Acetone Metabolism Research Articles

Elevated blood-ethanol concentration promotes reduction of aliphatic ketones (acetone and ethyl methyl ketone) to secondary alcohols along with slower oxidation to aliphatic diols.

Many people convicted for drunken driving suffer from an alcohol use disorder and some traffic offenders consume denatured alcohol for intoxication purposes. Venous blood samples from people arrested for driving under the influence of alcohol were analyzed in triplicate by headspace gas chromatography (HS-GC) using three different stationary phases. The gas chromatograms from this analysis sometimes showed peaks with retention times corresponding to acetone, ethyl methylketone (2-butanone), 2-propanol, and 2-butanol in addition to ethanol and the internal standard (1-propanol). Further investigations showed that these drink-driving suspects had consumed an industrial alcohol (T-Red) for intoxication purposes, which contained > 90% w/v ethanol, acetone (~ 2% w/v), 2-butanone (~ 5% w/v) as well as Bitrex to impart a bitter taste. In n = 75 blood samples from drinkers of T-Red, median concentrations of ethanol, acetone, 2-butanone, 2-propanol and 2-butanol were 2050mg/L (2.05g/L), 97mg/L, 48mg/L, 26mg/L and 20mg/L, respectively. In a separate GC analysis, 2,3-butanediol (median concentration 87mg/L) was identified in blood samples containing 2-butanone. When the redox state of the liver is shifted to a more reduced potential (excess NADH), which occurs during metabolism of ethanol, this favors the reduction of low molecular ketones into secondary alcohols via the alcohol dehydrogenase (ADH) pathway. Routine toxicological analysis of blood samples from apprehended drivers gave the opportunity to study metabolism of acetone and 2-butanone without having to administer these substances to human volunteers.

Micromycete Responses to Acetone: Electrochemical Express Evaluation of Acetone Metabolism.

The biosensor method has not yet been applied to study the fungus-acetone interaction. The first electrochemical (amperometric) study of Fusarium oxysporum f. sp. vasinfectum cells' responses to acetone was performed to evaluate the initial stages of the metabolism of acetone in the cells of the micromycete. Using a laboratory model of a membrane microbial sensor based on the micromycete cells, it was found that the fungus had constitutive enzyme systems that were involved in acetone transport into fungal cells. The research showed that uninduced by acetone cells had degradative activity against acetone. A positive cooperativity of acetone binding with enzymes initiating acetone degradation was revealed. Oxygen concentration affected the activation of cell enzymes initiating acetone degradation, but the cells' activity in the presence of acetone maintained even at low oxygen concentration. Kinetic parameters (the maximum rate of the cells' response to substrate and the half-saturation constant) of the processes causing the fungal cells' response to acetone were calculated. The results demonstrated the convenience of the biosensor method for assessing the potential of the micromycete as a substrate-degrading culture. In the future, the mechanism of response to acetone for microbial cells will be studied.

Endogenous De Novo Synthesis of Isopropanol Following Severe Non-Diabetic Ketoacidosis in a Patient With Duchenne Muscular Dystrophy

In Duchenne muscular dystrophy (DMD), patients seem prone to severe metabolic acidosis following excessive bicarbonate loss due to laxative use or gastrointestinal disorders. In addition, hypoalimentation and fasting can cause life-threatening non-diabetic ketoacidoses with acetonemia in these patients. Isopropanolemia instead is predominantly found following intoxication. Only few reports exist describing isopropanolemia in diabetic or alcoholic ketoacidosis. Here, we report on a patient suffering from DMD who developed life-threatening non-diabetic ketoacidosis, acetonemia, and endogenous de novo synthesis of isopropanol following glucose fasting. We hypothesize isopropanolemia being a byproduct of acetone metabolism, possibly in association with a metabolic predisposition in patients with DMD such as an altered NADH/NAD + ratio leading to a reduction from acetone to isopropyl alcohol whilst in severe ketoacidosis. J Endocrinol Metab. 2022;12(3):107-110 doi: https://doi.org/10.14740/jem804

Effects of Plant Sterols Intake on Systematic and Tissue Specific Lipid Metabolism in C57BL/6J Mice

Abstract Objectives To investigate the effects of plant sterols intake on systematic and tissue specific lipid metabolism in C57BL/6J mice. Methods Male C57BL/6J mice were randomly divided into control diet group (CS) and plant sterol group (PS, 2% plant sterols). After 28 weeks of continuous feeding, the serum of the mice were collected for biochemical and mass spectrometry tests. Serum levels of total cholesterol (TC), triglyceride (TG) and free sterols were determined. The livers and lungs were collected for free sterol quantification and RNA-seq analysis. Results Compared with the CS group, 2% plant sterols intake significantly reduced the levels of TC in the serum of mice (P < 0.05), with the TG level unchanged. The quantitative results of free sterols showed that the concentration of campesterol were increased, and the cholestanol levels were decreased significantly in the serum and liver of the PS group mice. The results of RNA-seq analysis were used to further evaluate its impact on the lipid metabolism related gene expression profile in the livers and lungs. The results showed that HMGCR, SQLE, HMGCS1, SREBF1, and other genes related to cholesterol synthesis in the PS group were significantly up-regulated in the liver, but not in the lung; Among the first 20 targeting pathways related to the action of plant sterols, the liver differentially expressed genes were enriched in lipid metabolism (steroid biosynthesis, terpenoid skeleton biosynthesis, peroxisome, bile acid secretion, PPAR, MAPK, fatty acid metabolism.), inflammation related (Cell adhesion molecules, leukocyte trans-endothelial migration) and amino acid metabolism (glutathione, valine, leucine and isoleucine metabolism). The differential genes in lung tissue are enriched in lipid metabolism (acetone metabolism, fatty acid metabolism, insulin resistance, terpenoid skeleton biosynthesis, iron death, PPAR), cell function (internal Swallowing, aging) and vascular smooth muscle contraction etc. Conclusions Differentially expressed gene networks reflect the multi-dimensional regulation of plant sterols on tissue specific lipid metabolism, which lays a good foundation for further revealing its mechanism. Funding Sources Yihaikerry Nutrition and Food Safety Foundation, Chinese Nutrition Society; Project of Technology Innovation and Application, Chongqing, China

Genetic requirements and transcriptomics of Helicobacter pylori biofilm formation on abiotic and biotic surfaces

Biofilm growth is a widespread mechanism that protects bacteria against harsh environments, antimicrobials, and immune responses. These types of conditions challenge chronic colonizers such as Helicobacter pylori but it is not fully understood how H. pylori biofilm growth is defined and its impact on H. pylori survival. To provide insights into H. pylori biofilm growth properties, we characterized biofilm formation on abiotic and biotic surfaces, identified genes required for biofilm formation, and defined the biofilm-associated gene expression of the laboratory model H. pylori strain G27. We report that H. pylori G27 forms biofilms with a high biomass and complex flagella-filled 3D structures on both plastic and gastric epithelial cells. Using a screen for biofilm-defective mutants and transcriptomics, we discovered that biofilm cells demonstrated lower transcripts for TCA cycle enzymes but higher ones for flagellar formation, two type four secretion systems, hydrogenase, and acetone metabolism. We confirmed that biofilm formation requires flagella, hydrogenase, and acetone metabolism on both abiotic and biotic surfaces. Altogether, these data suggest that H. pylori is capable of adjusting its phenotype when grown as biofilm, changing its metabolism, and re-shaping flagella, typically locomotion organelles, into adhesive structures.

What Constitutes a Gluconeogenic Precursor?

A gluconeogenic precursor is a biochemical compound acted on by a gluconeogenic pathway enabling the net synthesis of glucose. Recognized gluconeogenic precursors in fasting placental mammals include glycerol, lactate/pyruvate, certain amino acids, and odd-chain length fatty acids. Each of these precursors is capable of contributing net amounts of carbon to glucose synthesis via the tricarboxylic acid cycle (TCA cycle) because they are anaplerotic, that is, they are able to increase the pools of TCA cycle intermediates by the contribution of more carbon than is lost via carbon dioxide. The net synthesis of glucose from even-chain length fatty acids (ECFAs) in fasting placental mammals, via the TCA cycle alone, is not possible because equal amounts of carbon are lost via carbon dioxide as is contributed from fatty acid oxidation via acetyl-CoA. Therefore, ECFAs do not meet the criteria to be recognized as a gluconeogenic precursor via the TCA cycle alone. ECFAs are gluconeogenic precursors in organisms with a functioning glyoxylate cycle, which enables the net contribution of carbon to the intermediates of the TCA cycle from ECFAs and the net synthesis of glucose. The net conversion of ECFAs to glucose in fasting placental mammals via C3 metabolism of acetone may be a competent though inefficient metabolic path by which ECFA could be considered a gluconeogenic precursor. Defining a substrate as a gluconeogenic precursor requires careful articulation of the definition, organism, and physiologic conditions under consideration.

Insights into the unique carboxylation reactions in the metabolism of propylene and acetone.

Alkenes and ketones are two classes of ubiquitous, toxic organic compounds in natural environments produced in several biological and anthropogenic processes. In spite of their toxicity, these compounds are utilized as primary carbon and energy sources or are generated as intermediate metabolites in the metabolism of other compounds by many diverse bacteria. The aerobic metabolism of some of the smallest and most volatile of these compounds (propylene, acetone, isopropanol) involves novel carboxylation reactions resulting in a common product acetoacetate. Propylene is metabolized in a four-step pathway involving five enzymes where the penultimate step is a carboxylation reaction catalyzed by a unique disulfide oxidoreductase that couples reductive cleavage of a thioether linkage with carboxylation to produce acetoacetate. The carboxylation of isopropanol begins with conversion to acetone via an alcohol dehydrogenase. Acetone is converted to acetoacetate in a single step by an acetone carboxylase which couples the hydrolysis of MgATP to the activation of both acetone and bicarbonate, generating highly reactive intermediates that are condensed into acetoacetate at a Mn2+ containing the active site. Acetoacetate is then utilized in central metabolism where it is readily converted to acetyl-coenzyme A and subsequently converted into biomass or utilized in energy metabolism via the tricarboxylic acid cycle. This review summarizes recent structural and biochemical findings that have contributed significant insights into the mechanism of these two unique carboxylating enzymes.

Effects of oral health instructions on glycemic control and oral health status of periodontitis patients with type 2 diabetes mellitus: A preliminary observation

Diabetes mellitus (DM) is a group of metabolic diseases,1 and several studies have demonstrated a relationship between DM and periodontitis.2, 3, 4, 5 Diabetic complications include an increase in oxidative stress, inflammation, and advanced glycation end product formation. Poorly controlled DM is strongly associated with clinical attachment loss and edentulism in people with DM,6 and periodontal therapy influences glycemic control in type 2 DM (T2DM).7 A meta-analysis showed that scaling and root planning (SRP) improved the levels of glycated hemoglobin (HbA1c) and fasting plasma glucose (FPG), although statistically nonsignificant.3 Other systematic reviews and meta-analysis have reported that nonsurgical periodontal therapy (NSPT) significantly reduces mean HbA1c levels and also FPG levels.4,5 As periodontitis is caused due to dental plaque accumulation, maintaining oral hygiene is essential for preventing inflammation from periodontitis. Poor oral hygiene increases the risk of periodontitis by 2- to 5-fold,8 and plaque removal and control has been shown to be fundamentally important in preventing periodontal disease occurrence and progression.9 However, only a few studies have reported that oral hygiene instructions (OHIs) improve glycemic control in patients with T2DM.10, 11, 12 Furthermore, patients with DM recognize the characteristic oral malodor as “a disagreeable odor that emanates from the mouth.”13,14 Oral malodor is socially awkward for others to point out. As diabetes causes abnormal liver metabolism, abnormal ketone metabolism in the body results in acetone metabolism.15 We hypothesized that HbA1c level and oral malodor may decrease by regulating a patient's oral hygiene instructed by a dental hygienist. The aim of our study was to investigate the effects of OHIs on glycemic control and oral malodor in patients with T2DM.

Effect of cis-9, trans-11 Conjugated Linoleic Acid (CLA) on the Metabolism Profile of Breast Cancer Cells Determined by 1 H HR-MAS NMR Spectroscopy

Conjugated linoleic acid (CLA), a fatty acid found in ruminant food products, has been associated with anticarcinogenic activity. However, its effect on cancer metabolism is unclear. In this paper we evaluated the effects of cis-9, trans-11 CLA on the metabolic profile of MCF-7 and MDA-MB-231 breast cancer cells using high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. The NMR spectra showed that phosphocholine level, a cancer malignance biomarker, was reduced in both cells treated with CLA, but the reduction was more pronounced in MCF-7 cells. The NMR spectra also showed that CLA has opposite effect on MCF-7 and MDA-MB-231 acetone metabolism. Acetone signal has been observed in the spectra of MDA-MB-231 control cells, but not in the spectra of the cells treated with 50 and 100 µM CLA. Conversely, the acetone signal is very small or not observed in the NMR spectra of MCF-7 control cells and in cells treated with 50 µM of CLA, but is very strong in the spectra of the cells treated with 100 µM of CLA. Therefore, this CLA concentration is causing a ketosis in MCF-7 cells by inducing the use of fatty acids as an energy source or by reducing acetone catabolism. These results indicate that CLA interfere in the metabolism of both cells. However, the strongest effect has been observed on the metabolism of MCF-7 cells cultivated in the presence of 100 µM CLA. Therefore, CLA could be a potential anticarcinogenic drug, especially for cells with positive estrogen receptor, such as MCF-7.

Microbial diversity of a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon and shotgun metagenomic sequencing.

The 16S rRNA gene amplicon and whole‐genome shotgun metagenomic (WGSM) sequencing approaches were used to investigate wide‐spectrum profiles of microbial composition and metabolic diversity from a full‐scale UASB reactor applied to poultry slaughterhouse wastewater treatment. The data were generated by using MiSeq 2 × 250 bp and HiSeq 2 × 150 bp Illumina sequencing platforms for 16S amplicon and WGSM sequencing, respectively. Each approach revealed a distinct microbial community profile, with Pseudomonas and Psychrobacter as predominant genus for the WGSM dataset and Clostridium and Methanosaeta for the 16S rRNA gene amplicon dataset. The virome characterization revealed the presence of two viral families with Bacteria and Archaea as host, Myoviridae, and Siphoviridae. A wide functional diversity was found with predominance of genes involved in the metabolism of acetone, butanol, and ethanol synthesis; and one‐carbon metabolism (e.g., methanogenesis). Genes related to the acetotrophic methanogenesis pathways were more abundant than methylotrophic and hydrogenotrophic, corroborating the taxonomic results that showed the prevalence of the acetotrophic genus Methanosaeta. Moreover, the dataset indicated a variety of metabolic genes involved in sulfur, nitrogen, iron, and phosphorus cycles, with many genera able to act in all cycles. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) revealed that microbial community contained 43 different types of antibiotic resistance genes, some of them were associated with growth chicken promotion (e.g., bacitracin, tetracycline, and polymyxin).

Cloning, functional expression and characterization of a bifunctional 3-hydroxybutanal dehydrogenase /reductase involved in acetone metabolism by Desulfococcus biacutus.

BackgroundThe strictly anaerobic, sulfate-reducing bacterium Desulfococcus biacutus can utilize acetone as sole carbon and energy source for growth. Whereas in aerobic and nitrate-reducing bacteria acetone is activated by carboxylation with CO2 to acetoacetate, D. biacutus involves CO as a cosubstrate for acetone activation through a different, so far unknown pathway. Proteomic studies indicated that, among others, a predicted medium-chain dehydrogenase/reductase (MDR) superfamily, zinc-dependent alcohol dehydrogenase (locus tag DebiaDRAFT_04514) is specifically and highly produced during growth with acetone.ResultsThe MDR gene DebiaDRAFT_04514 was cloned and overexpressed in E. coli. The purified recombinant protein required zinc as cofactor, and accepted NADH/NAD+ but not NADPH/NADP+ as electron donor/acceptor. The pH optimum was at pH 8, and the temperature optimum at 45 °C. Highest specific activities were observed for reduction of C3 - C5-aldehydes with NADH, such as propanal to propanol (380 ± 15 mU mg−1 protein), butanal to butanol (300 ± 24 mU mg−1), and 3-hydroxybutanal to 1,3-butanediol (248 ± 60 mU mg−1), however, the enzyme also oxidized 3-hydroxybutanal with NAD+ to acetoacetaldehyde (83 ± 18 mU mg−1).ConclusionThe enzyme might play a key role in acetone degradation by D. biacutus, for example as a bifunctional 3-hydroxybutanal dehydrogenase/reductase. Its recombinant production may represent an important step in the elucidation of the complete degradation pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0899-9) contains supplementary material, which is available to authorized users.

Measuring breath acetone for monitoring fat loss: Review.

ObjectiveEndogenous acetone production is a by‐product of the fat metabolism process. Because of its small size, acetone appears in exhaled breath. Historically, endogenous acetone has been measured in exhaled breath to monitor ketosis in healthy and diabetic subjects. Recently, breath acetone concentration (BrAce) has been shown to correlate with the rate of fat loss in healthy individuals. In this review, the measurement of breath acetone in healthy subjects is evaluated for its utility in predicting fat loss and its sensitivity to changes in physiologic parameters.ResultsBrAce can range from 1 ppm in healthy non‐dieting subjects to 1,250 ppm in diabetic ketoacidosis. A strong correlation exists between increased BrAce and the rate of fat loss. Multiple metabolic and respiratory factors affect the measurement of BrAce. BrAce is most affected by changes in the following factors (in descending order): dietary macronutrient composition, caloric restriction, exercise, pulmonary factors, and other assorted factors that increase fat metabolism or inhibit acetone metabolism. Pulmonary factors affecting acetone exchange in the lung should be controlled to optimize the breath sample for measurement.ConclusionsWhen biologic factors are controlled, BrAce measurement provides a non‐invasive tool for monitoring the rate of fat loss in healthy subjects.

Catalytic function of the mycobacterial binuclear iron monooxygenase in acetone metabolism.

Mycobacteria such as Mycobacterium smegmatis strain mc(2)155 and Mycobacterium goodii strain 12523 are able to grow on acetone and use it as a source of carbon and energy. We previously demonstrated by gene deletion analysis that the mimABCD gene cluster, which encodes a binuclear iron monooxygenase, plays an essential role in acetone metabolism in these mycobacteria. In the present study, we determined the catalytic function of MimABCD in acetone metabolism. Whole-cell assays were performed using Escherichia coli cells expressing the MimABCD complex. When the recombinant E. coli cells were incubated with acetone, a product was detected by gas chromatography (GC) analysis. Based on the retention time and the gas chromatography-mass spectrometry (GC-MS) spectrum, the reaction product was identified as acetol (hydroxyacetone). The recombinant E. coli cells produced 1.02 mM of acetol from acetone within 24 h. Furthermore, we demonstrated that MimABCD also was able to convert methylethylketone (2-butanone) to 1-hydroxy-2-butanone. Although it has long been known that microorganisms such as mycobacteria metabolize acetone via acetol, this study provides the first biochemical evidence for the existence of a microbial enzyme that catalyses the conversion of acetone to acetol.

Antiglycative activity of sulforaphane: a new avenue to counteract neurodegeneration?

Antiglycative activity of sulforaphane: a new avenue to counteract neurodegeneration?

Blood, urine and vitreous isopropyl alcohol as biochemical markers in forensic investigations

Isopropyl alcohol (IPA) is widely used as an industrial solvent and cleaning fluid. After ingestion or absorption, IPA is converted into acetone by alcohol dehydrogenase. However, in ketosis, acetone can be reduced to IPA. The aim of this study was to investigate blood IPA and acetone concentrations in a series of 400 medico-legal autopsies, including cases of diabetic ketoacidosis, hypothermia and alcohol misuse-related deaths, to illustrate the extent of ketosis at the time of death. Vitreous glucose, blood 3-β-hydroxybutyrate (3HB) and acetoacetate (AcAc) concentrations were also determined systematically. Additionally, vitreous and urine IPA, acetone, 3HB and AcAc concentrations as well as other biochemical markers, including glycated hemoglobin and carbohydrate-deficient transferrin (CDT) were also determined in selected cases. The results of this study indicate that ketosis is characterized by the presence of IPA resulting from the acetone metabolism and that IPA can be detected in several substrates. These findings confirm the importance of the systematic determination of IPA and acetone levels that is used to quantify biochemical disturbances and the importance of ketosis at the time of death.

Acetone and butanone metabolism of the denitrifying bacterium "Aromatoleum aromaticum" demonstrates novel biochemical properties of an ATP-dependent aliphatic ketone carboxylase.

The anaerobic and aerobic metabolism of acetone and butanone in the betaproteobacterium "Aromatoleum aromaticum" is initiated by their ATP-dependent carboxylation to acetoacetate and 3-oxopentanoic acid, respectively. Both reactions are catalyzed by the same enzyme, acetone carboxylase, which was purified and characterized. Acetone carboxylase is highly induced under growth on acetone or butanone and accounts for at least 5.5% of total cell protein. The enzyme consists of three subunits of 85, 75, and 20 kDa, respectively, in a (αβγ)(2) composition and contains 1 Zn and 2 Fe per heterohexamer but no organic cofactors. Chromatographic analysis of the ATP hydrolysis products indicated that ATP was exclusively cleaved to AMP and 2 P(i). The stoichiometry was determined to be 2 ATP consumed per acetone carboxylated. Purified acetone carboxylase from A. aromaticum catalyzes the carboxylation of acetone and butanone as the only substrates. However, the enzyme shows induced (uncoupled) ATPase activity with many other substrates that were not carboxylated. Acetone carboxylase is a member of a protein family that also contains acetone carboxylases of various other organisms, acetophenone carboxylase of A. aromaticum, and ATP-dependent hydantoinases/oxoprolinases. While the members of this family share several characteristic features, they differ with respect to the products of ATP hydrolysis, subunit composition, and metal content.

Identification of the Monooxygenase Gene Clusters Responsible for the Regioselective Oxidation of Phenol to Hydroquinone in Mycobacteria

Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc(2)155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc(2)155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc(2)155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc(2)155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.

Analysis of Protein Expression Regulated by theHelicobacter pyloriArsRS Two-Component Signal Transduction System

Previous studies have shown that the Helicobacter pylori ArsRS two-component signal transduction system contributes to acid-responsive gene expression. To identify additional members of the ArsRS regulon and further investigate the regulatory role of the ArsRS system, we analyzed protein expression in wild-type and arsS null mutant strains. Numerous proteins were differentially expressed in an arsS mutant strain compared to a wild-type strain when the bacteria were cultured at pH 5.0 and also when they were cultured at pH 7.0. Genes encoding 14 of these proteins were directly regulated by the ArsRS system, based on observed binding of ArsR to the relevant promoter regions. The ArsRS-regulated proteins identified in this study contribute to acid resistance (urease and amidase), acetone metabolism (acetone carboxylase), resistance to oxidative stress (thioredoxin reductase), quorum sensing (Pfs), and several other functions. These results provide further definition of the ArsRS regulon and underscore the importance of the ArsRS system in regulating expression of H. pylori proteins during bacterial growth at both neutral pH and acidic pH.

Comparative Genome-Wide Transcriptional Profiling of Azorhizobium caulinodans ORS571 Grown under Free-Living and Symbiotic Conditions

The whole-genome sequence of the endosymbiotic bacterium Azorhizobium caulinodans ORS571, which forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, was recently determined. The sizes of the genome and symbiosis island are 5.4 Mb and 86.7 kb, respectively, and these sizes are the smallest among the sequenced rhizobia. In the present study, a whole-genome microarray of A. caulinodans was constructed, and transcriptomic analyses were performed on free-living cells grown in rich and minimal media and in bacteroids isolated from stem nodules. Transcriptional profiling showed that the genes involved in sulfur uptake and metabolism, acetone metabolism, and the biosynthesis of exopolysaccharide were highly expressed in bacteroids compared to the expression levels in free-living cells. Some mutants having Tn5 transposons within these genes with increased expression were obtained as nodule-deficient mutants in our previous study. A transcriptomic analysis was also performed on free-living cells grown in minimal medium supplemented with a flavonoid, naringenin, which is one of the most efficient inducers of A. caulinodans nod genes. Only 18 genes exhibited increased expression by the addition of naringenin, suggesting that the regulatory mechanism responding to the flavonoid could be simple in A. caulinodans. The combination of our genome-wide transcriptional profiling and our previous genome-wide mutagenesis study has revealed new aspects of nodule formation and maintenance.

What Is the Antiseizure Activity of Acetone Due To?

To the Editors: Recently, Gasior and associates examined the effects of acetone metabolites to evaluate their antiseizure activities and came to the conclusion that acetone itself, and not its metabolites, are likely capable of exerting antiepileptic activity (Gasior et al., 2007). Herewith this conclusion is disputed and the following analysis will address some significant points. The place of the antiseizure activity of any chemical compound is the brain. And this rule applies to acetone metabolites, too. Therefore, the first issue is the lack of evidence for brain distribution of metabolites. While acetone freely crosses blood–brain barrier, and its concentrations in blood and cerebrospinal fluid are similar in rats (Likhodii and Burnham, 2006), such an information is not available for its metabolites and the authors did not present data in this regard, either (Gasior et al., 2007). Studies with intravenously administered tracer amounts of [11C]-acetone to baboons proved that acetone exhibited a rapid uptake into and a relatively slow clearance from the brain (Gerasimov et al., 2005). And patients successfully treated with ketogenic diet have been reported to present elevated levels of acetone in their brains, too (Seymour et al., 1999). In contrast, methylglyoxal seems not to cross biological membranes as it binds to macromolecules the intracellular concentration of free methylglyoxal is estimated only 5% of the total (Chaplen et al., 1996). For the brain distribution of either acetol or 1,2-propanediol data are not known. Unless either the uptake of an acetone metabolite into the brain or its formation in the brain and its failure to exert antiseizure activity parallel to its uptake or production are proven, it cannot be stated that the indexed metabolite does not have antiseizure activity. The second issue is the application of acetone metabolites. All chemicals were administered intraperitoneally (Gasior et al., 2007). If some of the metabolites are unable to cross membranes then the intraperitoneal application of metabolites does not make sense. In addition, the mode of administration has an influence on the toxicity of a given compound as detailed for 1,2-propanediol and methylglyoxal (Ruddick, 1972; Kalapos, 1999). The third issue is data interpretation. The authors stated that acetol failed to protect animals against seizure, while it is seen in figure 3 that acetol exerted antiseizure activity in all the three seizure models (Gasior et al., 2007). Investigating data for acetone and acetol closely, it becomes obvious that the curves for acetol are shifted toward higher concentrations but run parallel to the curves gained for acetone (Gasior et al., 2007). Motor impairment caused by higher doses reflects toxicity and means a limitation to the application of acetol. For 1,2-propanediol, similar shift is seen in two of the three models (Gasior et al., 2007). The fourth issue is the contamination of commercially available chemicals. This particularly applies to methylglyoxal, which is contaminated with formaldehyde, pyruvate, lactate, and formate (Pourmotabbed and Creighton, 1986). Finally, it needs to be addressed whether animal models of this kind are suitable tools to shed light to antiseizure activity of acetone. Acetone metabolism is a complex network (Kalapos, 2006). The conversion of acetone to methylglyoxal needs the action of cytochrome P450 2E1 gene products, designated CYP2E1 (Casazza et al., 1984; Myksis and Tyndale, 2004). CYP2E1 gene products are most abundant in the liver but are present in the brain, too (Myksis and Tyndale, 2004). In animals, transgenic, knockout and viral vector methodologies represent powerful tools, thus the use of transgenic mouse line lacking CYP2E1 would be a unique model to test the role of acetone in seizure control (Bondoc et al., 1999; Kalapos, 2007).