Alcohol Dehydrogenase Protein Research Articles

Cinnamyl Alcohol Dehydrogenase Gene Regulates Bursaphelenchus xylophilus Reproduction and Development

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is a globally distributed destructive disease of pine forest. To study the PWD pathogenic mechanism, the cinnamyl alcohol dehydrogenase gene (BxCAD-1) from B. xylophilus was selected. The BxCAD-1 gene was amplified by PCR from the cDNA library of B. xylophilus and cloned into the expression vector pET-15b to construct the recombinant vector pET-15b-BxCAD-1. The recombinant cinnamyl alcohol dehydrogenase protein was purified by Ni-NTA affinity chromatography from Escherichia coli BL21 (DE3) harboring pET-15b-BxCAD-1 induced by IPTG. The effects of pH, temperature, metal ions and substrates on the activity of BxCAD-1 were determined, showing the highest catalytic activity at pH 8.0 and 40 °C with cinnamyl alcohol as substrate and Zn2+ as an activator. To elucidate the functions of BxCAD-1 in B. xylophilus, the expression of the gene was down-regulated by RNA interference. Results showed that the movement, feeding, reproduction, spawning rate, hatching rate, lifespan, infectivity and sensitivity to ethanol decreased compared with negative controls. RNA interference also affected the development of B. xylophilus from the larval stage to the adult stage. In situ hybridization showed that the gene was expressed in the digestive tract of male and female adults. This study revealed a promising target for PWD control.

Changes in the proteomic and polyamine profile induced by seed aging of <i>Cariniana legalis</i> (Martius) O. Kuntze

Studies on metabolic processes and seed germinability of native endangered species are essential for its conservation. The objective of this work was to evaluate the effect of accelerated aging on germination and on the proteomic and polyamine (PAs) profile in seeds of Cariniana legalis. The seeds were aged at 41, 43, 45 and 47ºC for 24, 48, 72 and 96 h, under 100 % relative humidity using a completely randomized factorial design. The data were submitted to analysis of variance followed by Tukey’s test. The temperature of 47ºC was efficient in simulating seed aging, with no germination after 72 h of incubation. At 41ºC, the germination did not change significantly until 96 h. The reduction in the concentration of total free PAs, mainly spermidine, and the down-accumulation of heat shock proteins in seeds aged at 47°C were associated with induction of seed aging. Furthermore, the down-accumulation of the aconitate hydratase protein and the up-accumulation of the alcohol dehydrogenase protein were associated with loss of seed germinability and rapid deterioration in seeds aged at 47°C. The aging of C. legalis seeds is related to alteration in the contents of PAs and accumulation of some proteins involved with the loss of germinability.

The pathogenic biomarker alcohol dehydrogenase protein is involved in Bacillus cereus virulence and survival against host innate defence.

Bacillus cereus is a spore forming bacteria recognized among the leading agents responsible for foodborne outbreaks in Europe. B. cereus is also gaining notoriety as an opportunistic human pathogen inducing local and systemic infections. The real incidence of such infection is likely underestimated and information on genetic and phenotypic characteristics of the incriminated strains is generally scarce. We have recently analyzed a large strain collection of varying pathogenic potential. Screening for biomarkers to differentiate among clinical and non-clinical strains, a gene encoding an alcohol dehydrogenase-like protein was identified among the leading candidates. This family of proteins has been demonstrated to be involved in the virulence of several bacterial species. The relevant gene was knocked out to elucidate its function with regards to resistance to host innate immune response, both in vitro and in vivo. Our results demonstrate that the adhB gene plays a significant role in resistance to nitric oxide and oxidative stress in vitro, as well as its pathogenic ability with regards to in vivo toxicity. These properties may explain the pathogenic potential of strains carrying this newly identified virulence factor.

Physiological and proteomic insights related to the loss of seed viability in Cariniana legalis (Lecythidaceae)

In seed conservation, proper storage conditions are crucial to prolonging viability, especially for seeds of endangered woody species, such as Cariniana legalis. We evaluated the influence of the storage temperature and time on the viability of C. legalis seeds and performed physiological and proteomic analyses. Seeds were stored at 6 and 25 °C for 24 months. At 4-month intervals, the germination (%), seedling growth, moisture content and electrical conductivity were evaluated. The proteomic profile was evaluated in seeds before and after 8 months of storage. The viability of the seeds was maintained for a longer time at 6 °C than at 25 °C. At 25 °C, seeds showed no germination at 8 months, which apparently was related to a higher seed moisture content and changes in accumulation of the proteins alcohol dehydrogenase, proteasome subunit alpha type and triosephosphate isomerase. Moreover, the absence of the aconitate hydratase 3 protein in seeds at 8 months of storage at both temperatures was related to the reduction of seed viability when stored at 6 °C and lack of germination of seeds stored at 25 °C, which can be associated with reduced mobilization of seed-stored reserves. In addition, the time spent in seed storage at both temperatures induced a reduction in the abundance of phosphoglycerate kinase and heat shock proteins, both which might be required for better seed longevity. Our results show that germination is negatively affected by the temperature and time of storage, which both affect the protein profile of C. legalis, an endangered species from the Brazilian Atlantic rainforest.

Evolutionary coupling-inspired engineering of alcohol dehydrogenase reveals the influence of distant sites on its catalytic efficiency for stereospecific synthesis of chiral alcohols.

Alcohol dehydrogenase (ADH) has attracted much attention due to its ability to catalyze the synthesis of important chiral alcohol pharmaceutical intermediates with high stereoselectivity. ADH protein engineering efforts have generally focused on reshaping the substrate-binding pocket. However, distant sites outside the pocket may also affect its activity, although the underlying molecular mechanism remains unclear. The current study aimed to apply evolutionary coupling-inspired engineering to the ADH CpRCR and to identify potential mutation sites. Through conservative analysis, phylogenic analysis and residues distribution analysis, the co-evolution hotspots Leu34 and Leu137 were confirmed to be highly evolved under the pressure of natural selection and to be possibly related to the catalytic function of the protein. Hence, Leu34 and Leu137, far away from the active center, were selected for mutation. The generated CpRCR-L34A and CpRCR-L137V variants showed high stereoselectivity and 1.24-7.81 fold increase in k cat /K m value compared with that of the wild type, when reacted with 8 aromatic ketones or β-ketoesters. Corresponding computational study implied that L34 and L137 may extend allosteric fluctuation in the protein structure from the distal mutational site to the active site. Moreover, the L34 and L137 mutations modified the pre-reaction state in multiple ways, in terms of position of the hydride with respect to the target carbonyl. These findings provide insights into the catalytic mechanism of the enzyme and facilitate its regulation from the perspective of the site interaction network.

A single nucleotide mutation in the fourth exon of RBH1 is responsible for brown hull phenotype in rice

As one of the important traits, hull color is the morphological marker of rice, which plays an important role in the mechanized color selection of hybrid rice seed production but lacks good application. Here, we obtained a reddish-brown hull 1 (rbh1) mutant from an Indica maintainer material H9808 by aerospace mutagenesi. In the rbh1 mutant, the hull color was reddish brown, and the grain width and 1000-grain weight decreased significantly, but the other agronomic traits did not change significantly. Furthermore, the total flavonoids and anthocyanin content in the rbh1 hulls deposition was remarkably higher than WT, and the lignin level in the rbh1 hull was reduced. Genetic analysis indicated that the reddish-brown hull trait was controlled by a pair of recessive nuclear genes. Map-based cloning indicated that RBH1 was located within the physical distance of 48 kb on the short arm of chromosome 2. The comparative analysis of genome DNA sequence between rbh1 and WT found that a substitution from T to C (+ 1001) occurred in the fourth exon of LOC_Os02g09490 in rbh1 mutant. Genetic complementation experiments indicate that RBH1 is an allele of the previously reported GH2, which encoding a cinnamyl alcohol dehydrogenase protein involving in lignin biosynthesis. qRT-PCR showed that the relative expression of lignin and flavonoid-related genes in the hulls of rbh1 mutant was significantly upregulated, confirming that GH2/RBH1 is an important gene in the metabolism of lignin and flavonoids; and provides material basis for further studying the mechanism of GH2/RBH1 regulating the rice hull color.. In addition, the hybrid F1 combination analysis indicated that the rbh1 mutation site did not affect the agronomic traits and yield of the hybrid rice, which was to cultivate the rbh1 locus into a new sterile line or restorer line with reddish-brown hulls and rice breeding application of rbh1 locus on mechanized seed production of hybrid rice provides a theoretical basis.

Aldo-keto reductase and alcohol dehydrogenase contribute to benznidazole natural resistance in Trypanosoma cruzi.

The improvement of Chagas disease treatment is focused not only on the development of new drugs but also in understanding mechanisms of action and resistance to drugs conventionally used. Thus, some strategies aim to detect specific changes in proteins between sensitive and resistant parasites and to evaluate the role played in these processes by functional genomics. In this work, we used a natural Trypanosoma cruzi population resistant to benznidazole, which has clones with different susceptibilities to this drug without alterations in the NTR I gene. Using 2DE-gel electrophoresis, the aldo-keto reductase and the alcohol dehydrogenase proteins were found up regulated in the natural resistant clone and therefore their possible role in the resistance to benznidazole and glyoxal was investigated. Both genes were overexpressed in a drug sensitive T. cruzi clone and the biological changes in response to these compounds were evaluated. The results showed that the overexpression of these proteins enhances resistance to benznidazole and glyoxal in T. cruzi. Moreover, a decrease in mitochondrial and cell membrane damage was observed, accompanied by a drop in the intracellular concentration of reactive oxygen species after treatment. Our results suggest that these proteins are involved in the mechanism of action of benznidazole.

Prevention of protein aggregation by extracellular fungal sucrase of Termitomyces clypeatus

Abstract Objective Extracellular sucrase from Termitomyces clypeatus is known to impart stability and enhance activity of cellobiase, another secreted glycosidase of the fungus through co-aggregation with cellobiase. To explore whether sucrase can bind with some proteins like Insulin, BSA, alcohol dehydrogenase (ADH), carbonic anhydrase and whey proteins and prevent their dithiothreitol (DTT) induced/thermal aggregation and/or loss of activity measuring by spectrophotometry, gel filtration assay and activity assays. Methods MALDI-TOF and dynamic light scattering were used to assess the monomeric and aggregated molecular size of sucrase. Thermostatted spectrophotometric assays, gel filtration assays were used to study protein aggregation. Fluorescence of bound ANS was used to monitor temperature induced structural changes in sucrase together with determination of melting temperature. Results The mass of the monomeric unit of sucrase as 6649 Da. Enzyme inhibited DTT induced aggregation of insulin and suppressed the thermal aggregation of carbonic anhydrase, ADH and whey proteins, respectively by 83%, 68% and 70% at 70°C. Sucrase also protected about 84% activity of ADH. Conclusion An extracellular fungal sucrase with a low monomeric size can efficiently prevent protein aggregation. The studies can impart knowledge about potential therapeutic applications of this industrially important enzyme in protein misfolding disorders.

Experimental test and refutation of a classic case of molecular adaptation in Drosophila melanogaster.

Identifying the genetic basis for adaptive differences between species requires explicit tests of historical hypotheses concerning the effects of past changes in gene sequence on molecular function, organismal phenotype and fitness. We address this challenge by combining ancestral protein reconstruction with biochemical experiments and physiological analysis of transgenic animals that carry ancestral genes. We tested a widely held hypothesis of molecular adaptation-that changes in the alcohol dehydrogenase protein (ADH) along the lineage leading to Drosophila melanogaster increased the catalytic activity of the enzyme and thereby contributed to the ethanol tolerance and adaptation of the species to its ethanol-rich ecological niche. Our experiments strongly refute the predictions of the adaptive ADH hypothesis and caution against accepting intuitively appealing accounts of historical molecular adaptation that are based on correlative evidence. The experimental strategy we employed can be used to decisively test other adaptive hypotheses and the claims they entail about past biological causality.

Alpha-ketoglutarate reduces ethanol toxicity in Drosophila melanogaster by enhancing alcohol dehydrogenase activity and antioxidant capacity.

Ethanol at low concentrations (<4%) can serve as a food source for fruit fly Drosophila melanogaster, whereas at higher concentrations it may be toxic. In this work, protective effects of dietary alpha-ketoglutarate (AKG) against ethanol toxicity were studied. Food supplementation with 10-mM AKG alleviated toxic effects of 8% ethanol added to food, and improved fly development. Two-day-old adult flies, reared on diet containing both AKG and ethanol, possessed higher alcohol dehydrogenase (ADH) activity as compared with those reared on control diet or diet with ethanol only. Native gel electrophoresis data suggested that this combination diet might promote post-translational modifications of ADH protein with the formation of a highly active ADH form. The ethanol-containing diet led to significantly higher levels of triacylglycerides stored in adult flies, and this parameter was not altered by AKG supplement. The influence of diet on antioxidant defenses was also assessed. In ethanol-fed flies, catalase activity was higher in males and the levels of low molecular mass thiols were unchanged in both sexes compared to control values. Feeding on a mixture of AKG and ethanol did not affect catalase activity but caused a higher level of low molecular mass thiols compared to ethanol-fed flies. It can be concluded that both a stimulation of some components of antioxidant defense and the increase in ADH activity may be responsible for the protective effects of AKG diet supplementation in combination with ethanol. The results suggest that AKG might be useful as a treatment option to neutralize toxic effects of excessive ethanol intake and to improve the physiological state of D.melanogaster and other animals, potentially including humans.

English

The aim of our study was to isolate the alcohol dehydrogenase (ADH) mRNA from Phoenix dactifera, and examine the molecular evolutionary history of this nuclear gene with others ADH genes from palms and other plants species. The DnADH gene has been isolated in silico by BLAST2GO from a cDNA library of date palm cv Deglet Nour. The prediction of candidate’s mRNA and protein for ADH gene from khalas were performed in silico from whole genome shotgun sequence (ACYX02009373.1) using FGENESH prediction program. Nucleotide polymorphism using DnaSPv5 was examined in four palm ADH mRNA sequences across the entire 1.098 kb length of ADH mRNA. A primary conclusion of the present study is that nucleotide diversity for ADH between palm species is very low. In order to assess selective pressure, we calculated the ratio of non-synonymous to synonymous substitutions. We conclude that ADH palms genes appear to be under very different selective constraints. Phylogenetic analyses using PHYLIP and Notung 2.8 programs suggest that ADH genes of some plants species resulted from relatively ancient duplication events. In this study, we present for the first time a molecular characterization of ADH protein of P. dactylifera L cv Deglet nour and a phylogeny analysis between plants ADH. Keys word: Alcohol dehydrogenase, palms species, evolution, duplication.

Novel role for carbohydrate responsive element binding protein in the control of ethanol metabolism and susceptibility to binge drinking.

Carbohydrate responsive element binding protein (ChREBP) is central for de novo fatty acid synthesis under physiological conditions and in the context of nonalcoholic fatty liver disease. We explored its contribution to alcohol-induced steatosis in a mouse model of binge drinking as acute ethanol (EtOH) intoxication has become an alarming health problem. Within 6 hours, ChREBP acetylation and its recruitment onto target gene promoters were increased in liver of EtOH-fed mice. Acetylation of ChREBP was dependent on alcohol metabolism because inhibition of alcohol dehydrogenase (ADH) activity blunted ChREBP EtOH-induced acetylation in mouse hepatocytes. Transfection of an acetylation-defective mutant of ChREBP (ChREBP(K672A) ) in HepG2 cells impaired the stimulatory effect of EtOH on ChREBP activity. Importantly, ChREBP silencing in the liver of EtOH-fed mice prevented alcohol-induced triglyceride accumulation through an inhibition of the lipogenic pathway but also led, unexpectedly, to hypothermia, increased blood acetaldehyde concentrations, and enhanced lethality. This phenotype was associated with impaired hepatic EtOH metabolism as a consequence of reduced ADH activity. While the expression and activity of the NAD(+) dependent deacetylase sirtuin 1, a ChREBP-negative target, were down-regulated in the liver of alcohol-fed mice, they were restored to control levels upon ChREBP silencing. In turn, ADH acetylation was reduced, suggesting that ChREBP regulates EtOH metabolism and ADH activity through its direct control of sirtuin 1 expression. Indeed, when sirtuin 1 activity was rescued by resveratrol pretreatment in EtOH-treated hepatocytes, a significant decrease in ADH protein content and/or acetylation was observed. our study describes a novel role for ChREBP in EtOH metabolism and unravels its protective effect against severe intoxication in response to binge drinking.

Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization

Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide–mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways.

The Adh1 gene of the fungus Metarhizium anisopliae is expressed during insect colonization and required for full virulence

Zymography of alcohol dehydrogenase (ADH) activity in the entomopathogenic fungus Metarhizium anisopliae grown under various conditions revealed that micro-aerobic growth was associated with increased ADH activity. The major ADH protein, AdhIp, was purified to homogeneity by affinity chromatography and has an estimated molecular weight of 41kDa and an isoelectric point (pI) of 6.4. Peptide mass fingerprint analysis allowed the identification and cloning of the gene that encodes this protein, Adh1, as annotated in the M. anisopliae genome database. AdhIp is related to the medium-chain dehydrogenase/reductase (MDR)/zinc-dependent alcohol dehydrogenase-like family and contains conserved ADH sequence motifs, such as the zinc-containing ADH signature, the FAD/NAD binding domain and amino acid residues that are conserved in most microbial ADHs. Semi-quantitative RT-PCR analysis revealed that Adh1 gene expression occurs at low levels during early Plutella xylostella infection and that the Adh1 gene was primarily expressed at larval death and as mycelia emerge from the insect cuticle before conidiation. Antisense-RNA experiments indicated that NAD+-dependent ADH activity was diminished by 20–75% in the transformants, and the transformants that had lower ADH activity showed allyl alcohol resistance, which indicates that reduction in ADH activity also occurs in vivo. Bioassays performed using antisense adh1 transformants, which have lower ADH activity, showed that LC50 values were two to five times higher than the wild-type, indicating that AdhIp is required for full capability of the fungus to penetrate and/or colonize the insect.

Pre-endurance training prevents acute alcoholic liver injury in rats through the regulation of damaged mitochondria accumulation and mitophagy balance.

The aim of this study is to investigate the effect of pre-endurance training on the prevention of alcohol-induced acute hepatic injury and on hepatic mitophagy. Forty-eight male Sprague-Dawley rats were randomly divided into four groups: (1) control group, (2) 12-week exercise training group, (3) 5-day alcohol intake group, and (4) 12-week exercise training plus 5-day alcohol intake group. The rats were examined to determine the following: BCL2/adenovirus E1B 19kDa protein-interacting protein 3 (BNIP3), hypoxia-inducible factor-1α (HIF-1α), cytochrome P450 2E1 (CYP2E1), alcohol dehydrogenase (ADH), microtubule-associated protein 1 light chain 3 (LC3II), Beclin1 mRNA and protein expressions, mitochondrial reactive oxygen species (ROS) production, mitochondrial thiobarbituric acid-reactive substances (TBARS) level, aconitase and ATP synthase activities, mitochondrial inner membrane potential, NADH/NAD(+) ratio, triglyceride (TG), the number of mtDNA and mitochondrial respiration functions in liver tissue, and serum ALT and AST. Pre-endurance training attenuated acute alcohol treatment-induced increase in mitochondrial TBARS, ROS production, NADH/NAD(+) ratio, state 4 respiration rate, TG, serum ALT and AST, as well as BNIP3, HIF-1α, LC3II, and Beclin 1 mRNA and protein levels, however, CYP2E1 and ADH mRNA and protein levels unchanged. Meanwhile, it attenuated the acute alcohol intake-induced decrease in aconitase activity, inner mitochondrial membrane potential (Δψ), ATP synthase activity, state 3 respiration rate, respiratory control ratio, and the number of mtDNA. Pre-endurance training can decrease acute alcohol intake-induced damaged mitochondria accumulation and reduced acute alcohol intake-induced mitophagy, which built a new balance between mitophagy and damaged mitochondria accumulation.

Alcohol dehydrogenase-specific T-cell responses are associated with alcohol consumption in patients with alcohol-related cirrhosis

Patients with alcohol-related liver disease (ALD) have antibodies directed to alcohol dehydrogenase (ADH), anti-ADH titers being associated with disease severity and active alcohol consumption. ADH-specific T-cell responses have not been characterized. We aimed to define anti-ADH cellular immune responses and their association with active alcohol consumption and disease severity. Using cultures of peripheral blood mononuclear cells (PBMCs) from 25 patients with alcohol-related cirrhosis (ARC; 12 were actively drinking or abstinent for <6 months, and 13 were abstinent for >6 months) and hepatic mononuclear cells (HMCs) from 14 patients with ARC who were undergoing transplantation, we investigated T-cell reactivity to 25 overlapping peptides representing the full human ADH protein (beta 1 subunit). ADH-specific peripheral T-cell responses were assessed by the quantification of T-cell proliferation and cytokine production and were correlated with the clinical course. In active alcohol consumers, proliferative T-cell responses targeted ADH31-95 and other discontinuous sequences in the ADH peptide, whereas only one sequence was targeted in abstinents. ADH peptides induced the production of interferon-γ (IFN-γ), interleukin-4 (IL-4), and IL-17. IL-4 production was lower in active drinkers versus abstinents, and IL-17 production was higher. Peptides inducing IFN-γ production outnumbered those inducing T-cell proliferation. The intensity of the predominantly T helper 1 (Th 1) responses directly correlated with disease severity. Similar to PBMCs in abstinents, ADH peptides induced weak T-cell proliferation and a similar level of IL-4 production in HMCs but less vigorous Th 1 and T helper 17 responses. This suggests that Th 1 responses to ADH in ARC are induced by alcohol consumption. A Th 1/T helper 2 imbalance characterizes T-cell responses in active drinkers with ARC, whereas IL-4 production prevails in abstinents. This identifies new targets for immunoregulatory therapies in ALD patients for halting detrimental effector T-cell responses, which may encourage liver fibrogenesis and progression to end-stage liver disease.

Induction and construct UV protective yeast plasmid

In this study, we apply concepts of synthetic biology in combination with conventional methods to assemble different genetic components to construct yeast resistant to UV radiation, and to induce production of anti-UV proteins. This work combines sequences of different promoters, STRESS-proteins, heat shock protein (HSP), kinase proteins, alcohol dehydrogenase protein (ADH), ribosomal binding sites, fluorescent reporter proteins, terminators, and a synthetic ribosomal switch. The aim of this investigation was to induce an anti-UV proteins, and to construct an anti-UV yeast plasmid to be used for protection of skin cells against UV radiation. This investigation demonstrates induction and construction of anti-UV genes and production of their corresponding proteins. Cultures of Saccharomyces cerevisiae (ATCC # 66348) were exposed to short-wave UV radiation and were then subjected to time-PCR to assess specific gene expression. Proteins were identified using two dimensional difference gel electrophoresis (2D DIGE) and LC–MS/MS. Different up-regulated and down-regulated proteins were identified. Highly expressed identified proteins were cloned into S. cerevisiae using a synthetic biology approach. Extracts from UV-induced genetically transformed yeasts were used to protect skin cell cultures (ATCC #2522-CRL) in vitro. Both microscopic analysis and an apoptosis assay showed protection of the skin cell cultures against UV radiation.

Neutron Spin-Echo and TOF Reveals Protein Dynamics in Solution

The ps to ns dynamics of the protein alcohol dehydrogenase (ADH) has been measured in dilute solution by time-of-flight (TOF) neutron spectroscopy. The investigation extends previous experiments on ADH solutions with neutron-spin-echo spectroscopy. The interpretation of TOF experiments used the diffusion data from NSE as input to decribe the observed slow component of the quasielastic spectra, in addition a fast component to be associated to mobile groups in the protein could be observed. The description within a simple model for translational and rotational diffusion and a fraction of protons with confined local mobility is given together with a novel method to accurately and efficiently account for the instrumental resolution.

ADH IB Expression, but Not ADH III, Is Decreased in Human Lung Cancer

Endogenous S-nitrosothiols, including S-nitrosoglutathione (GSNO), mediate nitric oxide (NO)-based signaling, inflammatory responses, and smooth muscle function. Reduced GSNO levels have been implicated in several respiratory diseases, and inhibition of GSNO reductase, (GSNOR) the primary enzyme that metabolizes GSNO, represents a novel approach to treating inflammatory lung diseases. Recently, an association between decreased GSNOR expression and human lung cancer risk was proposed in part based on immunohistochemical staining using a polyclonal GSNOR antibody. GSNOR is an isozyme of the alcohol dehydrogenase (ADH) family, and we demonstrate that the antibody used in those studies cross reacts substantially with other ADH proteins and may not be an appropriate reagent. We evaluated human lung cancer tissue arrays using monoclonal antibodies highly specific for human GSNOR with minimal cross reactivity to other ADH proteins. We verified the presence of GSNOR in ≥85% of specimens examined, and extensive analysis of these samples demonstrated no difference in GSNOR protein expression between cancerous and normal lung tissues. Additionally, GSNOR and other ADH mRNA levels were evaluated quantitatively in lung cancer cDNA arrays by qPCR. Consistent with our immunohistochemical findings, GSNOR mRNA levels were not changed in lung cancer tissues, however the expression levels of other ADH genes were decreased. ADH IB mRNA levels were reduced (>10-fold) in 65% of the lung cancer cDNA specimens. We conclude that the previously reported results showed an incorrect association of GSNOR and human lung cancer risk, and a decrease in ADH IB, rather than GSNOR, correlates with human lung cancer.

Comparative Root Protein Profiles of Korean Ginseng (Panax ginseng) and Indian Ginseng (Withania somnifera)

Ginsenosides and withanolides are the secondary metabolites from Panax ginseng and Withania somnifera, respectively. These compounds have similar biological properties. Two-dimensional electrophoresis (2-DE) analysis was utilized to reveal the protein profile in the roots of both plants, with the aim of clarifying similarly- and differentially-expressed proteins. Total proteins of Korea ginseng (P. ginseng) and Indian ginseng (W. somnifera) roots were separated by 2-DE using a pH 4-7 immobilized pH gradient strip in the first dimension and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. The protein spots were visualized by silver staining. Twenty-one P. ginseng proteins and 35 W. somnifera proteins were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry; of these, functions were ascribed to 14 and 22 of the P. ginseng and W. somnifera proteins, respectively. Functions mainly included general cell metabolism, defense and secondary metabolism. ATPase and alcohol dehydrogenase proteins were expressed in both plants. The results of this study, to our knowledge, are the first to provide a reference 2-DE map for the W. somnifera root proteome, and will aid in the understanding of the expression and functions of proteins in the roots of Korean ginseng and Indian ginseng.