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PubMed1996

Kinetics and specificity of human liver aldehyde dehydrogenases toward aliphatic, aromatic, and fused polycyclic aldehydes.

Human mitochondrial aldehyde dehydrogenase (ALDH-2) has a Km for acetaldehyde that is 900-fold lower than that for the cytosolic isozyme, ALDH-1. An increase in aliphatic aldehyde chain length decreases the ALDH-2 Km by up to 10-fold but decreases that of ALDH-1 by 5 orders of magnitude. As a consequence, the Km of ALDH-1 for decanal is 8 times lower than that of ALDH-2, i.e. 2.9 +/- 0.4 and 22 +/- 3 nM, respectively. Determination of these low Km values required kinetic analysis of the simultaneous enzymatic conversion of two aldehyde substrates, an approach also applied to aromatic and fused polycyclic aldehydes. For most of these substrates, maximum velocities are 5-100 times lower than those for acetaldehyde. Addition of one of these tight-binding, slow-turnover substrates to a reaction mixture containing ALDH, NAD+, and a "reference" aldehyde substrate (e.g. acetaldehyde) blocks the principal (reference) enzymatic reaction temporarily and reversibly. Once the first substrate is converted to product, the enzyme can act on the reference substrate. In terms of apparent affinity and blocking capacity, naphthalene and phenanthrene aldehydes were the most potent effectors. Other aromatic and fused polycyclic and heterocyclic aldehydes, as well as derivatives of coumarin, quinoline, indole, and pyridine, are tight-binding, slow-turnover substrates for ALDH-2 and relatively weak inhibitors of ALDH-1. The hydrophobicity of substituents of benzaldehydes, and particularly of naphthaldehydes, correlates with their binding constants toward ALDH-2. Vitamin A1 aldehydes are specific natural substrates for ALDH-1; at pH 7.5, for all-trans- and 13-cis-retinal, Km = 1.1 and 0.37 micromolar, respectively, and kcat/Km is 50-100 times higher than that for acetaldehyde. At the same time, the retinals are inhibitors of ALDH-2, all-trans-retinal being a particularly potent inhibitor (competitive Ki = 43 nM, noncompetitive Ki = 316 nM). These properties suggest that all-trans-retinal is a possible regulatory compound for ALDH-2 in vivo. The data in general point to specialized roles for both major human liver ALDH isozymes in the oxidation of bulky/hydrophobic natural compounds, with Km values in the low nanomolar range.

Acetaldehyde↗

PubMed2006

In vivo and scanning electron microscopy imaging of up-converting nanophosphors in Caenorhabditis elegans.

We show here that upconversion phosphors can be imaged both by infrared excitation and in a scanning electron microscope. We have synthesized and characterized for this work up-converting phosphor nanoparticles nonaggregated nanocrystals of size range 50-200 nm. We have investigated the optical properties of 50-200 nm nanoparticles and found a square dependence of the emitted visible fluorescence on the infrared excitation and verified that under electron excitation similar narrow band emission spectra can be obtained as is seen with IR upconversion. The viability of the nanoparticles for biological imaging was confirmed by imaging the digestive system of the nematode worm Caenorhabditis elegans, and we have confirmed using energy-dispersive X-ray analysis that the up-conversion nanoparticles can be identified in a scanning electron microscope at high spatial resolution.

Animals↗

PubMed1981

Investigations of rat liver uroporphyrinogen decarboxylase. Comparisons of porphyrinogens I and III as substrates and the inhibition by porphyrins.

1. The decarboxylations of uroporphyrinogens, hepta-, hexa- and penta-carboxyporphyrinogens I and III by porphyrinogen carboxy-lyase (EC 4.1.1.37) in rat liver supernatant have been compared as functions of substrate concentrations. Although Km and Vmax. (for total porphyrinogens formed) were estimated, prophyrinogens and CO2 produced at 1 microM were considered to be a better indication of real relative rates, owing to substrate/product inhibitions. Uroporphyrinogen III was the best substrate by the criteria of Km/Vmax. and decarboxylation at 1 microM and was converted into coproporphyrinogen more quickly than its series-I isomer. 2. The difference between uroporphyrinogens I and III as substrates was confirmed by using a mixture of [14C8]uroporphyrinogens, the discrimination occurring principally in the first decarboxylation. 3. Porphyrins, especially oxidation products of the substrates, inhibited the enzyme. Heptacarboxyporphyrin III was the most effective inhibitor of both uroporphyrinogen III and heptacarboxyporphyrinogen III conversion into coproporphyrinogen. 4. Rapid analysis of the livers from rats made porphyric with hexachlorobenzene demonstrated that substantial quantities of the tetrapyrroles were present in vivo as the porphyrinogens (21-42%). 5. Enzymic decarboxylation of uroporphyrinogen III in 2H2O-containing buffer gave [2H4]coproporphyrinogen. 6. Rats treated with cycloheximide for 10h showed no decrease in uroporphyrinogen decarboxylase activity/mg of protein, suggesting a relatively slow turnover of the enzyme.

Animals↗

PubMed2003

Genomic identification and biochemical characterization of the mammalian polyamine oxidase involved in polyamine back-conversion.

In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N1 -acetyltransferase (SSAT) and then oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine respectively. Although PAO was first purified more than two decades ago, the protein has not yet been linked to genomic sequences. In the present study, we apply a BLAST search strategy to identify novel oxidase sequences located on human chromosome 10 and mouse chromosome 7. Homologous mammalian cDNAs derived from human brain and mouse mammary tumour were deduced to encode proteins of approx. 55 kDa having 82% sequence identity. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by approx. 30%, whereas spermidine increased 2-4-fold. Lysates of human PAO cDNA-transfected HEK-293 cells, but not vector-transfected cells, rapidly oxidized N1-acetylspermine to spermidine. Substrate specificity determinations with the lysate assay revealed a preference ranking of N1-acetylspermine= N1-acetylspermidine> N1,N12-diacetylspermine>>spermine; spermidine was not acted upon. This ranking is identical to that reported for purified PAO and distinctly different from the recently identified spermine oxidase (SMO), which prefers spermine over N1-acetylspermine. Monoethyl- and diethylspermine analogues also served as substrates for PAO, and were internally cleaved adjacent to a secondary amine. We deduce that the present oxidase sequences are those of the FAD-dependent PAO involved in the polyamine back-conversion pathway. In Northern blot analysis, PAO mRNA was much less abundant in HEK-293 cells than SMO or SSAT mRNA, and all three were differentially induced in a similar manner by selected polyamine analogues. The identification of PAO sequences, together with the recently identified SMO sequences, provides new opportunities for understanding the dynamics of polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically-relevant polyamine analogues and inhibitors.

Amino Acid Sequence↗

PubMed2006

Structure-function relationships for Schizophyllum commune trehalose phosphorylase and their implications for the catalytic mechanism of family GT-4 glycosyltransferases.

The cDNA encoding trehalose phosphorylase, a family GT-4 glycosyltransferase from the fungus Schizophyllum commune, was isolated and expressed in Escherichia coli to yield functional recombinant protein in its full length of 737 amino acids. Unlike the natural phosphorylase that was previously obtained as a truncated 61 kDa monomer containing one tightly bound Mg2+, the intact enzyme produced in E. coli is a dimer and not associated with metal ions [Eis, Watkins, Prohaska and Nidetzky (2001) Biochem. J. 356, 757-767]. MS analysis of the slow spontaneous conversion of the full-length enzyme into a 61 kDa fragment that is fully active revealed that critical elements of catalysis and specificity of trehalose phosphorylase reside entirely in the C-terminal protein part. Intact and truncated phosphorylases thus show identical inhibition constants for the transition state analogue orthovanadate and alpha,alpha-trehalose (K(i) approximately 1 microM). Structure-based sequence comparison with retaining glycosyltransferases of fold family GT-B reveals a putative active centre of trehalose phosphorylase, and results of site-directed mutagenesis confirm the predicted crucial role of Asp379, His403, Arg507 and Lys512 in catalysis and also delineate a function of these residues in determining the large preference of the wild-type enzyme for the phosphorolysis compared with hydrolysis of alpha,alpha-trehalose. The pseudo-disaccharide validoxylamine A was identified as a strong inhibitor of trehalose phosphorylase (K(i)=1.7+/-0.2 microM) that displays 350-fold tighter binding to the enzyme-phosphate complex than the non-phosphorolysable substrate analogue alpha,alpha-thio-trehalose. Structural and electronic features of the inhibitor that may be responsible for high-affinity binding and their complementarity to an anticipated glucosyl oxocarbenium ion-like transition state are discussed.

Amino Acid Sequence↗

PubMed2000

Comparison of QT dispersion during atrial fibrillation and sinus rhythm in the same patients, at normal and prolonged ventricular repolarization.

AIMS: Drug-induced increase in QT dispersion has been associated with increased risk of ventricular proarrhythmia. The aim of the present study was to compare QT dispersion during atrial fibrillation and sinus rhythm in the same patients at normal and prolonged ventricular repolarization. METHODS AND RESULTS: Sixty-one patients who had had chronic atrial fibrillation for 8 +/- 14 months received a 6 h infusion of the Ikr-blocker almokalant, the first 90 min of which are used for this analysis. The following day, after conversion to sinus rhythm, by almokalant (n = 19) or direct current cardioversion (n=42), an identical 90 min infusion was administered. Prior to infusion, there was no difference in precordial QT dispersion between atrial fibrillation and sinus rhythm (29 +/- 12 vs 36 +/- 17 ms, P=ns). During infusion, at prolonged repolarization, the increase in QT dispersion was greater during sinus rhythm than during atrial fibrillation (58 +/- 49 vs 30 +/- 15 ms, P=0.0011, after 30 min infusion). No correlation was found between QT dispersion and the QT or RR interval. CONCLUSION: QT dispersion during atrial fibrillation does not differ from QT dispersion during sinus rhythm during normal repolarization. while measurement of QT dispersion during prolonged repolarization, induced by an Ikr-blocker, yielded larger values during sinus rhythm than during atrial fibrillation.

Action Potentials↗

PubMed1996

Characterization of serum amyloid A protein mRNA expression and secondary amyloidosis in the domestic duck.

Secondary amyloidosis is a common disease of water fowl and is characterized by the deposition of extracellular fibrils of amyloid A (AA) protein in the liver and certain other organs. Neither the normal role of serum amyloid A (SAA), a major acute phase response protein, nor the causes of secondary amyloidosis are well understood. To investigate a possible genetic contribution to disease susceptibility, we cloned and sequenced SAA cDNA derived from livers of domestic ducks. This revealed that the three C-terminal amino acids of SAA are removed during conversion to insoluble AA fibrils. Analysis of SAA cDNA sequences from several animals identified a distinct genetic dimorphism that may be relevant to susceptibility to secondary amyloid disease. The duck genome contained a single copy of the SAA gene that was expressed in liver and lung tissue of ducklings, even in the absence of induction of acute phase response. Genetic analysis of heterozygotes indicated that only one SAA allele is expressed in livers of adult birds. Immunofluorescence staining of livers from adult ducks displaying early symptoms of amyloidosis revealed what appear to be amyloid deposits within hepatocytes that are expressing unusually high amounts of SAA protein. This observation suggests that intracellular deposition of AA may represent an early event during development of secondary amyloidosis in older birds.

Amino Acid Sequence↗

PubMed1989

Body weight: relationship to conversational distance and self-actualization.

Body weight is a major concern for women. This study investigated the relationship between body weight deviation, perceived effect of weight, conversational distance, and self-actualization in healthy caucasian college women (N = 109) between the ages of 18 and 50. The perception of body weight was measured with a structured questionnaire. Conversational distance was measured by having the participant approach the investigator, and self-actualization was determined using the Personal Orientation Inventory (POI). Significant correlations were found between the Time Competence (TC) subscale of the POI and Perceived Effect (PE) and between conversational distance and TC. Further analysis of the data revealed a relationship between body weight deviation, using the actual deviation from the norm, and the combined effect (magnitude and direction) of body weight (r = .54, p less than .001). Path analysis revealed the multidimensional nature of the issue of body weight for women. The usual assumptions about body fat for women are questioned and implications for future research are discussed.

Adult↗

PubMed2004

The Loss Inventory: preliminary reliability and validity data for a new measure of emotional and cognitive responses to disability.

PURPOSE: To examine the utility, reliability and validity of a new scale, The Loss Inventory (LI) for measuring symptoms unique to grief following disablement. METHODS: Prospective survey study in which two self-report measures, the LI and Zung Self-Rating Depression Scale, were administered to 103 hospitalized rehabilitation patients. Correlational analyses, t tests, t score conversions of raw data, and analysis of variance used to determine reliability (internal consistency) of the LI and compare means of scores across several variables. RESULTS: Cronbach's Alpha=0.90 for the LI. The Zung and the LI correlated 0.59 but study subjects were significantly (p<0.001) more likely to score higher on the LI than on the Zung. Certain LI items were significantly more frequently endorsed by depressed patients. Significant gender and ethnic differences were found. CONCLUSIONS: Rehabilitation patients sampled in this study were no more likely to be depressed than most people. Grief symptoms reported following functional losses are similar to those reported by persons who have experienced the death of someone close. The Loss Inventory reliably measures these grief symptoms. Gender and ethnicity affect the manner in which grief symptoms are reported. The LI may improve mental health clinicians' diagnostic accuracy in rehabilitation and medical settings.

Brain Injuries↗

PubMed1991

Lipid traffic between high density lipoproteins and Plasmodium falciparum-infected red blood cells.

Several intraerythrocytic growth cycles of Plasmodium falciparum could be achieved in vitro using a serum free medium supplemented only with a human high density lipoprotein (HDL) fraction (d = 1.063-1.210). The parasitemia obtained was similar to that in standard culture medium containing human serum. The parasite development was incomplete with the low density lipoprotein (LDL) fraction and did not occur with the VLDL fraction. The lipid traffic from HDL to the infected erythrocytes was demonstrated by pulse labeling experiments using HDL loaded with either fluorescent NBD-phosphatidylcholine (NBD-PC) or radioactive [3H]palmitoyl-PC. At 37 degrees C, the lipid probes rapidly accumulated in the infected cells. After incubation in HDL medium containing labeled PC, a subsequent incubation in medium with either an excess of native HDL or 20% human serum induced the disappearance of the label from the erythrocyte plasma membrane but not from the intraerythrocytic parasite. Internalization of lipids did not occur at 4 degrees C. The mechanism involved a unidirectional flux of lipids but no endocytosis. The absence of labeling of P. falciparum, with HDL previously [125I]iodinated on their apolipoproteins or with antibodies against the apolipoproteins AI and AII by immunofluorescence and immunoblotting, confirmed that no endocytosis of the HDL was involved. A possible pathway of lipid transport could be a membrane flux since fluorescence videomicroscopy showed numerous organelles labeled with NBD-PC moving between the erythrocyte and the parasitophorous membranes. TLC analysis showed that a partial conversion of the PC to phosphatidylethanolamine was observed in P. falciparum-infected red cells after pulse with [3H]palmitoyl-PC-HDL. The intensity of the lipid traffic was stage dependent with a maximum at the trophozoite and young schizont stages (38th h of the erythrocyte life cycle). We conclude that the HDL fraction appears to be a major lipid source for Plasmodium growth.

Animals↗

PubMed1999

Pharmacoeconomics of antimicrobial therapy.

Switch therapy, sequential therapy, and step-down therapy are discussed in terms of their contribution to reducing antimicrobial expenditures. Pharmacoeconomics is the science used to identify and compare the costs and consequences of drug therapy in terms of efficacy, safety, and overall health care. Pharmacoeconomic studies of antimicrobials for respiratory-tract infections have identified significant cost savings associated with regimens that are optimized for a particular patient on the basis of a drug's pharmacokinetic profile. For fluoroquinolones, optimal therapy has been associated with targeting the specific pharmacodynamic variable known as the ratio of the area under the serum concentration-time curve from 0 to 24 hours (AUC) to the minimum inhibitory concentration, also referred to as the area under the inhibitory curve (AUIC). Several studies have shown that regimens that achieve targeted AUIC values of 125 to 250 against gram-negative aerobic bacteria are cost-effective; cost savings are linked to decreased time to bacterial eradication and higher AUICs. Additional cost-effective measures for hospitals and health care institutions include the implementation of formalized i.v.-to-oral conversions and streamlining programs. Pharmacoeconomic analysis of therapies for respiratory-tract and other infections demonstrates that reducing health care costs may best be achieved by curing the infection in the shortest possible time through dosage optimization individualized to the patient.

Anti-Bacterial Agents↗

PubMed1997

Evolution of SINE S1 retroposons in Cruciferae plant species.

The S1 element is a plant short interspersed element (SINE) that was first described and studied in Brassica napus. In this work, we investigated the distribution and the molecular phylogeny of the S1 element within the Cruciferae (= Brassicaceae). S1 elements were found to be widely distributed within the Cruciferae, especially in species of the tribe Brassiceae. The molecular phylogeny of S1 elements in eight Cruciferae species (Brassica oleracea, Brassica rapa, Brassica napus, Brassica nigra, Sinapis, arvensis, Sinapis pubescens, Coincya monensis, and Vella spinosa) was inferred using 14-36 elements per species. Significant neighbor-joining and maximum-parsimony phylogenetic clusters, supported by high bootstrap P values and/or represented in 100% of the most-parsimonious trees, were observed for each species. Most of these clusters probably correspond to recent species-specific bursts of S1 amplification. Since these species diverged recently, S1 amplification in Cruciferae plants is proposed to be a highly dynamic process that could contribute to genome rearrangements and eventually lead to reproductive isolation. S1 sequence analysis also revealed putative gene conversion events that occurred between different S1 elements of a given species. These events suggest that gene conversion is a minor but significant component of the molecular drive governing S1 concerted evolution.

Base Sequence↗

PubMed2002

Auxotrophic mutant strains of Rhizobium etli reveal new nodule development phenotypes.

We report here the isolation and characterization of amino acid-requiring mutant strains of Rhizobium etli. We observe that the phenotype of most mutations, even when causing a strict auxotrophy, is overcome by cross-feeding from the host plant Phaseolus vulgaris, thereby allowing bacterial production of Nod factors and, consequently, nodule induction. Conversely, light and electron microscopy analysis reveals that the nodules induced by all mutants, including those with normal external morphology, are halted or strongly altered at intermediate or late stages of development. Moreover, some mutants induce nodules that display novel symbiotic phenotypes, such as specific alterations of the invaded cells or the presence of a reduced number of abnormally shaped uninvaded cells. Other mutants induce nodules showing an early and vast necrosis of the central tissue, a phenotype not previously observed in bean nodules, not even in nodules induced by a Fix- mutant. These observations indicate that amino acid auxotrophs represent a powerful tool to study the development of globose determinate-type nodules and emphasize the importance of establishing their histology and cytology before considerations of metabolic exchange are made.

Amino Acids↗

PubMed2005

Resonance reflection of acoustic waves in piezoelectric bi-crystalline structures.

The paper studies the bulk wave reflection from internal interfaces in piezoelectric media. The interfaces of two types have been considered. Infinitesimally thin metallic layer inserted into homogeneous piezoelectric crystal of arbitrary symmetry. Rigidly bonded crystals whose piezoelectric coefficients differ by sign but the other material constants are identical. Analytic expressions for the coefficients of mode conversion have been derived. An analysis has been carried out of specific singularities arising when the angle of incidence is such that the resonance excitation of leaky interface acoustic waves occurs. The conditions for the resonance total reflection have been established. The computations performed for lithium niobate (LiNbO3) illustrate general conclusions.

Journal Article↗

PubMed1987

Metabolism of glyphosate in an Arthrobacter sp. GLP-1.

The metabolism of glyphosate [N-(phosphonomethyl)glycine] in a bacterium tentatively identified as an Arthrobacter sp., capable of growth on this herbicide as its sole phosphorus source, has been investigated using solid-state NMR techniques as well as radiotracer analysis. The pathway involves the conversion of glyphosate to glycine, a C1 unit and phosphate. The phosphonomethyl carbon is specifically incorporated into the amino acids serine, cysteine, methionine, and histidine, as well as into purine bases and thymine, indicating the involvement of tetrahydrofolate in single-carbon transfer reactions. Glycine derived from glyphosate is utilized in purine and protein biosynthesis. This pathway for glyphosate degradation in a gram-positive bacterium is similar to that previously reported for Pseudomonas sp. PG2982 [Jacob et al. (1985) J. Biol. Chem. 260, 5899-5905] and is distinct from that reported for soil metabolism of glyphosate where aminomethylphosphonic acid has been shown to be a major metabolite. Preliminary evidence is presented which indicates that the conversion of glyphosate to glycine and the C1 unit involves the intermediate formation of sarcosine. Thus, the primary event in glyphosate degradation by Arthrobacter sp. GLP-1 is the cleavage of its C-P bound. This report constitutes the first demonstration of the metabolism of glyphosate in a gram-positive bacterium.

Arthrobacter↗

PubMed2005

Mechanisms of azole resistance in clinical isolates of Candida glabrata collected during a hospital survey of antifungal resistance.

The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata infections has resulted in the selection and/or emergence of resistant strains. The main mechanisms of azole resistance include alterations in the C. glabrata ERG11 gene (CgERG11), which encodes the azole target enzyme, and upregulation of the CgCDR1 and CgCDR2 genes, which encode efflux pumps. In the present study, we evaluated these molecular mechanisms in 29 unmatched clinical isolates of C. glabrata, of which 20 isolates were resistant and 9 were susceptible dose dependent (S-DD) to fluconazole. These isolates were recovered from separate patients during a 3-year hospital survey for antifungal resistance. Four of the 20 fluconazole-resistant isolates were analyzed together with matched susceptible isolates previously taken from the same patients. Twenty other azole-susceptible clinical C. glabrata isolates were included as controls. MIC data for all the fluconazole-resistant isolates revealed extensive cross-resistance to the other azoles tested, i.e., itraconazole, ketoconazole, and voriconazole. Quantitative real-time PCR analyses showed that CgCDR1 and CgCDR2, alone or in combination, were upregulated at high levels in all but two fluconazole-resistant isolates and, to a lesser extent, in the fluconazole-S-DD isolates. In addition, slight increases in the relative level of expression of CgSNQ2 (which encodes an ATP-binding cassette [ABC] transporter and which has not yet been shown to be associated with azole resistance) were seen in some of the 29 isolates studied. Interestingly, the two fluconazole-resistant isolates expressing normal levels of CgCDR1 and CgCDR2 exhibited increased levels of expression of CgSNQ2. Conversely, sequencing of CgERG11 and analysis of its expression showed no mutation or upregulation in any C. glabrata isolate, suggesting that CgERG11 is not involved in azole resistance. When the isolates were grown in the presence of fluconazole, the profiles of expression of all genes, including CgERG11, were not changed or were only minimally changed in the resistant isolates, whereas marked increases in the levels of gene expression, particularly for CgCDR1 and CgCDR2, were observed in either the fluconazole-susceptible or the fluconazole-S-DD isolates. Finally, known ABC transporter inhibitors, such as FK506, were able to reverse the azole resistance of all the isolates. Together, these results provide evidence that the upregulation of the CgCDR1-, CgCDR2-, and CgSNQ2-encoded efflux pumps might explain the azole resistance in our set of isolates.

ATP-Binding Cassette Transporters↗

PubMed1986

Isolation and genetic characterizations of Bacillus megaterium cobalamin biosynthesis-deficient mutants.

Ethanolamine is deaminated by the action of ethanolamine ammonia-lyase (EC 4.3.1.7), an adenosylcobalamin-dependent enzyme. Consequently, to grow on ethanolamine as a sole nitrogen source, Bacillus megaterium requires vitamin B12. Identification of B. megaterium mutants deficient for growth on ethanolamine as the sole nitrogen source yielded a total of 34 vitamin B12 auxotrophs. The vitamin B12 auxotrophs were divided into two major phenotypic groups: Cob mutants, which could use cobinamide or vitamin B12 to grow on ethanolamine, and Cbl mutants, which could be supplemented only by vitamin B12. The Cob mutants were resolved into six classes and the Cbl mutants were resolved into three, based on the spectrum of cobalt-labeled corrinoid compounds which they accumulated. Although some radiolabeled cobalamin was detected in the wild type, little or none was evident in the auxotrophs. The results indicate that Cob mutants contain lesions in biosynthetic steps before the synthesis of combinamide, while Cbl mutants are defective in the conversion of cobinamide to cobalamin. Analysis of phage-mediated transduction experiments revealed tight genetic linkage within the Cob class and within the Cbl class. Similar transduction analysis indicated the Cob and Cbl classes are weakly linked. In addition, cross-feeding experiments in which extracts prepared from mutants were examined for their effect on growth of various other mutants allowed a partial ordering of mutations within the cobalamin biosynthetic pathway.

Bacillus megaterium↗

PubMed1990

Ethanol formation in adh0 mutants reveals the existence of a novel acetaldehyde-reducing activity in Saccharomyces cerevisiae.

A strain of Saccharomyces cerevisiae has been constructed which is deficient in the four alcohol dehydrogenase (ADH) isozymes known at present. This strain (adh0), being irreversibly mutated in the genes ADH1, ADH3, and ADH4 and carrying a point mutation in the gene ADH2 coding for the glucose-repressible isozyme ADHII, still produces up to one third of the theoretical maximum yield of ethanol in a homofermentative conversion of glucose to ethanol. Analysis of the glucose metabolism of adh0 cells shows that the lack of all known ADH isozymes results in the formation of glycerol as a major fermentation product, accompanied by a significant production of acetaldehyde and acetate. Treatment of glucose-growing adh0 cells with the respiratory-chain inhibitor antimycin A leads to an immediate cessation of ethanol production, demonstrating that ethanol production in adh0 cells is dependent on mitochondrial electron transport. Reduction of acetaldehyde to ethanol in isolated mitochondria could also be demonstrated. This reduction is apparently linked to the oxidation of acetaldehyde to acetate. Preliminary data suggest that this novel type of ethanol formation in S. cerevisiae is associated with the inner mitochondrial membrane.

Acetaldehyde↗