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.     

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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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.     

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.     

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.     

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.     

Nitro-fatty Acid Metabolome: Saturation, Desaturation, ��-Oxidation, and Protein Adduction

Nitrated derivatives of fatty acids (NO₂-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO₂-FA signal transduction lacks insight into how or if NO₂-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO₂) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO₂ or metabolites were detected under basal conditions, whereas administered 18:1-NO₂ is rapidly adducted to plasma thiol-containing proteins and glutathione. NO₂-FA are also metabolized via β-oxidation, with high performance liquid chromatography-tandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO₂ as metabolites. Additionally, a significant proportion of 18:1-NO₂ and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO₂ hydration or nitric oxide (•NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO₂ detected by exchange to added β-mercaptoethanol. This, coupled with the observation of 18:1-NO₂ release from glutathione-18:1-NO₂ adducts, supports that reversible and exchangeable NO₂-FA-thiol adducts occur under biological conditions. After administration of [³H]18:1-NO₂, 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet identified. In aggregate, these findings show that electrophilic FA nitroalkene derivatives (a) acquire an extended half-life by undergoing reversible and exchangeable electrophilic reactions with nucleophilic targets and (b) are metabolized predominantly via saturation of the double bond and β-oxidation reactions that terminate at the site of acyl-chain nitration.The reaction of unsaturated fatty acids with nitric oxide (•NO)- and nitrite species, including nitrogen dioxide (•NO₂), peroxynitrite (ONOO^–), and nitrous acid (HNO₂), yields a complex array of oxidized and nitrated products (1–4). The mechanisms of biological fatty acid nitration, the structural isomer distribution of nitrated fatty acids (NO₂-FAs)² and the signaling actions of specific NO₂-FA regioisomers remain incompletely characterized. Current data reveal that, during fatty acid oxidation and nitration, vinyl nitro regioisomers represent a component of these products that display distinctive chemical reactivities and receptor-dependent signaling actions. Here, we investigate the metabolic fate of the nitroalkene derivative of oleic acid (1, 2).Unsaturated fatty acid nitration was first described in model studies of air-pollutant-induced lipid oxidation where lipids were exposed to high concentrations of •NO₂ (5, 6). More recently nitrated unsaturated fatty acids have been reported as products of acidic reactions of , radical chain termination reactions induced by •NO (7–10), and the oxidation of to •NO₂ by the leukocyte-derived enzyme myeloperoxidase (1). Various mechanisms can mediate the formation of nitroalkene derivatives of unsaturated fatty acids (11), including homolytic attack of •NO₂ (12), reaction of •NO₂ with a pre-existing fatty acid carbon-centered radical (2, 13), and the protonation of nitrite under acidic conditions (pH 5.5 and lower) to yield an array of HNO₂-derived nitrating species (3, 14). The conditions promoting fatty acid nitration by •NO and species (low oxygen tension, radical formation, and low pH) are not expected to be broadly distributed systemically (e.g. in plasma or extracellular fluids). Rather, nitration reactions will preferably occur during inflammatory or metabolic stress in microenvironments such as the intermembrane space of mitochondria, the low pH environment of the digestive tract, and activated macrophage and neutrophil-rich compartments. Moreover, the acidic, replete and low O₂ tension conditions that promote nitration reactions are characteristic of inflammatory loci. Although multiple reactions leading to accelerated formation of nitrating species occur at specific anatomic sites, plasma levels of nitrated fatty acids are expected to be low due to events described herein.Robust electrophilic reactivity and avid nuclear lipid receptor ligand activity have conferred to the class of fatty acid nitroalkene derivatives potent anti-inflammatory properties that occur predominantly via non-cGMP-dependent mechanisms. Nitro derivatives of oleic and linoleic acid inhibit leukocyte and platelet activation (15), vascular smooth muscle proliferation (16), lipopolysaccharide-stimulated macrophage cytokine secretion (17), activate peroxisome proliferator-activated receptor-γ (1, 18), and induce endothelial heme oxygenase 1 expression (19). NO₂-FA also potently modulate nuclear factor-erythroid 2-related factor 2/Kelch-like ECH-associating protein 1 (Nrf2/Keap1) (16, 17) and nuclear factor κB (NFκB)-regulated inflammatory signaling (17). Previous observations of the •NO-mediated, cGMP-dependent vessel relaxation induced by NO₂-FA were made under serum- and lipid-free conditions. More recently, it has been appreciated that micellar and membrane stabilization of NO₂-FA prevents Nef-like aqueous decay reactions and consequent •NO release, supporting that the predominant signaling actions mediated by NO₂-FA are •NO and cGMP-independent (20, 21).Current data indicate that electrophilic adduction of biological targets primarily accounts for NO₂-FA signal transduction. The high electronegativity of NO₂ substituents, when bound to an alkenyl carbon of fatty acids, confers an electrophilic nature to the adjacent β-carbon and enables Michael addition reaction with nucleophiles such as protein His and Cys residues. This process, termed nitroalkylation (22), results in the clinically detectable and reversible adduction of the nucleophilic thiol of glutathione (GSH) and both cysteine and histidine residues of glyceraldehyde-3-phosphate dehydrogenase (23). Furthermore, inhibition of NFκB signaling occurs via nitroalkylation of p65 subunit thiols (17), and recent findings reveal that NO₂-FA activation of peroxisome proliferator-activated receptor-γ is uniquely induced by covalent nitroalkylation of the ligand binding domain Cys-285.³Multiple reports support the endogenous generation and presence of nitrated fatty acids (1, 24), first observed in bovine papillary muscles as a vicinal nitrohydroxyeicosatetraenoic acid (25). Nitrolinoleate has been detected in human blood plasma and cholesteryl nitrolinoleate in human plasma and lipoproteins (4, 26), with hyperlipidemic and post-prandial conditions elevating plasma levels of NO₂-FA. Further support for the inflammatory generation of NO₂-FA comes from lipopolysaccharide and interferon-γ-activated murine J774.1 macrophages, where increased nitration of the acyl chain of cholesteryl linoleate was paralleled by increased macrophage expression and activity of nitric-oxide synthase 2 (27).To date, insight into the mechanisms of nitroalkene signaling actions overshadows knowledge of the generation, trafficking, and metabolism of nitroalkenes in vivo. Appreciating that NO₂-FA derivatives are detectable clinically, and that their levels increase following •NO-dependent oxidative reactions (4, 28), challenges still exist in their routine detection. Because the in vivo administration of NO₂-FA may exert anti-inflammatory benefit, the disposition and metabolite profiles of these species in vivo is of relevance. Here we report that only 2.4% of nitrooctadecenoic acid (18:1-NO₂) is immediately detectable in the vascular compartment as native 18:1-NO₂ upon intravenous injection in mice, with the remaining pool of 18:1-NO₂ (a) reversibly bound to plasma and tissue thiols via Michael addition; (b) metabolized to nitro-octadecanoic acid (18:0-NO₂) and nitro-octadecadienoic acid (18:2-NO₂); and (c) catabolized by hepatic β-oxidation following thioester formation with coenzyme A.     

4-Aminotetrolic Acid : New Conformational-restricted Analogue of γ-Aminobutyric Acid

RECENT studies strongly support a role for γ-aminobutyric acid (GABA) as an inhibitory transmitter at certain synapses in the mammalian central nervous system. Structure activity correlations of many GABA analogues implicate both the intramolecular distance between the zwitterionic centres and the rotational freedom of the molecule as important factors governing the synaptic activity of these substances¹. The following observations provide pertinent information about the active conformation(s) of GABA recognized by the receptor. (1) Muscimol, an isoxazole isolated from Amanita muscaria, seems to function as a GABA analogue as its inhibitory action on central neurones is comparable with that of GABA both in potency² and with respect to antagonism by bicuculline³. Molecular orbital calculations suggest that GABA and muscimol can assume similar conformations as zwitterions with the charged centres (N⁺ and 0^?) at least 5 and, more likely, 6 Å apart⁴. (2) The selective GABA antagonist bicuculline exhibits some degree of structural similarity with particular conformations of GABA and muscimol³. (3) X-ray crystallography indicates that GABA exists in a partially folded conformation in the solid state^5,6. (4) A model of the GABA receptor proposes that GABA adopts a folded conformation with a distance of less than 4.4 Å between the charged centres⁷. Observations (1) and (2) suggest extended conformations for GABA, while (3) and (4) suggest folded conformations.     

Influence of Fatty Acid Precursors,Including Food Preservatives,on the Growth and Fatty Acid Composition of Listeria monocytogenes at 37 and 10°C

Listeria monocytogenes is a food-borne pathogen that grows at refrigeration temperatures and increases its content of anteiso-C_15:0 fatty acid, which is believed to be a homeoviscous adaptation to ensure membrane fluidity, at these temperatures. As a possible novel approach for control of the growth of the organism, the influences of various fatty acid precursors, including branched-chain amino acids and branched- and straight-chain carboxylic acids, some of which are also well-established food preservatives, on the growth and fatty acid composition of the organism at 37°C and 10°C were studied in order to investigate whether the organism could be made to synthesize fatty acids that would result in impaired growth at low temperatures. The results indicate that the fatty acid composition of L. monocytogenes could be modulated by the feeding of branched-chain amino acid, C₄, C₅, and C₆ branched-chain carboxylic acid, and C₃ and C₄ straight-chain carboxylic acid fatty acid precursors, but the growth-inhibitory effects of several preservatives were independent of effects on fatty acid composition, which were minor in the case of preservatives metabolized via acetyl coenzyme A. The ability of a precursor to modify fatty acid composition was probably a reflection of the substrate specificities of the first enzyme, FabH, in the condensation of primers of fatty acid biosynthesis with malonyl acyl carrier protein.Listeriosis is a severe and life-threatening human infection encompassing meningoencephalitis, meningitis, focal infections in the immunocompromised, and stillbirths and neonatal sepsis due to infection of pregnant women (2). The disease is caused by the Gram-positive food-borne pathogen Listeria monocytogenes, which is responsible for common-source and sporadic disease involving a variety of different foods (27). Listeriosis has a high fatality rate (24). The U.S. Department of Agriculture has a zero tolerance policy for L. monocytogenes in ready-to-eat products, and high costs are associated with product recalls.L. monocytogenes has a remarkably low minimum growth temperature, e.g., −0.1°C (34), and thus the organism can multiply to dangerous levels when food is kept at refrigeration temperatures. We are interested in the molecular mechanisms of L. monocytogenes psychrotolerance, with a view to applying this knowledge to improve the control of the growth of the organism. Although the adaptations involved in low-temperature tolerance are global in scope, we have focused on changes in fatty acid composition that result in homeoviscous adjustments of membrane fluidity (31, 36). L. monocytogenes has a fatty acid composition that is dominated to an unusual extent (90% or more) by branched-chain fatty acids (BCFAs); the major fatty acids are anteiso-C_15:0, anteiso-C_17:0, and iso-C_15:0. Numerous studies have shown that the major change in fatty acid composition when L. monocytogenes is grown at low temperatures is an increase in the content of anteiso-C_15:0 fatty acid to 65% or more of the total (1, 12, 23, 25, 26, 28). Two cold-sensitive mutants with Tn917 insertions in the branched-chain α-keto acid dehydrogenase gene complex (bkd) were deficient in BCFAs, grew poorly at low temperatures, and had decreased membrane fluidity; all of these defects could be restored by growth in the presence of 2-methylbutyrate (2-MB), a precursor of odd-numbered anteiso fatty acids, including anteiso-C_15:0 fatty acid (1, 7, 13, 37). We believe that anteiso-C_15:0 fatty acid imparts fluidity to the cytoplasmic membrane, as revealed by its low phase transition temperature in model phospholipids (18) and disruption of the close packing of fatty acyl chains (21, 35).The amino acids isoleucine, leucine, and valine are the starting points for the biosynthesis of odd-numbered anteiso, odd-numbered iso, and even-numbered iso fatty acids, respectively (18, 37). The amino acids are converted to their corresponding α-keto acid derivatives through the activity of branched-chain amino acid transaminase. Branched-chain α-keto acid dehydrogenase (Bkd) then converts these α-keto compounds to branched-chain acyl coenzyme A (acyl-CoA) primers of fatty acid biosynthesis (18). These primers are then used to initiate fatty acid biosynthesis through the activity of β-ketoacyl-acyl carrier protein synthase III (FabH), which prefers branched-chain acyl-CoAs to acetyl-CoA as substrates (4, 22, 32). β-Keto-acyl carrier protein synthase II (FabF) is responsible for subsequent rounds of elongation until the acyl chain reaches 14 to 17 carbon atoms (36).We wished to ascertain whether we could manipulate the fatty acid composition of L. monocytogenes by feeding precursors that favored the production of fatty acids other than anteiso-C_15:0 and thereby inhibit the growth of the organism, especially at low temperatures. Kaneda (15, 16) has grouped Bacillus subtilis fatty acids into four pairs based on the precursors from which they are generated, i.e., anteiso-C_15:0 and C_17:0 from isoleucine, iso-C_15:0 and C_17:0 from leucine, iso-C_14:0 and C_16:0 from valine, and n-C_14:0 and n-C_16:0 from acetate or butyrate. The proportions of the fatty acids could be modulated by precursor feeding. We have studied the effects of feeding the potential fatty acid precursors branched-chain amino acids, branched-chain α-keto acids, short branched-chain carboxylic acids, short straight-chain carboxylic acids, medium-length straight-chain carboxylic acids, branched-chain C₆ carboxylic acids, and sodium diacetate (Fig. ​(Fig.1)1) on the growth and fatty acid composition of L. monocytogenes. Various short-chain carboxylic acids are used as food preservatives (5, 8, 29), and it was of interest to see whether any of them had an effect on the fatty acid composition of L. monocytogenes. Precursors giving rise to C₅ and C₆ branched-chain acyl-CoA derivatives, propionate, and butyrate had significant impacts on growth and fatty acid composition. Acetate and precursors that were metabolized to acetyl-CoA had minor effects on fatty acid composition, indicating that their preservative action is not due to effects on fatty acid composition.Open in a separate windowFIG. 1.Structures of potential fatty acid precursors.     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

Occurrence of Endogenous Pollen Growth Inhibitors, 4-Hydroxybenzoic Acid and 4-(β-D-Glucopyranosyloxy)benzoic Acid,in the Pollen of Pinus densiflora†

A Pseudomonas strain capable of using pyrazinamide as the sole source of nitrogen was isolated from soil. An aromatic amidase from the bacterium was purified 400-fold to homogeneous on polyacrylamide gel electrophoresis. The enzyme had a molecular weight of 43,000 by gel filtration on Sephadex G-150 and consisted of two identical subunits. The isoelectric point was at 4.45. Among the compounds tested, pyrazinamide (relative activity, 100%), nicotinamide (60%), and 5-methylpyrazinamide (3.4%) were hydrolyzed at considerable rates. Benzamide, picolinamide, and isonicotinamide were not substrates. Apparent Km of the enzyme for pyrazinamide and nicotinamide were 5.6 × 10 ^?5 m and below 5 × 10^?6 m, respectively. The enzyme was not able to hydrolyze aliphatic amides. The enzyme was most active between pH 6.5 and 10 and 75°C, and was stable between pH 5.5 and 8.5 and below 45°C.     

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.     

The Production of α-Ketoglutaric Acid from Salicylic Acid by Pseudomonas sp.†

Metabolites from salicylic acid by microorganisms were investigated. About eighty strains of bacteria which were able to utilize salicylic acid as a sole source of carbon were isolated from soil and other natural sources.

Among these bacteria, several strains produced a large amount of keto acids in the culture fluid during the cultivation. The acid was isolated from the culture fluid of strain K 102 in crystalline form. The crystal was identified as α-ketoglutaric acid by physicochemical methods. From the taxonomical studies, the isolated bacterial strains K 102 and K 362 were assumed to be Pseudomonas sp.