –500) of the maizeAdh1 promoter. Hyperreactive sites were identified bothin vitro andin vivo in an area that coincides with AT motifs similar to those found in scaffold attachment regions. Further, the region of OsO₄ hyperreactivity lies within a fragment of DNA that is associated with the nuclear scaffold in histone-depleted nuclei.     

Evolutionary genetics of the Drosophila alcohol dehydrogenase gene-enzyme system

Evolutionary genetics embodies a broad research area that ranges from the DNA level to studies of genetic aspects in populations. In all cases the purpose is to determine the impact of genetic variation on evolutionary change. The broad range of evolutionary genetics requires the involvement of a diverse group of researchers: molecular biologists, (population) geneticists, biochemists, physiologists, ecologists, ethologists and theorists, each of which has its own insights and interests. For example, biochemists are often not concerned with the physiological function of a protein (with respect to pH, substrates, temperature, etc.), while ecologists, in turn, are often not interested in the biochemical-physiological aspects underlying the traits they study. This review deals with several evolutionary aspects of the Drosophila alcohol dehydrogenase gene-enzyme system, and includes my own personal viewpoints. I have tried to condense and integrate the current knowledge in this field as it has developed since the comprehensive review by van Delden (1982). Details on specific issues may be gained from Sofer and Martin (1987), Sullivan, Atkinson and Starmer (1990); Chambers (1988, 1991); Geer, Miller and Heinstra (1991); and Winberg and McKinley-McKee (1992).Dedicated to Professor Billy W. Geer, because of his contributions to knowledge of the biochemical genetics of Drosophila.     

Second messenger and protein phosphorylation mechanisms underlying opiate addiction: Studies in the rat locus coeruleus

We have studied the role of second messenger and protein phosphorylation pathways in mediating changes in neuronal function associated with opiate addiction in the rat locus coeruleus. We have found that chronic opiates increase levels of the G-protein subunits Gi and Go, adenylate cyclase, cyclic AMP-dependent protein kinase, and a number of phosphoproteins (including tyrosine hydroxylase) in this brain region. Electrophysiological data have provided direct support for the view that this up-regulation of the cyclic AMP system contributes to opiate tolerance, dependence, and withdrawal exhibited by these neurons. As the adaptations in G-proteins and the cyclic AMP system appear to occur at least in part at the level of gene expression, current efforts are aimed at identifying the mechanisms, at the molecular level, by which opiates regulate the expression of these intracellular messenger proteins in the locus coeruleus. These studies will lead to an improved understanding of the biochemical basis of opiate addiction.Special issue dedicated to Dr. Paul Greengard     

Direct resolution of enantiomers of basic imidazol-2-yl-substituted alcohols by gas chromatography on chirasil-val

The direct resolution of enantiomers of a series of imidazol-2-yl-substituted alcohols has been achieved by gas chromatography on a well-deactivated fused-silica capillary column, coated with L -Chirasil-Val as the chiral stationary phase. The separation of these basic compounds is accomplished without exaggerated peak tailing. Compared to simpler alcohols the resolution factors (α) observed are extraordinarily large. While the imidazolyl substituent may contribute to the mechanism of the chiral discrimination, the crucial interaction is assumed to be through the hydroxy group, based on the observation that the resolution factors for the corresponding O-acetyl derivatives are markedly reduced. © 1993 Wiley-Liss, Inc.     

Substance use disorders: a comprehensive update of classification,epidemiology, neurobiology,clinical aspects,treatment and prevention

Substance use disorders (SUDs) are highly prevalent and exact a large toll on individuals’ health, well-being, and social functioning. Long-lasting changes in brain networks involved in reward, executive function, stress reactivity, mood, and self-awareness underlie the intense drive to consume substances and the inability to control this urge in a person who suffers from addiction (moderate or severe SUD). Biological (including genetics and developmental life stages) and social (including adverse childhood experiences) determinants of health are recognized factors that contribute to vulnerability for or resilience against developing a SUD. Consequently, prevention strategies that target social risk factors can improve outcomes and, when deployed in childhood and adolescence, can decrease the risk for these disorders. SUDs are treatable, and evidence of clinically significant benefit exists for medications (in opioid, nicotine and alcohol use disorders), behavioral therapies (in all SUDs), and neuromodulation (in nicotine use disorder). Treatment of SUDs should be considered within the context of a Chronic Care Model, with the intensity of intervention adjusted to the severity of the disorder and with the concomitant treatment of comorbid psychiatric and physical conditions. Involvement of health care providers in detection and management of SUDs, including referral of severe cases to specialized care, offers sustainable models of care that can be further expanded with the use of telehealth. Despite advances in our understanding and management of SUDs, individuals with these conditions continue to be stigmatized and, in some countries, incarcerated, highlighting the need to dismantle policies that perpetuate their criminalization and instead develop policies to ensure support and access to prevention and treatment.     

Rapid changes in oxidative metabolism as a consequence of elicitor treatment of suspension-cultured cells of French bean (Phaseolus vulgaris L.)

Stressed plant cells often show increased oxygen uptake which can manifest itself in the transient production of active oxygen species, the oxidative burst. There is a lack of information on the redox status of cells during the early stages of biotic stress. In this paper we measure oxygen uptake and the levels of redox intermediates NAD/NADH and ATP and show the transient induction of the marker enzyme for redox stress, alcohol dehydrogenase. Rapid changes in the redox potential of elicitor-treated suspension cultures of French bean cells indicate that, paradoxically, during the period of maximum oxygen uptake the levels of ATP and the NADH/NAD ratio fall in a way that indicates the occurrence of stress in oxidative metabolism. This period coincides with the maximum production of active oxygen species particularly H₂O₂. The cells recover and start producing ATP immediately upon the cessation of H₂O₂ production. This indicates that the increased O₂ uptake is primarily incorporated into active O₂ species. A second consequence of these changes is probably a transient compromising of the respiratory status of the cells as indicated in expression of alcohol dehydrogenase. Elicitor-induced bean ADH was purified to homogeneity and the M_r 40 000 polypeptide was subjected to amino acid sequencing. 15% of the whole protein was sequenced from three peptides and was found to have nearly 100% sequence similarity to the amino acid sequence for pea ADH1 (PSADH1). The cDNA coding for the pea enzyme was used to demonstrate the transient induction of ADH mRNA in elicitor-treated bean cells. Enzyme activity levels also increased transiently subsequently. Increased oxygen uptake has previously been thought to be associated with provision of energy for the changes in biosynthesis that occur rapidly after perception of the stress signal. However the present work shows that this rapid increase in oxygen uptake as a consequence of elicitor action is not wholly associated with respiration.