Microbial Degradation of α-Picolinic Acid, 2-Pyridinecarboxylic Acid

The biological effects of raw winged bean seeds were investigated with feeding experiments on rats, and the effects of lectin (phytohemagglutinin) present in the seeds are discussed. Administration of a 30% raw winged bean diet caused strong growth depression in young rats, and led to death within 10 ~ 20 days, inducing severe damage to the small intestine of the rats. Significant morphological changes of the intestinal mucosa were observed with a microscopic investigation. As the lethal effect was eliminated by autoclaving but not removed with supplementation of 0.5% l-methionine to the raw winged bean diet, the lectin was assumed to be closely related to the deleterious effects of raw winged bean. In vitro and in vivo digestion tests of the lectin revealed that the winged bean lectin had resistance to peptic, pancreatic and membrane digestions. The hemagglutinating activity was also detected in the intestinal mucosa and faeces from rats ingesting the raw winged bean or its purified lectin. The binding action of lection to mucosal epitheliums of the gastrointestinal tract is suggested to be the initial step of the deleterious effects induced by the winged bean lectin.     

Alcohol,Phospholipase A2-associated Neuroinflammation,and ω3 Docosahexaenoic Acid Protection

Chronic alcohol (ethanol) abuse causes neuroinflammation and brain damage that can give rise to alcoholic dementia. Insightfully, Dr. Albert Sun was an early proponent of oxidative stress as a key factor in alcoholism-related brain deterioration. In fact, oxidative stress has proven to be critical to the hippocampal and temporal cortical neurodamage resulting from repetitive “binge” alcohol exposure in adult rat models. Although the underlying mechanisms are uncertain, our immunoelectrophoretic and related assays in binge alcohol experiments in vivo (adult male rats) and in vitro (rat organotypic hippocampal-entorhinal cortical slice cultures) have implicated phospholipase A₂ (PLA₂)-activated neuroinflammatory pathways, release of pro-oxidative arachidonic acid (20:4 ω6), and elevated oxidative stress adducts (i.e., 4-hydroxynonenal-protein adducts). Also, significantly increased by the binge alcohol treatments was aquaporin-4 (AQP4), a water channel enriched in astrocytes that, when augmented, may trigger brain (esp. cellular) edema and neuroinflammation; of relevance, glial swelling is known to provoke increased PLA₂ activities or levels. Concomitant with PLA₂ activation, the results have further implicated binge alcohol-elevated poly (ADP-ribose) polymerase-1 (PARP-1), an oxidative stress-responsive DNA repair enzyme linked to parthanatos, a necrotic-like neuronal death process. Importantly, supplementation of the brain slice cultures with docosahexaenoic acid (22:6 ω3) exerted potent suppression of the induced changes in PLA₂ isoforms, AQP4, PARP-1 and oxidative stress footprints, and prevention of the binge alcohol neurotoxicity, by as yet unknown mechanisms. These neuroinflammatory findings from our binge alcohol studies and supportive rat binge studies in the literature are reviewed.     

New Phosphonoformic Acid Derivatives of 3′-Azido-3′-Deoxythymidine

New 5-alkyl ethoxy- and aminocarbonylphosphonates of 3-azido-3-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5-aminocarbonylphosphonates were shown to be significantly more stable in phosphate buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of some of the compounds exceeded that of the parent AZT due to their higher antiviral activity.     

Palmitic Acid Induces Osteoblastic Differentiation in Vascular Smooth Muscle Cells through ACSL3 and NF-κB,Novel Targets of Eicosapentaenoic Acid

Free fatty acids (FFAs), elevated in metabolic syndrome and diabetes, play a crucial role in the development of atherosclerotic cardiovascular disease, and eicosapentaenoic acid (EPA) counteracts many aspects of FFA-induced vascular pathology. Although vascular calcification is invariably associated with atherosclerosis, the mechanisms involved are not completely elucidated. In this study, we tested the hypothesis that EPA prevents the osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC) induced by palmitic acid (PA), the most abundant long-chain saturated fatty acid in plasma. PA increased and EPA abolished the expression of the genes for bone-related proteins, including bone morphogenetic protein (BMP)-2, Msx2 and osteopontin in human aortic smooth muscle cells (HASMC). Among the long-chain acyl-CoA synthetase (ACSL) subfamily, ACSL3 expression was predominant in HASMC, and PA robustly increased and EPA efficiently inhibited ACSL3 expression. Importantly, PA-induced osteoblastic differentiation was mediated, at least in part, by ACSL3 activation because acyl-CoA synthetase (ACS) inhibitor or siRNA targeted to ACSL3 completely prevented the PA induction of both BMP-2 and Msx2. Conversely, adenovirus-mediated ACSL3 overexpression enhanced PA-induced BMP-2 and Msx2 expression. In addition, EPA, ACSL3 siRNA and ACS inhibitor attenuated calcium deposition and caspase activation induced by PA. Notably, PA induced activation of NF-κB, and NF-κB inhibitor prevented PA-induction of osteoblastic gene expression and calcium deposition. Immunohistochemistry revealed the prominent expression of ACSL3 in VSMC and macrophages in human non-calcifying and calcifying atherosclerotic plaques from the carotid arteries. These results identify ACSL3 and NF-κB as mediators of PA-induced osteoblastic differentiation and calcium deposition in VSMC and suggest that EPA prevents vascular calcification by inhibiting such a new molecular pathway elicited by PA.     

Characteristics of the Deoxyribonucleic Acid of Tφ3, a Bacteriophage for Bacillus stearothermophilus

It is shown that the individual strands of bacteriophage Tphi3 DNA are intact and that heat-denatured Tphi3 DNA forms a bimodal distribution in a neutral CsCl density gradient.     

The Synthesis of Dimethyl Esters of dl-O,O′-Dimethylfukiic Acid and dl-O,O′-Dimethylepifukiic Acid

The synthesis of dimethyl esters of dl-O,O′-dimethylfukiic acid (11) and dl-O,O′-dimethylepifukiic acid (12) are described.     

β-aminoisobutyric Acid,l-BAIBA,Is a Muscle-Derived Osteocyte Survival Factor

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Arachidonic Acid Binds 14-3-3ζ, Releases 14-3-3ζ from Phosphorylated BAD and Induces Aggregation of 14-3-3ζ

Polyunsaturated fatty acids, like arachidonic acid, can bind proteins and affect their function. The 14-3-3 proteins bind phosphorylated sites on a diverse array of client proteins and, in this way, are involved in many intracellular signaling pathways. In this study, we used a novel approach to discover that 14-3-3ζ is able to directly bind arachidonic acid. Furthermore, arachidonic acid, at physiological concentrations, reduced the binding of 14-3-3ζ to phosphorylated BAD, an interaction that is important in regulating apoptosis. In addition, high concentrations of arachidonic acid caused the polymerization of 14-3-3ζ, an event observed in neurodegenerative disorders. Taken together, these results indicate that arachidonic acid directly interacts with 14-3-3ζ and that this interaction may be important in both normal and pathological cellular events. If so, then factors that mediate the release, metabolism and reacylation of arachidonic acid into membranes represent key points of regulation.     

Monodehydro-2,2′-iminodi-2(2′)-deoxy-l-ascorbic Acid,a Radical Product from the Reaction of Dehydro-l-ascorbic Acid with an α-Amino Acid

One of the radical species produced by the reaction of dehydro-l-ascorbic acid with an α-amino acid gave a very characteristic hyperfine structure in its electron spin resonance spectrum. The same spectrum was also obtained when l-scorbamic acid was oxidized with some oxidants, indicating the formation of the radical via the oxidation of l-scorbamic acid. From the results of deuterium exchange experiments, simulation spectra and the reduction of 2,2′-nitrilodi-2(2′)-deoxy-l-ascorbic acid monoammonium salt, the radical was concluded to be monodehydro-2,2′-iminodi-2(2′)-deoxy-l-ascorbic acid. Possible formation mechanism of the radical was also discussed.     

Dihydrolipoamide Dehydrogenases of Advenella mimigardefordensis and Ralstonia eutropha Catalyze Cleavage of 3,3′-Dithiodipropionic Acid into 3-Mercaptopropionic Acid

The catabolism of the disulfide 3,3′-dithiodipropionic acid (DTDP) is initiated by the reduction of its disulfide bond. Three independent Tn5::mob-induced mutants of Advenella mimigardefordensis strain DPN7^T were isolated that had lost the ability to utilize DTDP as the sole source of carbon and energy and that harbored the transposon insertions in three different sites of the same dihydrolipoamide dehydrogenase gene encoding the E3 subunit of the pyruvate dehydrogenase multi-enzyme complex of this bacterium (LpdA_Am). LpdA_Am was analyzed in silico and compared to homologous proteins, thereby revealing high similarities to the orthologue in Ralstonia eutropha H16 (PdhL_Re). Both bacteria are able to cleave DTDP into two molecules of 3-mercaptopropionic acid (3MP). A. mimigardefordensis DPN7^T converted 3MP to 3-sulfinopropionic acid, whereas R. eutropha H16 showed no growth with DTDP as the sole carbon source but was instead capable of synthesizing heteropolythioesters using the resulting cleavage product 3MP. Subsequently, the genes lpdA_Am and pdhL_Re were cloned, heterologously expressed in Escherichia coli applying the pET23a expression system, purified, and assayed by monitoring the oxidation of NADH. The physiological substrate lipoamide was reduced to dihydrolipoamide with specific activities of 1,833 mkat/kg of protein (LpdA_Am) or 1,667 mkat/kg of protein (PdhL_Re). Reduction of DTDP was also unequivocally detected with the purified enzymes, although the specific enzyme activities were much lower: 0.7 and 0.5 mkat/kg protein, respectively.In Advenella mimigardefordensis strain DPN7^T (15, 42), three independent mutants with an insertion of Tn5::mob in the lpdA gene coding for the E3 component of the pyruvate dehydrogenase multi-enzyme complex revealed an interesting phenotype: these mutants were fully impaired in utilizing 3,3′-dithiodipropionic acid (DTDP) as the sole carbon and energy source, whereas the growth on no other tested carbon sources was affected (41). Our main interest in the catabolism of DTDP is to unravel the pathway and to identify the involved enzymes. Furthermore, the application of this disulfide as precursor substrate for biotechnological production of polythioesters (PTE) (22) is of interest. Since poly(3-mercaptopropionate) (PMP) biosynthesis depends hitherto on supplying the harmful thiol 3-mercaptopropionic acid (3MP) (35), an improvement of the recombinant Escherichia coli system by heterologous expression of enzymes capable of cleaving the less toxic DTDP symmetrically into two molecules of 3MP, which are then polymerized, could be an important achievement toward large-scale biotechnological production of PMP.Two different enzyme systems catalyzing the conversion of disulfides into the corresponding thiols are already known and have been described in detail. (i) Enzymes belonging to the well-characterized family of pyridine-nucleotide disulfide oxidoreductases (25) contain a redox center formed by a disulfide bridge coupled to a flavin ring. They catalyze a simultaneous two-electron transfer via the enzymatic active disulfides associated with the pyridine nucleotides and flavin, toward the substrate (39, 40). (ii) An alternative disulfide reduction is catalyzed by enzymes using iron-sulfur clusters to cleave of disulfide substrates in two one-electron reduction steps (37). The disrupted gene in A. mimigardefordensis was designated lpdA_Am (EC 1.8.1.4), and it encodes a homodimeric flavoprotein, the dihydrolipoamide dehydrogenase LpdA_Am (i.e., the E3 component of the pyruvate dehydrogenase multi-enzyme complex of A. mimigardefordensis strain DPN7^T) belonging to the above-mentioned family of pyridine nucleotide-disulfide oxidoreductases. Enzymes of this class share high sequence and structural similarities and catalyze reduction of compounds which are linked by disulfide bonds (38). Alkylhydroperoxide reductases, coenzyme A disulfide reductases, glutathione reductases, mycothione reductases, thioredoxin reductases, and trypanothione reductases also, in addition to dihydrolipoamide dehydrogenases, belong to this family (3, 38). The physiological function of LpdA_Am is most probably the conversion of lipoamide to dihydrolipoamide, but the reduction of DTDP into two molecules of 3MP (Fig. ​(Fig.1)1) is also predicted, enabling the first step in DTDP catabolism in A. mimigardefordensis strain DPN7^T (41).Open in a separate windowFIG. 1.Reactions catalyzed by LpdA_Am and PdhL_Re. Presented are the enzymatic conversions of DTDP into two molecules of 3MP (A), lipoamide into dihydrolipoamide (B), and DTNB into two molecules of NTB (C). Abbreviations: DTDP, 3,3′-dithiodipropionic acid; 3MP, 3-mercaptopropionic acid; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); NTB, 2-nitro-5-thiobenzoic acid.Ralstonia eutropha H16 synthesizes copolymers of 3-hydroxybutyrate and 3MP, if 3MP (23) or DTDP (22) is supplied as a precursor in addition to a second utilizable carbon source. Although R. eutropha is not able to grow with DTDP as the sole carbon source, it must be capable of cleaving this organic disulfide symmetrically, because it synthesizes from it heteropolymers containing the resulting 3MP. Thus, R. eutropha must possess at least one gene encoding a DTDP-cleaving enzyme. Five genes coding for homologues of a dihydrolipoamide dehydrogenase (DHLDH), which in A. mimigardefordensis DPN7^T is obviously involved in DTDP degradation, are known to exist in the genome of R. eutropha H16 (27; M. Raberg, J. Bechmann, U. Brandt, J. Schlüter, B. Uischner, and A. Steinbüchel, unpublished data). Therefore, LpdA_Am and the five DHLDH paralogues of R. eutropha H16 were aligned and compared (Fig. ​(Fig.2).2). Subsequently, lpdA_Am and the gene encoding the DHLDH belonging to the pyruvate dehydrogenase complex of R. eutropha H16 (pdhL_Re) were cloned, heterologously expressed in Escherichia coli, purified, and assayed.Open in a separate windowFIG. 2.Phylogenetic relationships of the A. mimigardefordensis strain DPN7^T LpdA (boldface), R. eutropha H16 PdhL (boldface), and homologues. The neighbor-joining plot was derived from a CLUSTAL X alignment of amino acid sequences closely related to LpdA_Am. The amino acid sequence of the outer membrane protein P64K from Neisseria meningitidis was used as the outgroup. GenBank accession numbers are given in parentheses. Scale bar, 10% sequence divergence.     

Inhibition of D-amino Acid Oxidase by α-Keto-γ-Methiolbutyric Acid,product of the reaction with D-Methionine

Summary The inhibition of D-Amino Acid Oxidase by -Keto Acid, product of the reaction with D-Amino Acid is described for the first time. Inhibition of the enzyme by -Keto--Methiolbutyric Acid (4-methylthio-2-oxobutanoic acid), product of the reaction with D-Methionine, was studied. From the results obtained it is deduced that inhibition is competitive with the substrate, the value of the inhibition constant being 1.85 10^–3 M.     

PM3 and AM1 Study on β-N-acetyl-muramic Acid and 3 Murein Related Derivatives

The energetically favoured conformations of -N-acetyl-Muramic acid, its C6-O-acetylated form, the methylamide and the methyl-glycoside have been investigated using the semiempirical PM3 and AM1 methods. All these compounds are either components or fragmentary structures of the murein network. The atomic coordinates of the starting set of the -N-acetyl-Muramic acid molecule have been obtained by a PM3 minimization of one saccharide molecule cut out from the murein single strand model proposed by Barnickel at al. [1]. The sidegroups of the derivatives have been introduced by a molecular editor. These conformations served as starting points in conformational space for a grid search by scanning all sidechain torsional angles for non-hydrogen atoms with exception of the N-acetyl group which was held in cisoid position (i.e. N2-H bond is parallel to C1-H and C3-H bond) and only minimized. The PM3 method with an additional amide correction potential and the AM1 method were used. The torsional angle distributions of the lactyl sidechain (free acid and methylamide), the C6-O-acetylated sidechain and the C1-methoxy sidechain have been investigated, showing distinct energetically favoured torsional angle regions. The results are compared to earlier studies on -N-acetyl-Muramic acid by J.S. Yadav et al. [2,3] who were using the MNDO and PCILO methods and by P.N.S. Yadav et al. [4] who were using the empirical MM2 force-field.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089460020190     

Retinoic Acid Metabolism Inhibition by 3-Azolylmethyl-1H-indoles and 2, 3 or 5-(α-Azolylbenzyl)-1H-indoles

Among a library of 70 azoles, 8 indole derivatives substituted in the 2-, 3- or 5- position with an azolylmethyl or α-azolylbenzyl chain were evaluated for retinoic acid (RA) metabolism inhibitory activity. The most active inhibitors identified in this study were 5-bromo-1-ethyl-3-methyl-2-[(phenyl)(1H-1,2,4-triazol-1-yl)methyl]-1H-indole (3) (68.9% inhibition) and 5-bromo-1-ethyl-2-[(4-fluorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-3-methyl-1H-indole (6) (60.4% inhibition). At the same concentration (100 μM) ketoconazole exerted similar inhibitory effect (70% inhibition).     

α-Aminoisobutyric Acid as the Constituent Amino Acid of Protein

α-Aminoisobutyric acid is the only tertiary amino acid which is reported to occur in the proteins. Nevertheless, this amino acid has not been yet isolated from the proteins. Recently we succeeded in isolating this amino acid as white prismy crystalline substance from both acid and pepsin hydrolysate of horse hind leg muscle proteins, and this crystal was identified to be α-amino-isobutyric acid by elementary analysis, properties of this derivates, etc.     

Reverse Reaction of Malic Enzyme for HCO3 − Fixation into Pyruvic Acid to Synthesize L-Malic Acid with Enzymatic Coenzyme Regeneration

Malic enzyme [L-malate: NAD(P)⁺ oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)⁺). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO₃ ^? fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD⁺, and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO₃ ^? and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 °C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD⁺.