Identification of keto- and hydroxy-dicarboxylic acids in remote marine aerosols from the western North Pacific: GC and GC/TOF-MS measurements

NASA Astrophysics Data System (ADS)

Vani, D.; Kawamura, K.; Tachibana, E.; Boreddy, S. K. R.

2015-12-01

Dicarboxylic acids (diacids) are dominant components of organic aerosols in the atmosphere. They contribute significantly to the total aerosol mass and have a serious impacts on global climate changes. However, studies on keto- and hydroxy-diacids in marine aerosols are limited. Compare to diacids, keto- and hydroxy-diacids are more hygroscopic due to the additional polar groups (OH and CO) and, hence, acts as cloud condensation nuclei (CCN). Molecular characterization of these compounds provides insight into organic aerosols sources and transformation pathways. We collected marine aerosols from remote Chichijima Island in the western North Pacific from December 2010 to November 2011 and studied for water-soluble keto- and hydroxy-diacids. Carboxyl groups were derivatized to dibutyl esters with 14% boron trifluoride/n-butanol, whereas hydroxyl groups were derivatized to trimethylsilyl ethers using N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA). After two-step derivatization, samples were injected to GC, GC/MS and GC/TOF-MS. In the GC chromatogram, we detected several new peaks after BSTFA derivatization of dibutyl ester fraction. Based on mass spectral interpretation, we found these peaks as homologues series of hydroxy-diacids and keto-diacids. Some of these hydroxy-diacids have been individually reported in literature in the laboratory photo-oxidation experiments and forest environments samples. But, there are no evidences to prove their sources and formation mechanism in the atmosphere. Here, we report for the first time homologous series of hydroxy-diacids (hC3di-hC6di) and keto-diacid (oxaloacetic acid, enol and keto forms) in remote marine atmosphere. Molecular distributions of hydroxy-diacids generally showed the predominance of malic acid followed by tartronic acid. Both hydroxy- and keto-diacids show significant positive correlation with oxalic acid and SO42-, suggesting that they are generated in the atmosphere and play an important role in the

Effect of short-term low-protein diet supplemented with keto acids on hyperphosphatemia in maintenance hemodialysis patients.

PubMed

Li, Haiming; Long, Quan; Shao, Chunhai; Fan, Hong; Yuan, Li; Huang, Bihong; Gu, Yong; Lin, Shanyan; Hao, Chuanming; Chen, Jing

2011-01-01

To evaluate the effects of short-term restriction of dietary protein intake (DPI) supplemented with keto acids on hyperphosphatemia in maintenance hemodialysis (MHD) patients. Forty MHD patients with uncontrolled hyperphosphatemia were randomized to either low DPI with keto acid-supplemented (sLP) or normal DPI (NP) group for 8 weeks. After 8 weeks, the sLP group was shifted to NP for another 8 weeks. Low-protein diet (LPD) was individualized with total caloric intake 30-35 kcal/kg/day, protein intake of 0.8 g/kg/day and phosphate intake of 500 mg/day. Keto acids were supplied in a dosage of 12 pills per day. Calcium phosphorous metabolism index and nutritional index (serum albumin, total protein, somatometric measurements, 3-day diaries and Mini-Nutritional Assessment score) were recorded. C-reactive protein, CO(2) combining power and Kt/V were measured to evaluate the inflammation, metabolic acidosis and dialysis adequacy, respectively. Serum phosphorus level and calcium-phosphate product were significantly decreased at the end of the first 8 weeks in the sLP group compared to the basal value and the NP group (p < 0.001). No difference was observed in C-reactive protein, Kt/V and nutritional index, while CO(2) combining power was significantly higher at week 8 in the sLP group (p < 0.001). Short-term restriction of DPI supplemented with keto acids could decrease hyperphosphatemia and calcium-phosphate product, while keeping stable nutritional status among MHD patients. Copyright © 2010 S. Karger AG, Basel.

Effect of a keto acid-amino acid supplement on the metabolism and renal elimination of branched-chain amino acids in patients with chronic renal insufficiency on a low protein diet.

PubMed

Teplan, V; Schück, O; Horácková, M; Skibová, J; Holecek, M

2000-10-27

The aim of our study was to evaluate the effect of a low-protein diet supplemented with keto acids-amino acids on renal function and urinary excretion of branched-chain amino acids (BCAA) in patients with chronic renal insufficiency (CRI). In a prospective investigation 28 patients with CRI (16 male, 12 female, aged 28-66 yrs, CCr 18.6 +/- 10.2 ml/min) on a low-protein diet (0.6 g of protein /kg BW/day and energy intake 140 kJ/kg BW/day) for a period of one month were included. Subsequently, this low protein diet was supplemented with keto acids-amino acids at a dose of 0.1 g/kg BW/day orally for a period of 3 months. Examinations performed at baseline and at the end of the follow-up period revealed significant increase in the serum levels of BCAA leucine (p < 0.02), isoleucine (p < 0.03), and valine (p < 0.02) while their renal fractional excretion declined (p < 0.02, p < 0.01 resp.). Keto acid-amino acid administration had no effect on renal function and on the clearance of inulin, para-aminohippuric acid. Endogenous creatinine and urea clearance remained unaltered. A significant correlation between fractional excretion of sodium and leucine (p < 0.05) and a hyperbolic relationship between inulin clearance and fractional excretion of BCAA (p < 0.01) were seen. Moreover, a significant decrease in proteinuria (p < 0.02), plasma urea concentration and renal urea excretion and a rise in albumin level (p < 0.03) were noted. We conclude that in patients with CRI on a low protein diet the supplementation of keto acids-amino acids does not affect renal hemodynamics, but is associated--despite increases in plasma concentrations--with a reduction of renal amino acid and protein excretion suggesting induction of alterations in the tubular transport mechanisms.

The role of keto acids in the supportive treatment of children with chronic renal failure.

PubMed

Mir, Sevgi; Ozkayin, Nese; Akgun, Aysegul

2005-07-01

According to the hyperfiltration theory of renal diseases characterized by a decrease in the number of functional nephrons, increased arterial blood pressure, excessive protein intake in the diet, high levels of calcium (Ca) and phosphorus (P), secondary hyperparathyroidism, hypertriglyceridemia and/or hypercholesterolemia, proteinuria and metabolic acidosis are some factors that impair the prognosis of the disease. The amount of protein in the diet is the most important of these factors. A protein-restricted diet administered to patients with chronic renal failure results in the risk of inadequate amino acid intake. To overcome this problem, the use of dysaminated alpha-keto analogues has been considered to reduce the risk of nitrogenemia resulting from the continuous intake of essential amino acids. Currently, the necessity of essential amino acids even in adult patients with chronic renal failure is controversial; besides, trials on the use of these amino acids in pediatric patients are scarce. The aim of this study is to investigate the efficacy and applicability of conservative therapy with a protein-restricted diet supplemented with keto acids in the management of chronic renal insufficiency or failure.

Interactions among the branched-chain amino acids and their effects on methionine utilization in growing pigs: effects on plasma amino- and keto-acid concentrations and branched-chain keto-acid dehydrogenase activity.

PubMed

Langer, S; Scislowski, P W; Brown, D S; Dewey, P; Fuller, M F

2000-01-01

The present experiment was designed to elucidate the mechanism of the methionine-sparing effect of excess branched-chain amino acids (BCAA) reported in the previous paper (Langer & Fuller, 2000). Twelve growing gilts (30-35 kg) were prepared with arterial catheters. After recovery, they received for 7 d a semipurified diet with a balanced amino acid pattern. On the 7th day blood samples were taken before (16 h postabsorptive) and after the morning meal (4 h postprandial). The animals were then divided into three groups and received for a further 7 d a methionine-limiting diet (80% of requirement) (1) without any amino acid excess; (2) with excess leucine (50% over requirement); or (3) with excesses of all three BCAA (leucine, isoleucine, valine, each 50% over the requirement). On the 7th day blood samples were taken as in the first period, after which the animals were killed and liver and muscle samples taken. Plasma amino acid and branched-chain keto acid (BCKA) concentrations in the blood and branched-chain keto-acid dehydrogenase (BCKDH; EC 1.2.4.4) activity in liver and muscle homogenates were determined. Compared with those on the balanced diet, pigs fed on methionine-limiting diets had significantly lower (P < 0.05) plasma methionine concentrations in the postprandial but not in the postabsorptive state. There was no effect of either leucine or a mixture of all three BCAA fed in excess on plasma methionine concentrations. Excess dietary leucine reduced (P < 0.05) the plasma concentrations of isoleucine and valine in both the postprandial and postabsorptive states. Plasma concentrations of the BCKA reflected the changes in the corresponding amino acids. Basal BCKDH activity in the liver and total BCKDH activity in the biceps femoris muscle were significantly (P < 0.05) increased by excesses of leucine or all BCAA.

Clustered Genes Encoding 2-Keto-l-Gulonate Reductase and l-Idonate 5-Dehydrogenase in the Novel Fungal d-Glucuronic Acid Pathway

PubMed Central

Kuivanen, Joosu; Arvas, Mikko; Richard, Peter

2017-01-01

D-Glucuronic acid is a biomass component that occurs in plant cell wall polysaccharides and is catabolized by saprotrophic microorganisms including fungi. A pathway for D-glucuronic acid catabolism in fungal microorganisms is only partly known. In the filamentous fungus Aspergillus niger, the enzymes that are known to be part of the pathway are the NADPH requiring D-glucuronic acid reductase forming L-gulonate and the NADH requiring 2-keto-L-gulonate reductase that forms L-idonate. With the aid of RNA sequencing we identified two more enzymes of the pathway. The first is a NADPH requiring 2-keto-L-gulonate reductase that forms L-idonate, GluD. The second is a NAD+ requiring L-idonate 5-dehydrogenase forming 5-keto-gluconate, GluE. The genes coding for these two enzymes are clustered and share the same bidirectional promoter. The GluD is an enzyme with a strict requirement for NADP+/NADPH as cofactors. The kcat for 2-keto-L-gulonate and L-idonate is 21.4 and 1.1 s-1, and the Km 25.3 and 12.6 mM, respectively, when using the purified protein. In contrast, the GluE has a strict requirement for NAD+/NADH. The kcat for L-idonate and 5-keto-D-gluconate is 5.5 and 7.2 s-1, and the Km 30.9 and 8.4 mM, respectively. These values also refer to the purified protein. The gluD deletion resulted in accumulation of 2-keto-L-gulonate in the liquid cultivation while the gluE deletion resulted in reduced growth and cessation of the D-glucuronic acid catabolism. PMID:28261181

New Complexity-Building Reactions of Alpha-Keto Esters

NASA Astrophysics Data System (ADS)

Bartlett, Samuel L.

I. Introduction: Importance of Asymmetric Catalysis and the Reactivity Patterns of alpha-Keto Esters. II. Synthesis of Complex Tertiary Glycolates by Enantioconvergent Arylation of Stereochemically Labile alpha-Keto Esters. Enantioconvergent arylation reactions of boronic acids and racemic ?-stereogenic alpha-keto esters have been developed. The reactions are catalyzed by a chiral (diene)Rh(I) complex and provide a wide array of beta-stereogenic tertiary aryl glycolate derivatives with high levels of diastereo- and enantioselectivity. Racemization studies employing a series of sterically differentiated tertiary amines suggest that the steric nature of the amine base additive exerts a significant influence on the rate of substrate racemization. III. Palladium-Catalyzed beta-Arylation of alpha-Keto Esters . A catalyst system derived from commercially available Pd2(dba) 3 and PtBu3 has been applied to the coupling of alpha-keto ester enolates and aryl bromides. The reaction provides access to an array of beta-stereogenic alpha-keto ester derivatives. When the air stable ligand precursor PtBu 3˙HBF4 is employed, the reaction can be carried out without use of a glovebox. The derived products are of broad interest given the prevalence of the alpha-keto acid substructure in biologically important molecules. IV. Catalytic Enantioselective [3+2] Cycloaddition of alpha-Keto Ester Enolates and Nitrile Oxides. An enantioselective [3+2] cycloaddition reaction between nitrile oxides and transiently generated enolates of alpha-keto esters has been developed. The catalyst system was found to be compatible with in situ nitrile oxide generation conditions. A versatile array of nitrile oxides and alpha-keto esters could participate in the cycloaddition, providing novel 5-hydroxy-2-isoxazolines in high chemical yield with high levels of diastereo- and enantioselectivity. Notably, the optimal reaction conditions circumvented concurrent reaction via O-imidoylation and hetero-[3

Production of α-keto acids Part I. Immobilized cells ofTrigonopsis variabilis containing D-amino acid oxidase.

PubMed

Brodelius, P; Nilsson, K; Mosbach, K

1981-12-01

Whole cells ofTrigonopsis variabilis were immobilized by entrapment in Ca(2+)-alginate and used for the production of α-keto acids from the corresponding D-amino acids. The D-amino acid oxidase within the immobilized cells has a broad substrate specificity. Hydrogen peroxide formed in the enzymatic reaction was efficiently hydrolyzed by manganese oxide co-immobilized with the cells. The amino acid oxidase activity was assayed with a new method based on reversed-phase HPLC. Oxygen requirements, bead size, concentration of cells in the beads, flow rate, and other factors were investigated in a " trickle-bed " reactor.

A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate*

PubMed Central

Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

2015-01-01

Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-d-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-d-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18–60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. PMID:26555267

A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-L-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-D-gluconate.

PubMed

Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

2015-12-25

Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼ 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of hepatic branched-chain alpha-keto acid dehydrogenase complex in rats fed a high-fat diet

USDA-ARS?s Scientific Manuscript database

Objective: Branched-chain alpha-keto acid dehydrogenase complex (BCKDC) regulates branched-chain amino acid (BCAA) metabolism at the level of branched chain alpha-ketoacid (BCKA) catabolism. It has been demonstrated that the activity of hepatic BCKDC is markedly decreased in type 2 diabetic animal...

[Effects of keto/amino acids and a low-protein diet on the nutritional status of patients with Stages 3B-4 chronic kidney disease].

PubMed

Milovanova, S Yu; Milovanov, Yu S; Taranova, M V; Dobrosmyslov, I A

To evaluate the efficacy of keto/amino acids in maintaining protein balance and preventing mineral metabolic disturbances and the development of uremic hyperparathyroidism in the long-term use of a low-protein diet (LPD) in patients with Stages 3B-4 chronic kidney disease (CKD). Ninety patients with CKD caused by chronic latent glomerulonephritis in 65 patients and chronic tubulointerstitial nephritis of various etiologies (gout, drug-induced, and infection) in 25 were examined. The investigators conducted clinical, laboratory, and instrumental examinations, including bioelectrical impedance analysis (body mass index (BMI), the percentages of lean and fat mass), echocardiography and radiography of the abdominal aorta in the lateral projection (the presence of cardiac valvular and aortic calcification), and pulse wave velocity measurements using a Sphygmocor apparatus (vessel stiffness estimation). The stages of CKD were defined according to the 2012 Kidney Disease: Improving Global Outcomes (KDIGO) criteria; glomerular filtration rate was calculated using the CKD EPI equation. According to the diet used, all the patients were divided into 3 groups: 1) 30 patients who took LPD (0.6 g of protein per kg of body weight/day) in combination with the keto/amino acid ketosteril (1 tablet per 5 kg of body weight/day; Diet One); 2) 30 patients who used LPD in combination with the other keto/amino acid ketoaminol at the same dose (Diet Two); 3) 30 patients had LPD without using the keto/amino acids (Diet Three) (a control group). During a follow-up, there were no signs of malnutrition in Groups 1 and 2 patients receiving LPD (0.6 g protein per kg/day) in combination with the keto/amino acids ketosteril and ketaminol, respectively. At the same time, 11 (36.6%) patients in Group 3 (a control group) who did not take the keto/amino acids showed a BMI decrease from 24 (23; 26) kg/m2 to 18.5 (17; 19.2) kg/m2 (p < 0.05), including that of lean body mass from 37.4 (36; 38.8) to 30

Multi-enzymatic one-pot reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid with whole-cell biocatalysts.

PubMed

Sun, Boqiao; Kantzow, Christina; Bresch, Sven; Castiglione, Kathrin; Weuster-Botz, Dirk

2013-01-01

Ursodeoxycholic acid (UDCA) is a bile acid of industrial interest as it is used as an agent for the treatment of primary sclerosing cholangitis and the medicamentous, non-surgical dissolution of gallstones. Currently, it is prepared industrially from cholic acid following a seven-step chemical procedure with an overall yield of <30%. In this study, we investigated the key enzymatic steps in the chemo-enzymatic preparation of UDCA-the two-step reduction of dehydrocholic acid (DHCA) to 12-keto-ursodeoxycholic acid using a mutant of 7β-hydroxysteroid dehydrogenase (7β-HSDH) from Collinsella aerofaciens and 3α-hydroxysteroid dehydrogenase (3α-HSDH) from Comamonas testosteroni. Three different one-pot reaction approaches were investigated using whole-cell biocatalysts in simple batch processes. We applied one-biocatalyst systems, where 3α-HSDH, 7β-HSDH, and either a mutant of formate dehydrogenase (FDH) from Mycobacterium vaccae N10 or a glucose dehydrogenase (GDH) from Bacillus subtilis were expressed in a Escherichia coli BL21(DE3) based host strain. We also investigated two-biocatalyst systems, where 3α-HSDH and 7β-HSDH were expressed separately together with FDH enzymes for cofactor regeneration in two distinct E. coli hosts that were simultaneously applied in the one-pot reaction. The best result was achieved by the one-biocatalyst system with GDH for cofactor regeneration, which was able to completely convert 100 mM DHCA to >99.5 mM 12-keto-UDCA within 4.5 h in a simple batch process on a liter scale. Copyright © 2012 Wiley Periodicals, Inc.

Binding pattern of intermediate UDP-4-keto-xylose to human UDP-xylose synthase: Synthesis and STD NMR of model keto-saccharides.

PubMed

Puchner, Claudia; Eixelsberger, Thomas; Nidetzky, Bernd; Brecker, Lothar

2017-01-02

Human UDP-xylose synthase (hUXS1) exclusively converts UDP-glucuronic acid to UDP-xylose via intermediate UDP-4-keto-xylose (UDP-Xyl-4O). Synthesis of model compounds like methyl-4-keto-xylose (Me-Xyl-4O) is reported to investigate the binding pattern thereof to hUXS1. Hence, selective oxidation of the desired hydroxyl function required employment of protecting group chemistry. Solution behavior of synthesized keto-saccharides was studied without enzyme via 1 H and 13 C NMR spectroscopy with respect to existent forms in deuterated potassium phosphate buffer. Keto-enol tautomerism was observed for all investigated keto-saccharides, while gem-diol hydrate forms were only observed for 4-keto-xylose derivatives. Saturation transfer difference (STD) NMR was used to study binding of synthesized keto-gylcosides to wild type hUXS1. Resulting epitope maps were correlated to earlier published molecular modeling studies of UDP-Xyl-4O. STD NMR results of Me-Xyl-4O are in good agreement with simulations of the intermediate UDP-Xyl-4O indicating a strong interaction of proton H3 with the enzyme, potentially caused by active site residue Ala 79 . In contrast, pyranoside binding pattern studies of methyl uronic acids showed some differences compared to previously published STD NMR results of UDP-glycosides. In general, obtained results can contribute to a better understanding in binding of UDP-glycosides to other UXS enzyme family members, which have high structural similarities in the active site. Copyright © 2016. Published by Elsevier Ltd.

[Effects of carbon and nitrogen sources on 5-keto-gluconic acid production].

PubMed

Tan, Zhilei; Wang, Hongcui; Wei, Yuqiao; Li, Yanyan; Zhong, Cheng; Jia, Shiru

2014-01-01

Gluconobacter oxydans is known to oxidize glucose to gluconic acid (GA), and subsequently, to 2-keto-gluconic acid (2KGA) and 5-keto-gluconic acid (5KGA), while 5KGA can be converted to L-(+)-tartaric acid. In order to increase the production of 5KGA, Gluconobacter oxydans HGI-1 that converts GA to 5KGA exclusively was chosen in this study, and effects of carbon sources (lactose, maltose, sucrose, amylum and glucose) and nitrogen sources (yeast extract, fish meal, corn steep liquor, soybean meal and cotton-seed meal) on 5KGA production were investigated. Results of experiment in 500 mL shake-flask show that the highest yield of 5KGA (98.20 g/L) was obtained using 100 g/L glucose as carbon source. 5KGA reached 100.20 g/L, 109.10 g/L, 99.83 g/L with yeast extract, fish meal and corn steep liquor as nitrogen source respectively, among which the optimal nitrogen source was fish meal. The yield of 5KGA by corn steep liquor is slightly lower than that by yeast extract. For the economic reason, corn steep liquor was selected as nitrogen source and scaled up to 5 L stirred-tank fermentor, and the final concentration of 5KGA reached 93.80 g/L, with its maximum volumetric productivity of 3.48 g/(L x h) and average volumetric productivity of 1.56 g/(L x h). The result obtained in this study showed that carbon and nitrogen sourses for large-scale production of 5KGA by Gluconobacter oxydans HGI-1 were glucose and corn steep liquor, respectively, and the available glucose almost completely (85.93%) into 5KGA.

Expression of mitochondrial branched-chain aminotransferase and α-keto-acid dehydrogenase in rat brain: implications for neurotransmitter metabolism

PubMed Central

Cole, Jeffrey T.; Sweatt, Andrew J.; Hutson, Susan M.

2012-01-01

In the brain, metabolism of the essential branched chain amino acids (BCAAs) leucine, isoleucine, and valine, is regulated in part by protein synthesis requirements. Excess BCAAs are catabolized or excreted. The first step in BCAA catabolism is catalyzed by the branched chain aminotransferase (BCAT) isozymes, mitochondrial BCATm and cytosolic BCATc. A product of this reaction, glutamate, is the major excitatory neurotransmitter and precursor of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). The BCATs are thought to participate in a α-keto-acid nitrogen shuttle that provides nitrogen for synthesis of glutamate from α-ketoglutarate. The branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC) catalyzes the second, irreversible step in BCAA metabolism, which is oxidative decarboxylation of the branched-chain α-keto acid (BCKA) products of the BCAT reaction. Maple Syrup Urine Disease (MSUD) results from genetic defects in BCKDC, which leads to accumulation of toxic levels of BCAAs and BCKAs that result in brain swelling. Immunolocalization of BCATm and BCKDC in rats revealed that BCATm is present in astrocytes in white matter and in neuropil, while BCKDC is expressed only in neurons. BCATm appears uniformly distributed in astrocyte cell bodies throughout the brain. The segregation of BCATm to astrocytes and BCKDC to neurons provides further support for the existence of a BCAA-dependent glial-neuronal nitrogen shuttle since the data show that BCKAs produced by glial BCATm must be exported to neurons. Additionally, the neuronal localization of BCKDC suggests that MSUD is a neuronal defect involving insufficient oxidation of BCKAs, with secondary effects extending beyond the neuron. PMID:22654736

Keto acid-supplemented low-protein diet for treatment of adult patients with hepatitis B virus infection and chronic glomerulonephritis.

PubMed

Mou, Shan; Li, Jialin; Yu, Zanzhe; Wang, Qin; Ni, Zhaohui

2013-02-01

An open-label, randomized, controlled, single-centre clinical trial to evaluate the effects of low-protein intake, with or without keto acid supplementation, on nutritional status and proteinuria, in patients with hepatitis B virus (HBV) and early stage chronic glomerulonephritis. Patients with chronic glomerulonephritis and HBV infection were randomized to receive a low-protein diet (0.6-0.8 g/kg ideal body weight [IBW] per day) either without (LP group) or with (sLP group) keto acid supplementation (0.1 g/kg IBW per day), for 12 months. Nutritional, clinical and safety parameters were recorded. The study included 17 patients (LP group n = 9; sLP group n = 8). Proteinuria and microalbuminuria were significantly lower in the sLP group at 6 and 12 months compared with baseline, and at 12 months compared with the LP group. There were no significant differences in serum creatinine level or estimated glomerular filtration rate. Nutritional parameters (serum albumin and prealbumin) were significantly improved at 12 months, compared with baseline, in the sLP group. Restriction of dietary protein intake to 0.6-0.8 g/kg IBW per day appears to have an acceptable safety profile. Supplementation with keto acids is associated with decreased urine protein excretion.

Acute supplementation with keto analogues and amino acids in rats during resistance exercise.

PubMed

de Almeida, Rosemeire Dantas; Prado, Eduardo Seixas; Llosa, Carlos Daniel; Magalhães-Neto, Anibal; Cameron, Luiz-Claudio

2010-11-01

During exercise, ammonia levels are related to the appearance of both central and peripheral fatigue. Therefore, controlling the increase in ammonia levels is an important strategy in ameliorating the metabolic response to exercise and in improving athletic performance. Free amino acids can be used as substrates for ATP synthesis that produces ammonia as a side product. Keto analogues act in an opposite way, being used to synthesise amino acids whilst decreasing free ammonia in the blood. Adult male rats were divided into four groups based on receiving either keto analogues associated with amino acids (KAAA) or a placebo and resistance exercise or no exercise. There was an approximately 40% increase in ammonaemia due to KAAA supplementation in resting animals. Exercise increased ammonia levels twofold with respect to the control, with a smaller increase (about 20%) in ammonia levels due to exercise. Exercise itself causes a significant increase in blood urea levels (17%). However, KAAA reduced blood urea levels to 75% of the pre-exercise values. Blood urate levels increased 28% in the KAAA group, independent of exercise. Supplementation increased glucose levels by 10% compared with control animals. Exercise did not change glucose levels in either the control or supplemented groups. Exercise promoted a 57% increase in lactate levels in the control group. Supplementation promoted a twofold exercise-induced increase in blood lactate levels. The present results suggest that an acute supplementation of KAAA can decrease hyperammonaemia induced by exercise.

Major urinary metabolites of 6-keto-prostaglandin F2α in mice[S

PubMed Central

Kuklev, Dmitry V.; Hankin, Joseph A.; Uhlson, Charis L.; Hong, Yu H.; Murphy, Robert C.; Smith, William L.

2013-01-01

Western diets are enriched in omega-6 vs. omega-3 fatty acids, and a shift in this balance toward omega-3 fatty acids may have health benefits. There is limited information about the catabolism of 3-series prostaglandins (PG) formed from eicosapentaenoic acid (EPA), a fish oil omega-3 fatty acid that becomes elevated in tissues following fish oil consumption. Quantification of appropriate urinary 3-series PG metabolites could be used for noninvasive measurement of omega-3 fatty acid tone. Here we describe the preparation of tritium- and deuterium-labeled 6-keto-PGF2α and their use in identifying urinary metabolites in mice using LC-MS/MS. The major 6-keto-PGF2α urinary metabolites included dinor-6-keto-PGF2α (∼10%) and dinor-13,14-dihydro-6,15-diketo-PGF1α (∼10%). These metabolites can arise only from the enzymatic conversion of EPA to the 3-series PGH endoperoxide by cyclooxygenases, then PGI3 by prostacyclin synthase and, finally, nonenzymatic hydrolysis to 6-keto-PGF2α. The 6-keto-PGF derivatives are not formed by free radical mechanisms that generate isoprostanes, and thus, these metabolites provide an unbiased marker for utilization of EPA by cyclooxygenases. PMID:23644380

Low-Protein Diet Supplemented with Keto Acids Is Associated with Suppression of Small-Solute Peritoneal Transport Rate in Peritoneal Dialysis Patients

PubMed Central

Jiang, Na; Qian, Jiaqi; Lin, Aiwu; Fang, Wei; Zhang, Weiming; Cao, Liou; Wang, Qin; Ni, Zhaohui; Yao, Qiang

2011-01-01

Objective. We investigate whether low-protein diet would show benefits in suppressing peritoneal transport rate in peritoneal dialysis (PD) patients. Methods. This is a supplemented analysis of our previously published trial, which randomized 60 PD patients to receive low- (LP: dietary protein intake of 0.6–0.8 g/kg/d), keto-acid-supplemented low- (sLP: 0.6–0.8 g/kg/d with 0.12 g/kg/d of keto acids), or high- (HP: 1.0–1.2 g/kg/d) protein diet and lasted for one year. In this study, the variations of peritoneal transport rate were assessed. Results. While baseline D/Pcr (dialysate-to-plasma concentration ratio for creatinine at 4 hour) and D/D0glu (dialysate glucose at 4 hour to baseline dialysate glucose concentration ratio) were similar, D/Pcr in group sLP was lower, and D/D0glu was higher than those in the other two groups (P < 0.05) at 12th month. D/D0glu increased (P < 0.05), and D/Pcr tended to decrease, (P = 0.071) in group sLP. Conclusions. Low-protein diet with keto acids may benefit PD patients by maintaining peritoneum at a lower transport rate. PMID:21747999

Low-protein diet supplemented with keto acids is associated with suppression of small-solute peritoneal transport rate in peritoneal dialysis patients.

PubMed

Jiang, Na; Qian, Jiaqi; Lin, Aiwu; Fang, Wei; Zhang, Weiming; Cao, Liou; Wang, Qin; Ni, Zhaohui; Yao, Qiang

2011-01-01

Objective. We investigate whether low-protein diet would show benefits in suppressing peritoneal transport rate in peritoneal dialysis (PD) patients. Methods. This is a supplemented analysis of our previously published trial, which randomized 60 PD patients to receive low- (LP: dietary protein intake of 0.6-0.8 g/kg/d), keto-acid-supplemented low- (sLP: 0.6-0.8 g/kg/d with 0.12 g/kg/d of keto acids), or high- (HP: 1.0-1.2 g/kg/d) protein diet and lasted for one year. In this study, the variations of peritoneal transport rate were assessed. Results. While baseline D/P(cr) (dialysate-to-plasma concentration ratio for creatinine at 4 hour) and D/D0(glu) (dialysate glucose at 4 hour to baseline dialysate glucose concentration ratio) were similar, D/P(cr) in group sLP was lower, and D/D0(glu) was higher than those in the other two groups (P < 0.05) at 12th month. D/D0(glu) increased (P < 0.05), and D/P(cr) tended to decrease, (P = 0.071) in group sLP. Conclusions. Low-protein diet with keto acids may benefit PD patients by maintaining peritoneum at a lower transport rate.

[Effects of low-protein diet plus alpha-keto acid on micro-inflammation and the relationship between micro-inflammation and nutritional status in patients performing continuous ambulatory peritoneal dialysis: a randomized controlled trial].

PubMed

Chen, Wei; Guo, Zhi-Yong; Wu, Hao; Sun, Li-Jing; Cai, Li-Li; Xu, Hai-Yan

2008-05-01

To investigate the effects of the combination of alpha-keto acid and low-protein diet on the levels of serum cytokines in patients performing continuous ambulatory peritoneal dialysis (CAPD) and to explore the relationship between inflammation and malnutrition in CAPD patients. Eighty-nine CAPD patients were randomized into three groups, and 78 cases completed a one-year follow-up and with complete data. There were 31 cases in low-protein diet plus alpha-keto acid group, 26 cases in low-protein diet group and 21 cases in routine-protein diet group. The levels of serum albumin (Alb), prealbumin (PA), retinol-binding protein (RBP), transferrin (TRF), cholesterol (TC), triglycerides (TG), leptin, and triceps skinfold thickness (TSF), mid-arm muscle circumference (MAMC), body mass index (BMI) were measured. The changes of serum interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein (CRP) were also detected. Compared with low-protein diet group, serum levels of PA, RBP and TRF were significantly increased both in low-protein diet plus alpha-keto acid and routine-protein diet groups ( P<0.01), however, there was no significant difference in the levels of PA, RBP and TRF between low-protein diet plus alpha-keto acid group and routine-protein diet group. There was an increased tendency in the content of Alb, TC, TG, BMI, TSF and MAMC, but there were no significant differences. The plasma levels of IL-1alpha, IL-6 and TNF-alpha in low-protein diet plus alpha-keto acid group were decreased as compared with the routine-protein diet group, but there were no significant differences. The plasma level of CRP in low-protein diet plus alpha-keto acid group was lower than that in the routine-protein diet group ( P<0.01). The combination of alpha-keto acid and low-protein diet can ameliorate malnutrition and micro-inflammation in CAPD patients.

Silver-catalyzed double-decarboxylative cross-coupling of α-keto acids with cinnamic acids in water: a strategy for the preparation of chalcones.

PubMed

Zhang, Ning; Yang, Daoshan; Wei, Wei; Yuan, Li; Nie, Fafa; Tian, Laijin; Wang, Hua

2015-03-20

A silver-catalyzed double-decarboxylative protocol has been proposed for the construction of chalcone derivatives via cascade coupling of substituted α-keto acids with cinnamic acids under the mild aqueous conditions. The developed method for constructing C-C bonds via double-decarboxylative reactions is efficient, practical, and environmentally benign by using the readily available starting materials. It should provide a promising synthesis candidate for the formation of diverse and useful chalcone derivatives in the fields of synthetic and pharmaceutical chemistry.

Dynamic mechanistic modeling of the multienzymatic one-pot reduction of dehydrocholic acid to 12-keto ursodeoxycholic acid with competing substrates and cofactors.

PubMed

Sun, Boqiao; Hartl, Florian; Castiglione, Kathrin; Weuster-Botz, Dirk

2015-01-01

Ursodeoxycholic acid (UDCA) is a bile acid which is used as pharmaceutical for the treatment of several diseases, such as cholesterol gallstones, primary sclerosing cholangitis or primary biliary cirrhosis. A potential chemoenzymatic synthesis route of UDCA comprises the two-step reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid (12-keto-UDCA), which can be conducted in a multienzymatic one-pot process using 3α-hydroxysteroid dehydrogenase (3α-HSDH), 7β-hydroxysteroid dehydrogenase (7β-HSDH), and glucose dehydrogenase (GDH) with glucose as cosubstrate for the regeneration of cofactor. Here, we present a dynamic mechanistic model of this one-pot reduction which involves three enzymes, four different bile acids, and two different cofactors, each with different oxidation states. In addition, every enzyme faces two competing substrates, whereas each bile acid and cofactor is formed or converted by two different enzymes. First, the kinetic mechanisms of both HSDH were identified to follow an ordered bi-bi mechanism with EBQ-type uncompetitive substrate inhibition. Rate equations were then derived for this mechanism and for mechanisms describing competing substrates. After the estimation of the model parameters of each enzyme independently by progress curve analyses, the full process model of a simple batch-process was established by coupling rate equations and mass balances. Validation experiments of the one-pot multienzymatic batch process revealed high prediction accuracy of the process model and a model analysis offered important insight to the identification of optimum reaction conditions. © 2015 American Institute of Chemical Engineers.

Alpha-keto acid metabolites of organoselenium compounds inhibit histone deacetylase activity in human colon cancer cells.

PubMed

Nian, Hui; Bisson, William H; Dashwood, Wan-Mohaiza; Pinto, John T; Dashwood, Roderick H

2009-08-01

Methylselenocysteine (MSC) and selenomethionine (SM) are two organoselenium compounds receiving interest for their potential anticancer properties. These compounds can be converted to beta-methylselenopyruvate (MSP) and alpha-keto-gamma-methylselenobutyrate (KMSB), alpha-keto acid metabolites that share structural features with the histone deacetylase (HDAC) inhibitor butyrate. We tested the organoselenium compounds in an in vitro assay with human HDAC1 and HDAC8; whereas SM and MSC had little or no activity up to 2 mM, MSP and KMSB caused dose-dependent inhibition of HDAC activity. Subsequent experiments identified MSP as a competitive inhibitor of HDAC8, and computational modeling supported a mechanism involving reversible interaction with the active site zinc atom. In human colon cancer cells, acetylated histone H3 levels were increased during the period 0.5-48 h after treatment with MSP and KMSB, and there was dose-dependent inhibition of HDAC activity. The proportion of cells occupying G(2)/M of the cell cycle was increased at 10-50 microM MSP and KMSB, and apoptosis was induced, as evidenced by morphological changes, Annexin V staining and increased cleaved caspase-3, -6, -7, -9 and poly(adenosine diphosphate-ribose)polymerase. P21WAF1, a well-established target gene of clinically used HDAC inhibitors, was increased in MSP- and KMSB-treated colon cancer cells at both the messenger RNA and protein level, and there was enhanced P21WAF1 promoter activity. These studies confirm that in addition to targeting redox-sensitive signaling molecules, alpha-keto acid metabolites of organoselenium compounds alter HDAC activity and histone acetylation status in colon cancer cells, as recently observed in human prostate cancer cells.

[Autophagy-lysosome pathway in skeletal muscle of diabetic nephropathy rats and the effect of low-protein diet plus α-keto acids on it].

PubMed

Huang, Juan; Yuan, Wei-jie; Wang, Jia-lin; Gu, Li-jie; Yin, Jun; Dong, Ting; Bao, Jin-fang; Tang, Zhi-huan

2013-11-26

To explore the regulation of autophagy-lysosome pathway (ALP) in skeletal muscle of diabetic nephropathy and examine the effect of low protein diet plus α-keto acid on ALP. A total of 45 24-week-old Goto-Kakizaki rats were randomized to receive normal protein (22%) diet (NPD), low-protein (6%) diet (LPD) or low-protein (5%) plus α-keto acids (1%) diet (Keto) (n = 15 each). Wistar control rats had a normal protein diet. The mRNA and protein levels of ALP markers LC3B, Bnip3, Cathepsin L in soleus muscle were evaluated at 48 weeks. Electron microscopy was used to confirm the changes of autophagy. Compared with CTL group, the mRNA levels of LC3B, Bnip3, Cathepsin L in soleus muscle of rats on NPD were higher, and protein levels of LC3B-I, LC3B-II, Bnip3, Cathepsin L in soleus muscle of rats on NPD also higher than CTL group (0.82 ± 0.33 vs 0.25 ± 0.07, 0.76 ± 0.38 vs 0.20 ± 0.12, 1.25 ± 0.30 vs 0.56 ± 0.19, 1.29 ± 0.40 vs 0.69 ± 0.20). The mRNA levels of LC3B, Bnip3 and Cathepsin L in LPD group were slightly lower, compared with NPD group. However there was no statistical significance. Similarly the protein levels of LC3B-I, LC3B-II, Bnip3 and Cathepsin L in LPD group were slightly lower with no statistical significance. In contrast, the mRNA levels of LC3B, Bnip3 and Cathepsin L were greatly lower in Keto group in comparison with NPD and LPD. And protein levels of LC3B-I, LC3B-II, Bnip3 and Cathepsin L were also greatly lower in Keto group in comparison with NPD and LPD. Additionally, autophagosome or auto-lysosome was found in NPD and LPD groups by electron microscopy. ALP is activated in skeletal muscle of diabetic nephropathy rats. And low protein plus α-keto acid decrease the activation of ALP and improve muscle wasting.

[Comparison of the effects of alpha-keto/ amino acid supplemented low protein diet and diabetes diet in patients with diabetic nephropathy].

PubMed

Qiu, Hong-yu; Liu, Fang; Zhao, Li-jun; Huang, Song-min; Zuo, Chuan; Zhong, Hui; Chen, Feng

2012-05-01

To investigate if a-keto/amino acid supplemented low protein diet can slow down the progression of diabetic nephrophathy in comparison with non-supplemented diabetes diet. A prospective, randomized, controlled clinical study was conducted. Twenty three cases of type 2 diabetic nephropathy in IV stage were randomly divided into alpha-keto/amino acid supplemented diet group (trial group) and conventional diabetes diet group (control group), The treatment duration was 52 weeks. 24 h urine protein was measured at 0, 12, 20, 36 and 52 weeks. Before and after the 52 weeks treatment, all the patients received the measurement of glomerular filtration rate (GFR), blood glucose, blood lipids, inflammatory markers, as well as nutritional status. After the treatment for 20, 36, 52 weeks, mean 24 h urine protein decreased significantly in trial groups (P < 0.05), and 24 h urine protein in trial group were significantly decreased (P < 0.05) compared with control group in 20 weeks after treatment. Either in trial group or in control group, GFR remained relatively stable during the observation period. Nutrition status, inflammatory markers, and serum calcium, phosphorus levels between the two groups were no significantly difference. The adverse events experienced by the patients in trial group were similar and consistent with the patients underlying renal diseases. Alpha-keto/amino acid can reduce proteinuria more effectively, while improve renal function and nutritional status in diabetic nephropathy patients with well-toleration.

Structure-Guided Engineering of α-Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature.

PubMed

Sutiono, Samuel; Carsten, Jörg; Sieber, Volker

2018-06-28

Branched chain keto acid decarboxylases (KDCs) are a class of enzymes that catalyze the decarboxylation of α-keto acids. It is a key enzyme for production of higher alcohols in vivo and in vitro. However, the two most active KDCs (KivD and KdcA) have only moderate thermostability (<55 °C) hindering the production of the alcohols at high temperatures. In this study, structure-guided engineering toward improved thermostability of KdcA is outlined. Several strategies such as, stabilization of the catalytic center, surface engineering, and optimization of dimer interactions were applied. With 7 point mutations, our mutant (7M.D) showed an increase of T501h by 14.8 °C without compromising its substrate specificity. 7M.D exhibited >400-fold improvement of half-life at 70 °C and >600-fold increase in process stability in the presence of 4 % isobutanol at 50 °C. 7M.D is more promising for the production of higher alcohols in thermophiles (>65 °C) as well as in cell-free applications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Decreased enzyme activity and contents of hepatic branched-chain alpha-keto acid dehydrogenase complex subunits in a rat model for type 2 diabetes mellitus.

PubMed

Bajotto, Gustavo; Murakami, Taro; Nagasaki, Masaru; Sato, Yuzo; Shimomura, Yoshiharu

2009-10-01

The mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC) is responsible for the committed step in branched-chain amino acid catabolism. In the present study, we examined BCKDC regulation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats both before (8 weeks of age) and after (25 weeks of age) the onset of type 2 diabetes mellitus. Long-Evans Tokushima Otsuka (LETO) rats were used as controls. Plasma branched-chain amino acid and branched-chain alpha-keto acid concentrations were significantly increased in young and middle-aged OLETF rats. Although the hepatic complex was nearly 100% active in all animals, total BCKDC activity and protein abundance of E1alpha, E1beta, and E2 subunits were markedly lower in OLETF than in LETO rats at 8 and 25 weeks of age. In addition, hepatic BCKDC activity and protein amounts were significantly decreased in LETO rats aged 25 weeks than in LETO rats aged 8 weeks. In skeletal muscle, E1beta and E2 proteins were significantly reduced, whereas E1alpha tended to increase in OLETF rats. Taken together, these results suggest that (1) whole-body branched-chain alpha-keto acid oxidation capacity is extremely reduced in OLETF rats independently of diabetes development, (2) the aging process decreases BCKDC activity and protein abundance in the liver of normal rats, and (3) differential posttranscriptional regulation for the subunits of BCKDC may exist in skeletal muscle.

Better preservation of residual renal function in peritoneal dialysis patients treated with a low-protein diet supplemented with keto acids: a prospective, randomized trial.

PubMed

Jiang, Na; Qian, Jiaqi; Sun, Weilan; Lin, Aiwu; Cao, Liou; Wang, Qin; Ni, Zhaohui; Wan, Yanping; Linholm, Bengt; Axelsson, Jonas; Yao, Qiang

2009-08-01

While a low-protein diet may preserve residual renal function (RRF) in chronic kidney disease (CKD) patients before the start of dialysis, a high-protein intake is usually recommended in dialysis patients to prevent protein-energy wasting. Keto acids, which were often recommended to pre-dialysis CKD patients treated with a low-protein diet, had also been reported to be associated with both RRF and nutrition maintenance. We conducted a randomized trial to test whether a low-protein diet with or without keto acids would be safe and associated with a preserved RRF during peritoneal dialysis (PD). To assess the safety of low protein, we first conducted a nitrogen balance study in 34 incident PD patients randomized to receive in-centre diets containing 1.2, 0.9 or 0.6 g of protein/kg ideal body weight (IBW)/day for 10 days. Second, 60 stable PD patients [RRF 4.04 +/- 2.30 ml/ min/1.73 m(2), urine output 1226 +/- 449 ml/day, aged 53.6 +/- 12.8 years, PD duration 8.8 (1.5-17.8) months] were randomized to receive either a low- (LP: 0.6-0.8 g/kg IBW/day), keto acid-supplemented low- (sLP: 0.6-0.8 g/kg IBW/day with 0.12 g/kg IBW/day of keto acids) or high-protein (HP: 1.0-1.2 g/kg IBW/day) diet. The groups were followed for 1 year and RRF as well as nutritional status was evaluated serially. A neutral or positive nitrogen balance was achieved in all three groups. RRF remained stable in group sLP (3.84 +/- 2.17 to 3.39 +/- 3.23 ml/min/1.73 m(2), P = ns) while it decreased in group LP (4.02 +/- 2.49 to 2.29 +/- 1.72 ml/min/1.73 m(2), P < 0.05) and HP (4.25 +/- 2.34 to 2.55 +/- 2.29 ml/min/1.73 m(2), P < 0.05). There was no change from baseline on nutritional status in any of the groups during follow-up. A diet containing 0.6-0.8 g of protein/kg IBW/day is safe and, when combined with keto acids, is associated with an improved preservation of RRF in relatively new PD patients without significant malnutrition or inflammation.

Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation

PubMed Central

Smit, Bart A.; van Hylckama Vlieg, Johan E. T.; Engels, Wim J. M.; Meijer, Laura; Wouters, Jan T. M.; Smit, Gerrit

2005-01-01

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain α-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3′ terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain α-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions. PMID:15640202

Improved training tolerance by supplementation with α-Keto acids in untrained young adults: a randomized, double blind, placebo-controlled trial

PubMed Central

2012-01-01

Background Exercise causes a variety of physiological and metabolic changes that can in turn reduce exercise tolerance. One of the potential mechanisms responsible for fatigue is “exercise-induced hyperammonemia”. Previous studies have shown that supplementation with amino acids can increase training tolerance. The α-keto acids are biochemical analogs of amino acids and can be converted to amino acids through transamination, thus reducing the cellular ammonia level. This double blind, placebo-controlled study was designed to investigate the effects of α-keto acid supplementation (KAS) on training tolerance, training effect, and stress-recovery state. Methods Thirty-three untrained young male adults underwent four weeks of training (5 sessions/week; 30 minutes running at the individual anaerobic threshold followed by 3 x 3 minute sprints/each session). Throughout the 4 weeks of training and one week of recovery, subjects took α-ketoglutarate (AKG group, 0.2 g/kg/d, n = 9), branched-chain keto acids (BCKA group, 0.2 g/kg/d, n = 12) or isocaloric placebo (control group, n = 12) daily. Results The 4th week training volume, maximum power output and muscle torque were higher in the AKG group (175 ± 42 min, 412 ± 49 Watts and 293 ± 58 Newton meters, respectively, P<0.05) and the BCKA group (158 ± 35, 390 ± 29 and 273 ± 47, P<0.05) than in the control group (92 ± 70, 381 ± 67 and 233 ± 43). The general stress and emotional exhaustion as assessed by the rest-stress-questionnaire-sport after the 3rd week of training increased significantly in the control group (P<0.05), but not in the KAS groups. Conclusions Under KAS, subjects could bear a higher training volume and reach a higher power output and peak muscle torque, accompanied by a better stress-recovery-state. Thus, KAS improves exercise tolerance and training effects along with a better stress-recovery state. Whether the improved training tolerance

10-oxo-12(Z)-octadecenoic acid, a linoleic acid metabolite produced by gut lactic acid bacteria, potently activates PPARγ and stimulates adipogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goto, Tsuyoshi, E-mail: tgoto@kais.kyoto-u.ac.jp; Research Unit for Physiological Chemistry, The Center for the Promotion of Interdisciplinary Education and Research, Kyoto University; Kim, Young-Il

2015-04-17

Our previous study has shown that gut lactic acid bacteria generate various kinds of fatty acids from polyunsaturated fatty acids such as linoleic acid (LA). In this study, we investigated the effects of LA and LA-derived fatty acids on the activation of peroxisome proliferator-activated receptors (PPARs) which regulate whole-body energy metabolism. None of the fatty acids activated PPARδ, whereas almost all activated PPARα in luciferase assays. Two fatty acids potently activated PPARγ, a master regulator of adipocyte differentiation, with 10-oxo-12(Z)-octadecenoic acid (KetoA) having the most potency. In 3T3-L1 cells, KetoA induced adipocyte differentiation via the activation of PPARγ, and increasedmore » adiponectin production and insulin-stimulated glucose uptake. These findings suggest that fatty acids, including KetoA, generated in gut by lactic acid bacteria may be involved in the regulation of host energy metabolism. - Highlights: • Most LA-derived fatty acids from gut lactic acid bacteria potently activated PPARα. • Among tested fatty acids, KetoA and KetoC significantly activated PPARγ. • KetoA induced adipocyte differentiation via the activation of PPARγ. • KetoA enhanced adiponectin production and glucose uptake during adipogenesis.« less

2-Keto-3-fluoroglutarate: a useful mechanistic probe of 2-keto-glutarate-dependent enzyme systems.

PubMed

Grissom, C B; Cleland, W W

1987-12-18

2-Keto-3-fluoroglutaric acid prepared by acid hydrolysis of its diethyl ester is stable, as the free acid in aqueous solution at pH 2, and can be stored at -20 degrees C for several years. Both enantiomers are reduced by NADH in the presence of glutamate dehydrogenase (EC 1.4.1.2) to the two diastereomers of 3-fluoro-L-glutamate, which are stable at neutral pH and at high pH unless heated. 2-Keto-3-fluoroglutarate exists in solution almost entirely as a hydrate both at low and neutral pH. Both enantiomers of ketofluoroglutarate react with the pyridoxamine forms of aspartate, alanine and 4-aminobutyrate transaminases to give fluoride release. 2 mol of cosubstrate amino acid react for each mol of ketofluoroglutarate (KFG) when starting from the pyridoxamine form of the enzyme: 2 RCHNH2COOH + KFG + H2O----F- + NH4+ + glutamate + 2 RCOCOOH. Both diastereomers of fluoroglutamate are decarboxylated by glutamate decarboxylase (EC 4.1.1.15) with fluoride release: KFG + H2O----CO2 + F- + HCOCH2CH2COOH. By contrast, only one isomer of fluoroglutamate will react with the pyridoxal form of glutamate-oxalacetate transaminase to give fluoride release: HOOCCHNH2CHFCH2COOH + H2O----4F- + NH4+ + HOOCCOCH2CH2COOH. The enzymatic decarboxylation of 3-fluoroisocitrate produces only one enantiomer of ketofluoroglutarate, which is reduced to threo (2R,3R)-3-fluoroglutamate by NADH and glutamate dehydrogenase: [2R,3S]-HOOCCH(OH)CF(COOH)CH2COOH + NADP+----[3R]-KFG + CO2 + NADPH + H+. The proton, 13C, and 19F-NMR parameters of ketofluoroglutarate and the two fluoroglutamate diastereomers are presented. These molecules are useful probes of enzymatic mechanisms thought to involve carbanion intermediates.

Branched-Chain Amino and Keto Acid Biochemistry and Cellular Biology in Central Nervous System Diseases

DTIC Science & Technology

2009-05-21

pyruvate dehydrogenase complex (PDC) and 2-oxo- glutarate dehydrogenase complex. These dehydrogenase complexes share the same basic structure, perform the...Science 312 (2006) 927-930. [20] J. Dancis, M. Levitz, R.G. Westall, Maple syrup urine disease: branched- chain keto- aciduria , Pediatrics 25 (1960...2127 2128 Dancis J, Levitz M, Westall RG. 1960. Maple syrup urine disease: branched-chain keto- aciduria . Pediatrics 25:72-9. Danner DJ, Lemmon

10-Oxo-trans-11-octadecenoic acid generated from linoleic acid by a gut lactic acid bacterium Lactobacillus plantarum is cytoprotective against oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furumoto, Hidehiro; Nanthirudjanar, Tharnath; Kume, Toshiaki

Oxidative stress is a well-known cause of multiple diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a central role in cellular antioxidative responses. In this study, we investigated the effects of novel fatty acid metabolite derivatives of linoleic acid generated by the gut lactic acid bacteria Lactobacillus plantarum on the Nrf2-ARE pathway. 10-Oxo-trans-11-octadecenoic acid (KetoC) protected HepG2 cells from cytotoxicity induced by hydrogen peroxide. KetoC also significantly increased cellular Nrf2 protein levels, ARE-dependent transcription, and the gene expression of antioxidative enzymes such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H:quinone oxidoreductasemore » 1 (NQO1) in HepG2 cells. Additionally, a single oral dose administration of KetoC also increased antioxidative gene expression and protein levels of Nrf2 and HO-1 in mouse organs. Since other fatty acid metabolites and linoleic acid did not affect cellular antioxidative responses, the cytoprotective effect of KetoC may be because of its α,β-unsaturated carbonyl moiety. Collectively, our data suggested that KetoC activated the Nrf2-ARE pathway to enhance cellular antioxidative responses in vitro and in vivo, which further suggests that KetoC may prevent multiple diseases induced by oxidative stress. - Highlights: • We evaluated the effect of modified fatty acids generated by Lactobacillus plantarum. • 10-Oxo-trans-11-ocatadecenoic acid (KetoC) protected cells from oxidative stress. • KetoC activated the Nrf2-ARE pathway to promote antioxidative gene expression. • KetoC promoted the expression of antioxidative enzymes in mice organs. • The cytoprotective effect of KetoC was because of α,β-unsaturated carbonyl moiety.« less

Dynamic Kinetic Asymmetric Transformations of β-Stereogenic-α-Keto Esters via Direct Aldolization

PubMed Central

Corbett, Michael T.; Johnson, Jeffrey S.

2014-01-01

Dynamic kinetic asymmetric transformations (DyKAT) of racemic β-bromo-α-keto esters via direct aldolization of nitromethane and acetone provide access to fully substituted α-glycolic acid derivatives bearing a β-stereocenter. The aldol adducts are obtained in excellent yield with high relative and absolute stereocontrol under mild reaction conditions. Mechanistic studies determined that the reactions proceed through a facile catalyst-mediated racemization of the β-bromo-α-keto esters under a DyKAT Type I manifold. PMID:24222195

The use of nutritional supplements to induce ketosis and reduce symptoms associated with keto-induction: a narrative review.

PubMed

Harvey, Cliff J D C; Schofield, Grant M; Williden, Micalla

2018-01-01

Adaptation to a ketogenic diet (keto-induction) can cause unpleasant symptoms, and this can reduce tolerability of the diet. Several methods have been suggested as useful for encouraging entry into nutritional ketosis (NK) and reducing symptoms of keto-induction. This paper reviews the scientific literature on the effects of these methods on time-to-NK and on symptoms during the keto-induction phase. PubMed, Science Direct, CINAHL, MEDLINE, Alt Health Watch, Food Science Source and EBSCO Psychology and Behavioural Sciences Collection electronic databases were searched online. Various purported ketogenic supplements were searched along with the terms "ketogenic diet", "ketogenic", "ketosis" and ketonaemia (/ ketonemia). Additionally, author names and reference lists were used for further search of the selected papers for related references. Evidence, from one mouse study, suggests that leucine doesn't significantly increase beta-hydroxybutyrate (BOHB) but the addition of leucine to a ketogenic diet in humans, while increasing the protein-to-fat ratio of the diet, doesn't reduce ketosis. Animal studies indicate that the short chain fatty acids acetic acid and butyric acid, increase ketone body concentrations. However, only one study has been performed in humans. This demonstrated that butyric acid is more ketogenic than either leucine or an 8-chain monoglyceride. Medium-chain triglycerides (MCTs) increase BOHB in a linear, dose-dependent manner, and promote both ketonaemia and ketogenesis. Exogenous ketones promote ketonaemia but may inhibit ketogenesis. There is a clear ketogenic effect of supplemental MCTs; however, it is unclear whether they independently improve time to NK and reduce symptoms of keto-induction. There is limited research on the potential for other supplements to improve time to NK and reduce symptoms of keto-induction. Few studies have specifically evaluated symptoms and adverse effects of a ketogenic diet during the induction phase. Those that

Theoretical study on keto-enol tautomerisation of glutarimide for exploration of the isomerisation reaction pathway of glutamic acid in proteins using density functional theory

NASA Astrophysics Data System (ADS)

Fukuyoshi, Shuichi; Nakayoshi, Tomoki; Takahashi, Ohgi; Oda, Akifumi

2017-03-01

In order to elucidate the reason why glutamic acid residues have lesser racemisation reactivity than asparaginic acid, we investigated the racemisation energy barrier of piperidinedione, which is the presumed intermediate of the isomerisation reaction of L-Glu to D-Glu, by density functional theory calculations. In two-water-molecule-assisted racemisation, the activation barrier for keto-enol isomerisation was 28.1 kcal/mol. The result showed that the activation barrier for the racemisation of glutamic acid residues was not different from that for the racemisation of aspartic acid residues. Thus, glutamic acid residues can possibly cause the racemisation reaction if the cyclic intermediate stably exists.

Modeling and parameters identification of 2-keto-L-gulonic acid fed-batch fermentation.

PubMed

Wang, Tao; Sun, Jibin; Yuan, Jingqi

2015-04-01

This article presents a modeling approach for industrial 2-keto-L-gulonic acid (2-KGA) fed-batch fermentation by the mixed culture of Ketogulonicigenium vulgare (K. vulgare) and Bacillus megaterium (B. megaterium). A macrokinetic model of K. vulgare is constructed based on the simplified metabolic pathways. The reaction rates obtained from the macrokinetic model are then coupled into a bioreactor model such that the relationship between substrate feeding rates and the main state variables, e.g., the concentrations of the biomass, substrate and product, is constructed. A differential evolution algorithm using the Lozi map as the random number generator is utilized to perform the model parameters identification, with the industrial data of 2-KGA fed-batch fermentation. Validation results demonstrate that the model simulations of substrate and product concentrations are well in coincidence with the measurements. Furthermore, the model simulations of biomass concentrations reflect principally the growth kinetics of the two microbes in the mixed culture.

The use of nutritional supplements to induce ketosis and reduce symptoms associated with keto-induction: a narrative review

PubMed Central

Schofield, Grant M.; Williden, Micalla

2018-01-01

Background Adaptation to a ketogenic diet (keto-induction) can cause unpleasant symptoms, and this can reduce tolerability of the diet. Several methods have been suggested as useful for encouraging entry into nutritional ketosis (NK) and reducing symptoms of keto-induction. This paper reviews the scientific literature on the effects of these methods on time-to-NK and on symptoms during the keto-induction phase. Methods PubMed, Science Direct, CINAHL, MEDLINE, Alt Health Watch, Food Science Source and EBSCO Psychology and Behavioural Sciences Collection electronic databases were searched online. Various purported ketogenic supplements were searched along with the terms “ketogenic diet”, “ketogenic”, “ketosis” and ketonaemia (/ ketonemia). Additionally, author names and reference lists were used for further search of the selected papers for related references. Results Evidence, from one mouse study, suggests that leucine doesn’t significantly increase beta-hydroxybutyrate (BOHB) but the addition of leucine to a ketogenic diet in humans, while increasing the protein-to-fat ratio of the diet, doesn’t reduce ketosis. Animal studies indicate that the short chain fatty acids acetic acid and butyric acid, increase ketone body concentrations. However, only one study has been performed in humans. This demonstrated that butyric acid is more ketogenic than either leucine or an 8-chain monoglyceride. Medium-chain triglycerides (MCTs) increase BOHB in a linear, dose-dependent manner, and promote both ketonaemia and ketogenesis. Exogenous ketones promote ketonaemia but may inhibit ketogenesis. Conclusions There is a clear ketogenic effect of supplemental MCTs; however, it is unclear whether they independently improve time to NK and reduce symptoms of keto-induction. There is limited research on the potential for other supplements to improve time to NK and reduce symptoms of keto-induction. Few studies have specifically evaluated symptoms and adverse effects of a

Evidence for the identity and some comparative properties of alpha-ketoglutarate and 2-keto-4-hydroxyglutarate dehydrogenase activity.

PubMed

Gupta, S C; Dekker, E E

1980-02-10

Enzyme preparations of pig heart and Escherichia coli are shown to catalyze a NAD+- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate. Several independent lines of evidence support the conclusion that this hydroxyketo acid is a substrate for the well known alpha-ketoglutarate dehydrogenase complex of the citric acid cycle. The evidence includes (a) a constant ratio of specific activity values for the two substrates through several steps of purification, (b) identical elution profiles from a calcium phosphate gel-cellulose column and a constant ratio of specific activity toward the two substrates throughout the activity peak, (c) identical inactivation curves in controlled heat denaturation studies, (d) the same pH activity curves, (e) no effect on the oxidation of either keto acid by repeated freezing and thawing of dehydrogenase preparations, and (f) the same activity pattern when the E. coli complex is distributed into several fractions by sucrose density gradient centrifugation. Additionally, the same cofactors are required for maximal activity and glyoxylate inhibits the oxidation of either substrate noncompetitively. Ferricyanide-linked oxidation of 2-keto-4-hydroxyglutarate yields malate as the product and a 1:2:1 stoichiometric relationship is obtained between the amount of hydroxyketo acid oxidized, ferricyanide reduced, and malate formed.

Role of Aldo-Keto Reductase Family 1 (AKR1) Enzymes in Human Steroid Metabolism

PubMed Central

Rižner, Tea Lanišnik; Penning, Trevor M.

2013-01-01

Human aldo-keto reductases AKR1C1-AKR1C4 and AKR1D1 play essential roles in the metabolism of all steroid hormones, the biosynthesis of neurosteroids and bile acids, the metabolism of conjugated steroids, and synthetic therapeutic steroids. These enzymes catalyze NADPH dependent reductions at the C3, C5, C17 and C20 positions on the steroid nucleus and side-chain. AKR1C1-AKR1C4 act as 3-keto, 17-keto and 20-ketosteroid reductases to varying extents, while AKR1D1 acts as the sole Δ4-3-ketosteroid-5β-reductase (steroid 5β-reductase) in humans. AKR1 enzymes control the concentrations of active ligands for nuclear receptors and control their ligand occupancy and trans-activation, they also regulate the amount of neurosteroids that can modulate the activity of GABAA and NMDA receptors. As such they are involved in the pre-receptor regulation of nuclear and membrane bound receptors. Altered expression of individual AKR1C genes is related to development of prostate, breast, and endometrial cancer. Mutations in AKR1C1 and AKR1C4 are responsible for sexual development dysgenesis and mutations in AKR1D1 are causative in bile-acid deficiency. PMID:24189185

Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism.

PubMed

Rižner, Tea Lanišnik; Penning, Trevor M

2014-01-01

Human aldo-keto reductases AKR1C1-AKR1C4 and AKR1D1 play essential roles in the metabolism of all steroid hormones, the biosynthesis of neurosteroids and bile acids, the metabolism of conjugated steroids, and synthetic therapeutic steroids. These enzymes catalyze NADPH dependent reductions at the C3, C5, C17 and C20 positions on the steroid nucleus and side-chain. AKR1C1-AKR1C4 act as 3-keto, 17-keto and 20-ketosteroid reductases to varying extents, while AKR1D1 acts as the sole Δ(4)-3-ketosteroid-5β-reductase (steroid 5β-reductase) in humans. AKR1 enzymes control the concentrations of active ligands for nuclear receptors and control their ligand occupancy and trans-activation, they also regulate the amount of neurosteroids that can modulate the activity of GABAA and NMDA receptors. As such they are involved in the pre-receptor regulation of nuclear and membrane bound receptors. Altered expression of individual AKR1C genes is related to development of prostate, breast, and endometrial cancer. Mutations in AKR1C1 and AKR1C4 are responsible for sexual development dysgenesis and mutations in AKR1D1 are causative in bile-acid deficiency. Copyright © 2013 Elsevier Inc. All rights reserved.

Stereochemistry and function of oxaloacetate keto-enol tautomerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Creighton, D.J.; Johnson, J.D.; Lambert, M.R.

1986-05-01

Oxaloacetate keto-enol tautomerase, partially purified from porcine kidney, catalyzes the conversions of enol- to keto-oxaloacetate by a mechanism in which solvent protons end up equally distributed between the two prochiral positions at C3 of keto-oxaloacetate. This conclusion is based upon the observation that when enzyme catalyzed ketonization is conducted in /sup 3/H/sub 2/O in the presence of excess malate dehydrogenase and NADH, only 50% of the /sup 3/H in the isolated (2S)-(3-/sup 3/H)malate is labilized to solvent upon treatment with fumarase. Either the tautomerase operates on the basis of a highly unusual stereomechanistic principle or tautomerase activity is not anmore » evolved property of the enzyme protein. As a result of an attempt to clarify the physiological importance of oxaloacetate tautomerase activity, keto-oxaloacetate was demonstrated to be directly transported across the inner membrane of rat liver mitochrondria, on the basis of the results of kinetic and isotope-trapping experiments.« less

Structure-based Mechanism of CMP-2-keto-3-deoxymanno-octulonic Acid Synthetase

PubMed Central

Heyes, Derren J.; Levy, Colin; Lafite, Pierre; Roberts, Ian S.; Goldrick, Marie; Stachulski, Andrew V.; Rossington, Steven B.; Stanford, Deborah; Rigby, Stephen E. J.; Scrutton, Nigel S.; Leys, David

2009-01-01

The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2β-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2β-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg2+ ion (Mg-B). Both ligands occupy conformations compatible with an Sn2-type attack on the α-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg2+ ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp100 and Asp235 in addition to the CTP α-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn2+-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg2+ to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the α-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group. PMID:19815542

Crystal Structures of Apo and Liganded 4-Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous β-Keto Acid.

PubMed

Guimarães, Samuel L; Coitinho, Juliana B; Costa, Débora M A; Araújo, Simara S; Whitman, Christian P; Nagem, Ronaldo A P

2016-05-10

The enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria Pseudomonas putida G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg(2+) as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent β-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous β-keto acid decarboxylase and the first crystal structure of a 4-OD.

Highly Functionalized 1,2–Diamino Compounds through Reductive Amination of Amino Acid-Derived β–Keto Esters

PubMed Central

Pérez-Faginas, Paula; Aranda, M. Teresa; García-López, M. Teresa; Infantes, Lourdes; Fernández-Carvajal, Asia; González-Ros, José Manuel; Ferrer-Montiel, Antonio; González-Muñiz, Rosario

2013-01-01

1,2-Diamine derivatives are valuable building blocks to heterocyclic compounds and important precursors of biologically relevant compounds. In this respect, amino acid-derived β–keto esters are a suitable starting point for the synthesis of β,γ–diamino ester derivatives through a two-step reductive amination procedure with either simple amines or α–amino esters. AcOH and NaBH3CN are the additive and reducing agents of choice. The stereoselectivity of the reaction is still an issue, due to the slow imine-enamine equilibria through which the reaction occurs, affording mixtures of diastereoisomers that can be chromatographically separated. Transformation of the β,γ–diamino esters into pyrrolidinone derivatives allows the configuration assignment of the linear compounds, and constitutes an example of their potential application in the generation of molecular diversity. PMID:23308167

Substituent Effects on Keto-Enol Equilibria Using NMR Spectroscopy

ERIC Educational Resources Information Center

Manbeck, Kimberly A.; Boaz, Nicholas C.; Bair, Nathaniel C.; Sanders, Allix M. S.; Marsh, Anderson L.

2011-01-01

In this extension to a classic physical chemistry experiment, students record the proton nuclear magnetic resonance spectra of the [beta]-diketones 2,4-pentanedione, 3-methyl-2,4-pentanedione, and 3-chloro-2,4-pentanedione to investigate the effect of substituents on keto-enol tautomerization equilibria. From the integrated intensities of keto and…

Keto analogue and amino acid supplementation affects the ammonaemia response during exercise under ketogenic conditions.

PubMed

Prado, Eduardo Seixas; de Rezende Neto, José Melquiades; de Almeida, Rosemeire Dantas; Dória de Melo, Marcelia Garcez; Cameron, Luiz-Claudio

2011-06-28

Hyperammonaemia is related to both central and peripheral fatigue during exercise. Hyperammonaemia in response to exercise can be reduced through supplementation with either amino acids or combined keto analogues and amino acids (KAAA). In the present study, we determined the effect of short-term KAAA supplementation on ammonia production in subjects eating a low-carbohydrate diet who exercise. A total of thirteen male cyclists eating a ketogenic diet for 3 d were divided into two groups receiving either KAAA (KEx) or lactose (control group; LEx) supplements. Athletes cycled indoors for 2 h, and blood samples were obtained at rest, during exercise and over the course of 1 h during the recovery period. Exercise-induced ammonaemia increased to a maximum of 35 % in the control group, but no significant increase was observed in the supplemented group. Both groups had a significant increase (approximately 35 %) in uraemia in response to exercise. The resting urate levels of the two groups were equivalent and remained statistically unchanged in the KEx group after 90 min of exercise; an earlier increase was observed in the LEx group. Glucose levels did not change, either during the trial time or between the groups. An increase in lactate levels was observed during the first 30 min of exercise in both groups, but there was no difference between the groups. The present results suggest that the acute use of KAAA diminishes exercise-induced hyperammonaemia.

A simple synthesis of 2-keto-3-deoxy-D-erythro-hexonic acid isopropyl ester, a key sugar for the bacterial population living under metallic stress.

PubMed

Grison, Claire M; Renard, Brice-Loïc; Grison, Claude

2014-02-01

2-Keto-3-deoxy-D-erythro-hexonic acid (KDG) is the key intermediate metabolite of the Entner Doudoroff (ED) pathway. A simple, efficient and stereoselective synthesis of KDG isopropyl ester is described in five steps from 2,3-O-isopropylidene-D-threitol with an overall yield of 47%. KDG isopropyl ester is studied as an attractive marker of a functional Entner Doudoroff pathway. KDG isopropyl ester is used to promote growth of ammonium producing bacterial strains, showing interesting features in the remediation of heavy-metal polluted soils. Copyright © 2013 Elsevier Inc. All rights reserved.

Aldo-keto Reductase 1B15 (AKR1B15)

PubMed Central

Weber, Susanne; Salabei, Joshua K.; Möller, Gabriele; Kremmer, Elisabeth; Bhatnagar, Aruni; Adamski, Jerzy; Barski, Oleg A.

2015-01-01

Aldo-keto reductases (AKRs) comprise a superfamily of proteins involved in the reduction and oxidation of biogenic and xenobiotic carbonyls. In humans, at least 15 AKR superfamily members have been identified so far. One of these is a newly identified gene locus, AKR1B15, which clusters on chromosome 7 with the other human AKR1B subfamily members (i.e. AKR1B1 and AKR1B10). We show that alternative splicing of the AKR1B15 gene transcript gives rise to two protein isoforms with different N termini: AKR1B15.1 is a 316-amino acid protein with 91% amino acid identity to AKR1B10; AKR1B15.2 has a prolonged N terminus and consists of 344 amino acid residues. The two gene products differ in their expression level, subcellular localization, and activity. In contrast with other AKR enzymes, which are mostly cytosolic, AKR1B15.1 co-localizes with the mitochondria. Kinetic studies show that AKR1B15.1 is predominantly a reductive enzyme that catalyzes the reduction of androgens and estrogens with high positional selectivity (17β-hydroxysteroid dehydrogenase activity) as well as 3-keto-acyl-CoA conjugates and exhibits strong cofactor selectivity toward NADP(H). In accordance with its substrate spectrum, the enzyme is expressed at the highest levels in steroid-sensitive tissues, namely placenta, testis, and adipose tissue. Placental and adipose expression could be reproduced in the BeWo and SGBS cell lines, respectively. In contrast, AKR1B15.2 localizes to the cytosol and displays no enzymatic activity with the substrates tested. Collectively, these results demonstrate the existence of a novel catalytically active AKR, which is associated with mitochondria and expressed mainly in steroid-sensitive tissues. PMID:25577493

Metabolic effects of keto acid--amino acid supplementation in patients with chronic renal insufficiency receiving a low-protein diet and recombinant human erythropoietin--a randomized controlled trial.

PubMed

Teplan, V; Schück, O; Votruba, M; Poledne, R; Kazdová, L; Skibová, J; Malý, J

2001-09-17

Supplement with keto acids/amino acids (KA) and erythropoietin can independently improve the metabolic sequels of chronic renal insufficiency. Our study was designed to establish whether a supplementation with keto acids/amino acids (KA) exerts additional beneficial metabolic effects in patients with chronic renal insufficiency (CRF) treated with a low-protein diet (LPD) and recombinant human erythropoietin (EPO). In a prospective randomized controlled trial over a period of 12 months, we evaluated a total of 38 patients (20 M/18 F) aged 32-68 years with a creatinine clearance (CCr) of 20-36 ml/min. All patients were receiving EPO (40 U/kg twice a week s.c.) and a low-protein diet (0.6 g protein/kg/day and 145 kJ/kg/day). The diet of 20 patients (Group I) was supplemented with KA at a dosage of 100 mg/kg/day while 18 patients (Group II) received no supplementation. During the study period, the glomerular filtration rate slightly decreased (CCr from 28.2 +/- 3.4 to 26.4 +/- 4.1 ml/min and 29.6 +/- 4.8 to 23.4 +/- 4.4 ml/min in groups I and II, respectively and Cin); this however was more marked in Group II (Group I vs. Group II, p < 0.01). The serum levels of urea also declined (p < 0.01), more pronouncedly in Group I (p < 0.025). In Group I, there was a significant rise in the levels of leucine (p < 0.01), isoleucine (p < 0.01), valine (p < 0.02) and albumin (p < 0.01) and a decrease in protein-uria (p < 0.01). Analysis of the lipid spectrum revealed a mild yet significant decrease in total cholesterol and LDL-cholesterol (p < 0.02), more pronounced in Group I. In Group I, there was a decrease in plasma triglycerides (from 4.2 +/- 0.8 down to values a low as 2.2 +/- 0.6 mmol/L; p < 0.01) whereas HDL-cholesterol levels increased (from 0.9 +/- 0.1 to 1.2 +/- 0.1 mmol/L, p < 0.01). A further remarkable finding was a reduction in the serum concentration of free radicals (p < 0.01). We conclude that a KA supplementation in patients with CRF receiving LPD and EPO

Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese.

PubMed

Broadbent, Jeffery R; Gummalla, Sanjay; Hughes, Joanne E; Johnson, Mark E; Rankin, Scott A; Drake, Mary Anne

2004-08-01

Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that alpha-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable alpha-keto acids to more-stable alpha-hydroxy acids via the action of alpha-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei d-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that alpha-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of d-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of alpha-keto acids, but sensory work indicated that this effect retarded cheese flavor development.

Field Observation of Heterogeneous Formation of Dicarboxylic acids, Keto-carboxylic acids, α-Dicarbonyls and Nitrate in Xi'an, China during Asian dust storm periods

NASA Astrophysics Data System (ADS)

Wang, G.; Wang, J.; Ren, Y.; Li, J.

2015-12-01

To understand the formation mechanism of secondary organic aerosols (SOA) on dust surfaces, this study investigated the concentrations and compositions of dicarboxylic acids (C2-C11), keto-carboxylic acids (C3-C7), α-dicarbonyls and inorganic ions in size-segregated aerosols (9-stages) collected in Xi'an, China during the nondust storm and dust storm periods of 2009 and 2011. During the events the ambient particulate dicarboxylic acids were 932-2240 ng m-3, which are comparable and even higher than those in nondust periods. Molecular compositions of the above SOA are similar to those in nondust periods with oxalic acid being the leading species. In the presence of the dust storms, all the above mentioned SOA species in Xi'an were predominantly enriched on the coarse particles (>2.1μm), and oxalic acid well correlated with NO3- (R2=0.72, p<0.001) rather than SO42-.This phenomenon differs greatly from the SOA in any other nondust period that is characterized by an enrichment of oxalic acid in fine particles and a strong correlation of oxalic acid with SO42-. Our results further demonstrate that NO3- in the dust periods in Xi'an was mostly derived from secondary oxidation, whereas SO42- during the events was largely derived from surface soil of Gobi deserts. We propose a formation pathway to explain these observations, in which nitric acid and/or nitrogen oxides react with dust to produce Ca(NO3)2 and form a liquid phase on the surface of dust aerosols via water vapor-absorption of Ca(NO3)2, followed by a partitioning of the gas-phase water-soluble organic precursors (e.g.,glyoxal and methylglyoxal) into the aqueous-phase and a subsequent oxidation into oxalic acid. To the best of our knowledge, we found for the first time the enrichment of glyoxal and methylglyoxal on dust surface. Our data suggest an important role of nitrate in the heterogeneous formation process of SOA on the surface of Asian dust.

Synthesis of Cyclic α-Diazo-β-keto Sulfoxides in Batch and Continuous Flow.

PubMed

McCaw, Patrick G; Buckley, Naomi M; Eccles, Kevin S; Lawrence, Simon E; Maguire, Anita R; Collins, Stuart G

2017-04-07

Diazo transfer to β-keto sulfoxides to form stable isolable α-diazo-β-keto sulfoxides has been achieved for the first time. Both monocyclic and benzofused ketone derived β-keto sulfoxides were successfully explored as substrates for diazo transfer. Use of continuous flow leads to isolation of the desired compounds in enhanced yields relative to standard batch conditions, with short reaction times, increased safety profile, and potential to scale up.

Dynamic kinetic asymmetric cross-benzoin additions of β-stereogenic α-keto esters.

PubMed

Goodman, C Guy; Johnson, Jeffrey S

2014-10-22

The dynamic kinetic resolution of β-halo α-keto esters via an asymmetric cross-benzoin reaction is described. A chiral N-heterocyclic carbene catalyzes the umpolung addition of aldehydes to racemic α-keto esters. The resulting fully substituted β-halo glycolic ester products are obtained with high levels of enantio- and diastereocontrol. The high chemoselectivity observed is a result of greater electrophilicity of the α-keto ester toward the Breslow intermediate. The reaction products are shown to undergo highly diastereoselective substrate-controlled reduction to give highly functionalized stereotriads.

Dynamic Kinetic Asymmetric Cross-Benzoin Additions of β-Stereogenic α-Keto Esters

PubMed Central

2015-01-01

The dynamic kinetic resolution of β-halo α-keto esters via an asymmetric cross-benzoin reaction is described. A chiral N-heterocyclic carbene catalyzes the umpolung addition of aldehydes to racemic α-keto esters. The resulting fully substituted β-halo glycolic ester products are obtained with high levels of enantio- and diastereocontrol. The high chemoselectivity observed is a result of greater electrophilicity of the α-keto ester toward the Breslow intermediate. The reaction products are shown to undergo highly diastereoselective substrate-controlled reduction to give highly functionalized stereotriads. PMID:25299730

Detection Identification and Quantification of Keto-Hydroperoxides in Low-Temperature Oxidation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hansen, Nils; Moshammer, Kai; Jasper, Ahren W.

2017-07-01

Keto-hydroperoxides are reactive partially oxidized intermediates that play a central role in chain-branching reactions during the low-temperature oxidation of hydrocarbons. In this Perspective, we outline how these short lived species can be detected, identified, and quantified using integrated experimental and theoretical approaches. The procedures are based on direct molecular-beam sampling from reactive environments, followed by mass spectrometry with single-photon ionization, identification of fragmentation patterns, and theoretical calculations of ionization thresholds, fragment appearance energies, and photoionization cross sections. Using the oxidation of neo-pentane and tetrahydrofuran as examples, the individual steps of the experimental approaches are described in depth together with amore » detailed description of the theoretical efforts. For neo-pentane, the experimental data are consistent with the calculated ionization and fragment appearance energies of the keto-hydroperoxide, thus adding confidence to the analysis routines and the employed levels of theory. For tetrahydrofuran, multiple keto-hydroperoxide isomers are possible due to the presence of nonequivalent O 2 addition sites. Despite this additional complexity, the experimental data allow for the identification of two to four keto-hydroperoxides. Mole fraction profiles of the keto-hydroperoxides, which are quantified using calculated photoionization cross sections, are provided together with estimated uncertainties as function of the temperature of the reactive mixture and can serve as validation targets for chemically detailed mechanisms.« less

The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis.

PubMed

Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

2002-07-16

Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.

Strong-acid, carboxyl-group structures in fulvic acid from the Suwannee River, Georgia. 1. Minor structures

USGS Publications Warehouse

Leenheer, J.A.; Wershaw, R. L.; Reddy, M.M.

1995-01-01

An investigation of the strong-acid characteristics (pKa 3.0 or less) of fulvic acid from the Suwannee River, Georgia, was conducted. Quantitative determinations were made for amino acid and sulfur-containing acid structures, oxalate half-ester structures, malonic acid structures, keto acid structures, and aromatic carboxyl-group structures. These determinations were made by using a variety of spectrometric (13C-nuclear magnetic resonance, infrared, and ultraviolet spectrometry) and titrimetric characterizations on fulvic acid or fulvic acid samples that were chemically derivatized to indicate certain functional groups. Only keto acid and aromatic carboxyl-group structures contributed significantly to the strong-acid characteristics of the fulvic acid; these structures accounted for 43% of the strong-acid acidity. The remaining 57% of the strong acids are aliphatic carboxyl groups in unusual and/or complex configurations for which limited model compound data are available.

Conservation of the 2-keto-3-deoxymanno-octulosonic acid (Kdo) biosynthesis pathway between plants and bacteria.

PubMed

Smyth, Kevin M; Marchant, Alan

2013-10-18

The increasing prevalence of multi-drug resistant bacteria is driving efforts in the development of new antibacterial agents. This includes a resurgence of interest in the Gram-negative bacteria lipopolysaccharide (LPS) biosynthesis enzymes as drug targets. The six carbon acidic sugar 2-keto-3-deoxymanno-octulosonic acid (Kdo) is a component of the lipid A moiety of the LPS in Gram-negative bacteria. In most cases the lipid A substituted by Kdo is the minimum requirement for cell growth, thus presenting the possibility of targeting either the synthesis or incorporation of Kdo for the development of antibacterial agents. Indeed, potent in vitro inhibitors of Kdo biosynthesis enzymes have been reported but have so far failed to show sufficient in vivo action against Gram-negative bacteria. As part of an effort to design more potent antibacterial agents targeting Kdo biosynthesis, the crystal structures of the key Kdo biosynthesis enzymes from Escherichia coli have been solved and their structure based mechanisms characterized. In eukaryotes, Kdo is found as a component of the pectic polysaccharide rhamnogalacturonan II in the plant primary cell wall. Interestingly, despite incorporating Kdo into very different macromolecules the Kdo biosynthesis and activation pathway is almost completely conserved between plants and bacteria. This raises the possibility for plant research to exploit the increasingly detailed knowledge and resources being generated by the microbiology community. Likewise, insights into Kdo biosynthesis in plants will be potentially useful in efforts to produce new antimicrobial compounds. Copyright © 2013 Elsevier Ltd. All rights reserved.

Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-d-galactarate (KDG) dehydratase from Agrobacterium tumefaciens

PubMed Central

Taberman, Helena; Andberg, Martina; Parkkinen, Tarja; Richard, Peter; Hakulinen, Nina; Koivula, Anu; Rouvinen, Juha

2014-01-01

d-Galacturonic acid is the main component of pectin. It could be used to produce affordable renewable fuels, chemicals and materials through biotechnical conversion. Keto-deoxy-d-galactarate (KDG) dehydratase is an enzyme in the oxidative pathway of d-galacturonic acid in Agrobacterium tumefaciens (At). It converts 3-deoxy-2-keto-l-threo-hexarate to α-ketoglutaric semialdehyde. At KDG dehydratase was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3 Å, β = 112.4° and an asymmetric unit of four monomers. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The three-dimensional structure of At KDG dehydratase will provide valuable information on the function of the enzyme and will allow it to be engineered for biorefinery-based applications. PMID:24419616

3-Acetyl-11-keto-beta-boswellic acid loaded-polymeric nanomicelles for topical anti-inflammatory and anti-arthritic activity.

PubMed

Goel, Amit; Ahmad, Farhan Jalees; Singh, Raman Mohan; Singh, Gyanendra Nath

2010-02-01

The aim of this study was to develop 3-acetyl-11-keto-beta-boswellic acid (AKBA)-loaded polymeric nanomicelles for topical anti-inflammatory and anti-arthritic activity. Polymeric nanomicelles of AKBA were developed by a radical polymerization method using N-isopropylacrylamide, vinylpyrrolidone and acrylic acid. The polymeric nanomicelles obtained were characterized by Fourier transform infrared (FTIR), transmission electron microscopy (TEM) and dynamic light scattering (DLS). In-vitro and in-vivo evaluations of AKBA polymeric nanomicelles gel were carried out for enhanced skin permeability and anti-inflammatory and anti-arthritic activity. TEM and DLS results demonstrated that polymeric nanomicelles were spherical with a mean diameter approximately 45 nm. FTIR data indicated a weak interaction between polymer and AKBA in the encapsulated system. The release of drug in aqueous buffer (pH 7.4) from the polymeric nanomicelles was 23 and 55% after 2 and 8 h, respectively, indicating sustained release. In-vitro skin permeation studies through excised abdominal skin indicated a threefold increase in skin permeability compared with AKBA gel containing the same amount of AKBA as the AKBA polymeric nanomicelles gel. The AKBA polymeric nanomicelle gel showed significantly enhanced anti-inflammatory and anti-arthritic activity compared with the AKBA gel. This study suggested that AKBA polymeric nanomicelle gel significantly enhanced skin permeability, and anti-inflammatory and anti-arthritic activity.

Structure-property study of keto-ether polyimides

NASA Technical Reports Server (NTRS)

Dezern, James F.; Croall, Catharine I.

1991-01-01

As part of an on-going effort to develop an understanding of how changes in the chemical structure affect polymer properties, an empirical study was performed on polyimides containing only ether and/or carbonyl connecting groups in the polymer backbone. During the past two decades the structure-property relationships in linear aromatic polyimides have been extensively investigated. More recently, work has been performed to study the effect of isomeric attachment of keto-ether polyimides on properties such as glass transition temperature and solubility. However, little work has been reported on the relation of polyimide structure to mechanical properties. The purpose of this study was to determine the effect of structural changes in the backbone of keto-ether polyimides on their mechanical properties, specifically, unoriented thin film tensile properties. This study was conducted in two stages. The purpose of the initial stage was to examine the physical and mechanical properties of a representative group (four) of polyimide systems to determine the optimum solvent and cure cycle requirements. These optimum conditions were then utilized in the second stage to prepare films of keto-ether polyimides which were evaluated for mechanical and physical properties. All of the polyimides were prepared using isomers of oxydianiline (ODA) and diaminobenzophenone (DABP) in combination with 3,3',4,4'-benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydiphthalic anhydride (ODPA).

Detection and formation scenario of citric acid, pyruvic acid, and other possible metabolism precursors in carbonaceous meteorites

PubMed Central

Cooper, George; Reed, Chris; Nguyen, Dang; Carter, Malika; Wang, Yi

2011-01-01

Carbonaceous meteorites deliver a variety of organic compounds to Earth that may have played a role in the origin and/or evolution of biochemical pathways. Some apparently ancient and critical metabolic processes require several compounds, some of which are relatively labile such as keto acids. Therefore, a prebiotic setting for any such individual process would have required either a continuous distant source for the entire suite of intact precursor molecules and/or an energetic and compact local synthesis, particularly of the more fragile members. To date, compounds such as pyruvic acid, oxaloacetic acid, citric acid, isocitric acid, and α-ketoglutaric acid (all members of the citric acid cycle) have not been identified in extraterrestrial sources or, as a group, as part of a “one pot” suite of compounds synthesized under plausibly prebiotic conditions. We have identified these compounds and others in carbonaceous meteorites and/or as low temperature (laboratory) reaction products of pyruvic acid. In meteorites, we observe many as part of three newly reported classes of compounds: keto acids (pyruvic acid and homologs), hydroxy tricarboxylic acids (citric acid and homologs), and tricarboxylic acids. Laboratory syntheses using 13C-labeled reactants demonstrate that one compound alone, pyruvic acid, can produce several (nonenzymatic) members of the citric acid cycle including oxaloacetic acid. The isotopic composition of some of the meteoritic keto acids points to interstellar or presolar origins, indicating that such compounds might also exist in other planetary systems. PMID:21825143

Detection and formation scenario of citric acid, pyruvic acid, and other possible metabolism precursors in carbonaceous meteorites.

PubMed

Cooper, George; Reed, Chris; Nguyen, Dang; Carter, Malika; Wang, Yi

2011-08-23

Carbonaceous meteorites deliver a variety of organic compounds to Earth that may have played a role in the origin and/or evolution of biochemical pathways. Some apparently ancient and critical metabolic processes require several compounds, some of which are relatively labile such as keto acids. Therefore, a prebiotic setting for any such individual process would have required either a continuous distant source for the entire suite of intact precursor molecules and/or an energetic and compact local synthesis, particularly of the more fragile members. To date, compounds such as pyruvic acid, oxaloacetic acid, citric acid, isocitric acid, and α-ketoglutaric acid (all members of the citric acid cycle) have not been identified in extraterrestrial sources or, as a group, as part of a "one pot" suite of compounds synthesized under plausibly prebiotic conditions. We have identified these compounds and others in carbonaceous meteorites and/or as low temperature (laboratory) reaction products of pyruvic acid. In meteorites, we observe many as part of three newly reported classes of compounds: keto acids (pyruvic acid and homologs), hydroxy tricarboxylic acids (citric acid and homologs), and tricarboxylic acids. Laboratory syntheses using (13)C-labeled reactants demonstrate that one compound alone, pyruvic acid, can produce several (nonenzymatic) members of the citric acid cycle including oxaloacetic acid. The isotopic composition of some of the meteoritic keto acids points to interstellar or presolar origins, indicating that such compounds might also exist in other planetary systems.

Determination of branched chain amino acids, methionine, phenylalanine, tyrosine and alpha-keto acids in plasma and dried blood samples using HPLC with fluorescence detection.

PubMed

Kand'ár, Roman; Záková, Pavla; Jirosová, Jana; Sladká, Michaela

2009-01-01

The determination of branched chain amino acids [BCAA; valine (Val), leucine (Leu), isoleucine (Ile)], alpha-keto acids derived from BCAA [BCKA; alpha-ketoisovaleric acid (KIV), alpha-ketoisocaproic acid (KIC), alpha-ketomethylvaleric acid (KMV)], methionine (Met), phenylalanine (Phe) and tyrosine (Tyr) is currently the most reliable approach for the diagnosis of maple syrup urine disease (MSUD), hypermethioninemia, phenylketonuria (PKU) and tyrosinemia. The aim of this study was to develop rapid and simple HPLC methods for measurement of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples. Samples of peripheral venous blood with EDTA as anticoagulant were obtained from a group of healthy blood donors (n=70, 35 females, 27-41 years of age and 35 males, 28-43 years of age). Blood-spot samples from a group of newborns (n=80, 40 girls and 40 boys 3-5 days of age) were collected onto #903 Specimen Collection Paper and allowed to dry for at least 24 h before analysis. Prior to separation, the amino acids (AA) were derivatized with o-phthaldialdehyde (OPA) and BCKA with o-phenylenediamine (OPD). Reverse phase column chromatography (LiChroCart 125-4 Purospher RP-18e, 5 microm) was used for separation and fluorescence detection used to monitoring of effluent. For AA analysis, 25 mmol/L sodium hydrogenphosphate-methanol (90:10, v/v), pH 6.5+/-0.1 was used as mobile phase A and 100% methanol was used as mobile phase B. Measurement of BCKA used a mixture of methanol and deionized water (55:45, v/v) as mobile phase A and mobile phase B consisted of 100% methanol. Analytical performance of these methods was satisfactory for the determination of all AA and BCKA. The intra-assay and inter-assay coefficients of variation were below 10% and recovery ranged from 90%-110%. We have developed simple, rapid and selective HPLC methods with fluorescence detection for the determination of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples.

Regulation of hepatic branched-chain alpha-keto acid dehydrogenase kinase in a rat model for type 2 diabetes mellitus at different stages of the disease.

PubMed

Doisaki, Masao; Katano, Yoshiaki; Nakano, Isao; Hirooka, Yoshiki; Itoh, Akihiro; Ishigami, Masatoshi; Hayashi, Kazuhiko; Goto, Hidemi; Fujita, Yuko; Kadota, Yoshihiro; Kitaura, Yasuyuki; Bajotto, Gustavo; Kazama, Shunsuke; Tamura, Tomohiro; Tamura, Noriko; Feng, Guo-Gang; Ishikawa, Naohisa; Shimomura, Yoshiharu

2010-03-05

Branched-chain alpha-keto acid dehydrogenase (BCKDH) kinase (BDK) is responsible for the regulation of BCKDH complex, which is the rate-limiting enzyme in the catabolism of branched-chain amino acids (BCAAs). In the present study, we investigated the expression and activity of hepatic BDK in spontaneous type 2 diabetes using hyperinsulinemic Zucker diabetic fatty rats aged 9weeks and hyperglycemic, but not hyperinsulinemic rats aged 18weeks. The abundance of hepatic BDK mRNA and total BDK protein did not correlate with changes in serum insulin concentrations. On the other hand, the amount of BDK bound to the complex and its kinase activity were correlated with alterations in serum insulin levels, suggesting that hyperinsulinemia upregulates hepatic BDK. The activity of BDK inversely corresponded with the BCKDH complex activity, which was suppressed in hyperinsulinemic rats. These results suggest that insulin regulates BCAA catabolism in type 2 diabetic rats by modulating the hepatic BDK activity. 2010 Elsevier Inc. All rights reserved.

Decarboxylative aldol reactions of allyl beta-keto esters via heterobimetallic catalysis.

PubMed

Lou, Sha; Westbrook, John A; Schaus, Scott E

2004-09-22

Mild and selective heterobimetallic-catalyzed decarboxylative aldol reactions involving allyl beta-keto esters have been developed. The reaction is promoted by Pd(0)- and Yb(III)-DIOP complexes at room temperature and involves the in situ formation of a ketone enolate from allyl beta-keto esters followed by addition of the enolate to aldehydes. The reaction is a new example of heterobimetallic catalysis in which the optimized reaction conditions require the addition of both metals.

Keto analogue and amino acid supplementation and its effects on ammonemia and performance under thermoneutral conditions.

PubMed

Camerino, Saulo Rodrigo Alves e Silva; Lima, Rafaela Carvalho Pereira; França, Thássia Casado Lima; Herculano, Edla de Azevedo; Rodrigues, Daniela Souza Araújo; Gouveia, Marcos Guilherme de Sousa; Cameron, L C; Prado, Eduardo Seixas

2016-02-01

Alterations of cerebral function, fatigue and disturbance in cognitive-motor performance can be caused by hyperammonemia and/or hot environmental conditions during exercise. Exercise-induced hyperammonemia can be reduced through supplementation with either amino acids or combined keto analogues and amino acids (KAAA) to improve exercise tolerance. In the present study, we evaluated KAAA supplementation on ammonia metabolism and cognitive-motor performance after high-intensity exercise under a low heat stress environment. Sixteen male cyclists received a ketogenic diet for 2 d and were divided into two groups, KAAA (KEx) or placebo (CEx) supplementation. The athletes performed a 2 h cycling session followed by a maximum test (MAX), and blood samples were obtained at rest and during exercise. Cognitive-motor tasks were performed before and after the protocol, and the exhaustion time was used to evaluate physical performance. The hydration status was also evaluated. The CEx group showed a significant increase (∼ 70%) in ammonia concentration at MAX, which did not change in the KEx group. The non-supplemented group showed a significant increase in uremia. Both the groups had a significant increase in blood urate concentrations at 120 min, and an early significant increase from 120 min was observed in the CEx group. There was no change in the glucose concentrations of the two groups. A significant increase in lactate was observed at the MAX moment in both groups. There was no significant difference in the exhaustion times between the groups. No changes were observed in the cognitive-motor tasks after the protocol. We suggest that KAAA supplementation decreases ammonia concentration during high-intensity exercise but does not affect physical or cognitive-motor performances under a low heat stress environment.

Providing a diet deficient in valine but with excess leucine results in a rapid decrease in feed intake and modifies the postprandial plasma amino acid and α-keto acid concentrations in pigs.

PubMed

Gloaguen, M; Le Floc'h, N; Corrent, E; Primot, Y; van Milgen, J

2012-09-01

Indispensable AA are involved in the control of feed intake. When a diet deficient in Val is offered to pigs, feed intake is typically reduced. This effect is aggravated when dietary Leu is supplied in excess of the requirement. If an unbalanced supply of branched-chain AA (BCAA) is harmful, an anorectic response may serve as a mechanism to prevent this situation. We verified this hypothesis by measuring the voluntary feed intake of a balanced diet offered during the 30-min period 1 h after ingestion of a test meal deficient or not in Val (Val- and Val+) with an excess of Leu. Twelve and four 6-wk-old crossbred female pigs were used in Exp. 1 and 2, respectively. Prior ingestion of the Val- test meal resulted in a 14% reduction in feed intake compared with that observed after ingestion of the Val+ test meal (P = 0.06) in Exp. 1, indicating that the signal to reduce feed intake occurred within 1 h. It is possible that the plasma concentration of the limiting AA serves as a signal for the dietary AA deficiency. We therefore determined the postprandial plasma concentrations of BCAA and their α-keto acids after ingestion of Val- and Val+ in 4 pigs in Exp. 2. After ingestion of the Val- diet, plasma concentrations of Val and its keto acid were reduced compared with values observed after ingestion of the Val+ diet. The peak concentration occurred earlier after ingestion of the Val- diet compared with that of the Val+ diet. Although the plasma concentration increased after the meal, it declined rapidly in pigs offered Val-, and the Val concentration 4 h after ingestion of the meal was even less than that observed in the fasted state. In conclusion, it appears that the pig is able to detect a deficient supply of Val within 1 h after ingestion. The plasma concentration of Val or its concentration relative to the other BCAA during the postprandial period may act as a signal indicating the AA deficiency.

Roles of pyruvate dehydrogenase and branched-chain α-keto acid dehydrogenase in branched-chain membrane fatty acid levels and associated functions in Staphylococcus aureus.

PubMed

Singh, Vineet K; Sirobhushanam, Sirisha; Ring, Robert P; Singh, Saumya; Gatto, Craig; Wilkinson, Brian J

2018-04-01

Membrane fluidity to a large extent is governed by the presence of branched-chain fatty acids (BCFAs). Branched-chain α-keto acid dehydrogenase (BKD) is the key enzyme in BCFA synthesis. A Staphylococcus aureus BKD-deficient strain still produced substantial levels of BCFAs. Pyruvate dehydrogenase (PDH) with structural similarity to BKD has been speculated to contribute to BCFAs in S. aureus. This study was carried out using BKD-, PDH- and BKD : PDH-deficient derivatives of methicillin-resistant S. aureus strain JE2. Differences in growth kinetics were evaluated spectrophotometrically, membrane BCFAs using gas chromatography and membrane fluidity by fluorescence polarization. Carotenoid levels were estimated by measuring A465 of methanol extracts from 48 h cultures. MIC values were determined by broth microdilution.Results/Key findings. BCFAs made up 50 % of membrane fatty acids in wild-type but only 31 % in the BKD-deficient mutant. BCFA level was ~80 % in the PDH-deficient strain and 38 % in the BKD : PDH-deficient strain. BKD-deficient mutant showed decreased membrane fluidity, the PDH-deficient mutant showed increased membrane fluidity. The BKD- and PDH-deficient strains grew slower and the BKD : PDH-deficient strain grew slowest at 37 °C. However at 20 °C, the BKD- and BKD : PDH-deficient strains grew only a little followed by autolysis of these cells. The BKD-deficient strain produced higher levels of staphyloxanthin. The PDH-deficient and BKD : PDH-deficient strains produced very little staphyloxanthin. The BKD-deficient strain showed increased susceptibility to daptomycin. The BCFA composition of the cell membrane in S. aureus seems to significantly impact cell growth, membrane fluidity and resistance to daptomycin.

Impaired growth and neurological abnormalities in branched-chain α-keto acid dehydrogenase kinase-deficient mice

PubMed Central

Joshi, Mandar A.; Jeoung, Nam Ho; Obayashi, Mariko; Hattab, Eyas M.; Brocken, Eric G.; Liechty, Edward A.; Kubek, Michael J.; Vattem, Krishna M.; Wek, Ronald C.; Harris, Robert A.

2006-01-01

The BCKDH (branched-chain α-keto acid dehydrogenase complex) catalyses the rate-limiting step in the oxidation of BCAAs (branched-chain amino acids). Activity of the complex is regulated by a specific kinase, BDK (BCKDH kinase), which causes inactivation, and a phosphatase, BDP (BCKDH phosphatase), which causes activation. In the present study, the effect of the disruption of the BDK gene on growth and development of mice was investigated. BCKDH activity was much greater in most tissues of BDK−/− mice. This occurred in part because the E1 component of the complex cannot be phosphorylated due to the absence of BDK and also because greater than normal amounts of the E1 component were present in tissues of BDK−/− mice. Lack of control of BCKDH activity resulted in markedly lower blood and tissue levels of the BCAAs in BDK−/− mice. At 12 weeks of age, BDK−/− mice were 15% smaller than wild-type mice and their fur lacked normal lustre. Brain, muscle and adipose tissue weights were reduced, whereas weights of the liver and kidney were greater. Neurological abnormalities were apparent by hind limb flexion throughout life and epileptic seizures after 6–7 months of age. Inhibition of protein synthesis in the brain due to hyperphosphorylation of eIF2α (eukaryotic translation initiation factor 2α) might contribute to the neurological abnormalities seen in BDK−/− mice. BDK−/− mice show significant improvement in growth and appearance when fed a high protein diet, suggesting that higher amounts of dietary BCAA can partially compensate for increased oxidation in BDK−/− mice. Disruption of the BDK gene establishes that regulation of BCKDH by phosphorylation is critically important for the regulation of oxidative disposal of BCAAs. The phenotype of the BDK−/− mice demonstrates the importance of tight regulation of oxidative disposal of BCAAs for normal growth and neurological function. PMID:16875466

5-(Tetradecyloxy)-2-furancarboxylic acid and related hypolipidemic fatty acid-like alkyloxyarylcarboxylic acids.

PubMed

Parker, R A; Kariya, T; Grisar, J M; Petrow, V

1977-06-01

5-(Tetradecyloxy)-2-furancarboxylic acid (91, RMI 14514) was found to lower blood lipids and to inhibit fatty acid synthesis with minimal effects on liver weight and liver fat content. This fatty acid-like compound represents a new class of hypolipidemic agent; it is effective in rats and monkeys. The compound resulted from discovery of hypolipidemic activity in certain beta-keto esters, postulation and confirmation of the corresponding benzoic acids as active metabolites, and systematic exploration of the structure--activity relationships.

N-Heterocyclic carbene-catalyzed direct cross-aza-benzoin reaction: Efficient synthesis of α-amino-β-keto esters.

PubMed

Uno, Takuya; Kobayashi, Yusuke; Takemoto, Yoshiji

2012-01-01

An efficient catalytic synthesis of α-amino-β-keto esters has been newly developed. Cross-coupling of various aldehydes with α-imino ester, catalyzed by N-heterocyclic carbene, leads chemoselectively to α-amino-β-keto esters in moderate to good yields with high atom efficiency. The reaction mechanism is discussed, and it is proposed that the α-amino-β-keto esters are formed under thermodynamic control.

Evaluation of a novel biodegradable thermosensitive keto-hydrogel for improving postoperative pain in a rat model.

PubMed

Wu, Meng-Huang; Shih, Ming-Hung; Hsu, Wei-Bin; Dubey, Navneet Kumar; Lee, Wen-Fu; Lin, Tsai-Yu; Hsieh, Meng-Yow; Chen, Chin-Fu; Peng, Kuo-Ti; Huang, Tsung-Jen; Shi, Chung-Sheng; Guo, Ren-Shyang; Cai, Chang-Jhih; Chung, Chiu-Yen; Wong, Chung-Hang

2017-01-01

This study evaluates the sustained analgesic effect of ketorolac-eluting thermosensitive biodegradable hydrogel in the plantar incisional pain model of the rat hind-paw. A ketorolac-embedded 2, 2'-Bis (2-oxazolin) (BOX) linking methoxy-poly(ethylene glycol) and poly(lactide-co-glycolide) (mPEG-PLGA) diblock copolymer (BOX copolymer) was synthesized as keto-hydrogel based on optimal sol-gel phase transition and in vitro drug release profile. The effect of keto-hydrogel on postoperative pain (POP) was assessed using the established plantar incisional pain model in hind-paw of rats and compared to that of ketorolac solution. Pain and sensory threshold, as well as pain scoring, were evaluated with behavioral tests by means of anesthesiometer and incapacitance apparatus, respectively. Pro-inflammatory cytokine levels (TNF-α, IL-6, VEGF, and IL-1β) around incisional wounds were measured by ELISA. Tissue histology was assessed using hematoxylin and eosin and Masson's trichrome staining. Ten mg/mL (25 wt%) keto-hydrogel showed a sol-gel transition at 26.4°C with a 10-day sustained drug release profile in vitro. Compared to ketorolac solution group, the concentration of ketorolac in tissue fluid was higher in the keto-hydrogel group during the first 18 h of application. Keto-hydrogel elevated pain and sensory threshold, increased weight-bearing capacity, and significantly reduced the levels of TNF-α, IL-6, and IL-1β while enhanced VEGF in tissue fluid. Histologic analysis reveals greater epithelialization and collagen deposition around wound treated with keto-hydrogel. In conclusion, our study suggests that keto-hydrogel is an ideal compound to treat POP with a secondary gain of improved incisional wound healing.

α-Keto phenylamides as P1'-extended proteasome inhibitors.

PubMed

Voss, Constantin; Scholz, Christoph; Knorr, Sabine; Beck, Philipp; Stein, Martin L; Zall, Andrea; Kuckelkorn, Ulrike; Kloetzel, Peter-Michael; Groll, Michael; Hamacher, Kay; Schmidt, Boris

2014-11-01

The major challenge for proteasome inhibitor design lies in achieving high selectivity for, and activity against, the target, which requires specific interactions with the active site. Novel ligands aim to overcome off-target-related side effects such as peripheral neuropathy, which is frequently observed in cancer patients treated with the FDA-approved proteasome inhibitors bortezomib (1) or carfilzomib (2). A systematic comparison of electrophilic headgroups recently identified the class of α-keto amides as promising for next generation drug development. On the basis of crystallographic knowledge, we were able to develop a structure-activity relationship (SAR)-based approach for rational ligand design using an electronic parameter (Hammett's σ) and in silico molecular modeling. This resulted in the tripeptidic α-keto phenylamide BSc4999 [(S)-3-(benzyloxycarbonyl-(S)-leucyl-(S)-leucylamino)-5-methyl-2-oxo-N-(2,4-dimethylphenyl)hexanamide, 6 a], a highly potent (IC50 = 38 nM), cell-permeable, and slowly reversible covalent inhibitor which targets both the primed and non-primed sites of the proteasome's substrate binding channel as a special criterion for selectivity. The improved inhibition potency and selectivity of this new α-keto phenylamide makes it a promising candidate for targeting a wider range of tumor subtypes than commercially available proteasome inhibitors and presents a new candidate for future studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pharmacological Characterization of a Novel Bifunctional Aldo-Keto Reductase 1C3 Inhibitor and Androgen Receptor Antagonist

DTIC Science & Technology

2013-10-01

Novel Bifunctional Aldo -Keto Reductase 1C3 Inhibitor and Androgen Receptor Antagonist” PRINCIPAL INVESTIGATOR: ADEGOKE ADENIJI, Ph.D...therapeutic benefit relative to targeting either mechanism alone. Aldo -keto reductase 1C3 (AKR1C3) is highly upregulated in APC and is localized within...therapy of Abi with MDV3100 has been proposed as a way to reduce resistance. 14, 15 Aldo -keto reductase IC3 (AKR1C3, type 5 17β hydroxysteroid

Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis.

PubMed

Kataoka, M; Delacruz-Hidalgo, A-R G; Akond, M A; Sakuradani, E; Kita, K; Shimizu, S

2004-04-01

The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

Heterogeneous enantioselective hydrogenation of beta-keto esters using chirally modified supported Ni nanoparticles

NASA Astrophysics Data System (ADS)

Acharya, Sushma

Enantioselective heterogeneous catalysis is an important and rapidly expanding research area. The two most heavily researched examples of this type of catalysis are the enantioselective hydrogenation of α-keto-esters over Pt-based catalysts and the enantioselective hydrogenation of β-keto-esters over Ni-based catalysts. These enantioselective surface reactions are controlled by the presence of adsorbed chiral molecules i.e. tartaric acid on the surface of the metal component of the catalyst. The work presented in this thesis focuses on two parts, the synthesis of pure nickel nanoparticles and enantioselective behavior of the modified nickel nanoparticles. The works on the synthesis of pure nickel nanoparticles were carried out using two methods, the reverse microemulsion and the reduction method. It was discovered that the reverse microemulsion method produced nickel oxide nanoparticles, whereas the reduction method produced pure nickel nanoparticles. Chiral modifications of Raney nickel (RNi) and C-supported catalysts were studied. The catalysts were employed in enantioselective hydrogenation of methyl acetoacetate (MAA) to (R) - and (S)-enantiomers of methyl 3-hydroxybutyrate (MHB). The effects of modification and hydrogenation parameters such as concentration of modifier temperature, pressure and solvent on the enantioselectivity of MAA hydrogenation were discussed. For RNi methanol was found to be the best solvent, with tartaric acid concentration 0.2 mol/L for achieving the highest enantiomeric excess under 8 bar at 70 oC. Characteristic features of the in-situ modification of Raney nickel and C-supported Ni were also evaluated and the results obtained were compared with the conventional (pre-modification) approach. Parameters for the conventional and in-situ methods were optimised in a series of experiments for both types of catalysts. The in-situ modified catalyst was found more active for both RNi and C-supported catalysts with 98 % and 42% enantiomeric

A single membrane-bound enzyme catalyzes the conversion of 2,5-diketo-d-gluconate to 4-keto-d-arabonate in d-glucose oxidative fermentation by Gluconobacter oxydans NBRC 3292.

PubMed

Tazoe, Masaaki; Oishi, Hiromi; Kobayashi, Setsuko; Hoshino, Tatsuo

2016-08-01

4-Keto-d-arabonate synthase (4KAS), which converts 2,5-diketo-d-gluconate (DKGA) to 4-keto-d-arabonate (4KA) in d-glucose oxidative fermentation by some acetic acid bacteria, was solubilized from the Gluconobacter oxydans NBRC 3292 cytoplasmic membrane, and purified in an electrophoretically homogenous state. A single membrane-bound enzyme was found to catalyze the conversion from DKGA to 4KA. The 92-kDa 4KAS was a homodimeric protein not requiring O2 or a cofactor for the conversion, but was stimulated by Mn(2+). N-terminal amino acid sequencing of 4KAS, followed by gene homology search indicated a 1,197-bp open reading frame (ORF), corresponding to the GLS_c04240 locus, GenBank accession No. CP004373, encoding a 398-amino acid protein with a calculated molecular weight of 42,818 Da. An Escherichia coli transformant with the 4kas plasmid exhibited 4KAS activity. Furthermore, overexpressed recombinant 4KAS was purified in an electrophoretically homogenous state and had the same molecular size as the natural enzyme.

Single step synthesis of strigolactone analogues from cyclic keto enols, germination stimulants for seeds of parasitic weeds.

PubMed

Mwakaboko, Alinanuswe S; Zwanenburg, Binne

2011-08-15

The single step synthesis of a newly designed series of strigolactones (SLs) from cyclic keto enols is described. The germinating activity of these SL analogues towards seeds of the parasitic weeds Striga and Orobanche spp. is reported. The first of these SL analogues are derived from the hydroxyl γ-pyrones kojic acid and maltol, the second type from hydroxyl α-pyrones, namely, 4-hydroxy-6-methyl-2H-pyran-2-one and 4-hydroxy-coumarin and the third type from 1,3-diketones, namely, 1,3-cyclohexane-dione (dimedone) and tricyclic 1,3-dione. All keto enols are coupled in a single step with the appropriate D-ring precursor in the presence of a base to give the desired SL analogues. All SL analogues are acceptably biologically active in inducing the germination of seeds of Striga hermonthica and Orobanchecernua. Most interesting are the analogues derived from 4-hydroxy coumarin and dimedone, as they have a remarkably high biological activity towards the seeds of parasitic weeds at relatively low concentrations, comparable with that of the general standard stimulant GR24. Copyright © 2011 Elsevier Ltd. All rights reserved.

Reduction of plasma asymmetric dimethylarginine in obese patients with chronic kidney disease after three years of a low-protein diet supplemented with keto-amino acids: a randomized controlled trial.

PubMed

Teplan, Vladimir; Schück, Otto; Racek, Jaroslav; Mareckova, Olga; Stollova, Milena; Hanzal, Vladimir; Malý, Jan

2008-01-01

Levels of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) are elevated in chronic kidney disease (CKD) and may contribute to vascular complications. In this study we tested the hypothesis that elevated ADMA can be reduced in obese CKD patients by long-term administration of a low-protein diet supplemented with keto-amino acids. In a long-term prospective double-blind placebo-controlled randomized trial, we evaluated for a period of 36 months a total of 111 CKD patients (54 men, 57 women) aged 22-76 years with obesity (BMI >or= 30 kg/m(2)) and an inulin clearance rate (C(in)) of 22-40 ml/min/1.73 m(2). All patients were on a low-protein diet containing 0.6 g protein/kg BW per day and 120-125 kJ/kg BW per day. The diet was randomly supplemented with keto-amino acids at a dosage of 100 mg/kg BW per day (66 patients, Group I); 65 patients received placebo (Group II). During the study period, the glomerular filtration rate decreased slightly in Group I (C(in) from 32.4 +/- 12.6 to 29.8 +/- 8.6 ml/min/1.73 m(2)) and more markedly in Group II (from 33.2 +/- 12.6 to 23.2 +/- 98.4 ml/min/1.73 m(2), P < 0.01). BMI decreased significantly in Group I (from 32.0 +/- 3.3 to 26.1 +/- 4.0 kg/m(2), P < 0.01) and was linked to reduced volume of visceral fat measured by MRI (P < 0.01). Reduction of BMI in Group II was not significant. In Group I, there was a significant decrease in the plasma level of ADMA (from 2.5 +/- 0.5 to 1.3 +/- 0.4 micromol/l, P < 0.01), but ADMA remained unchanged in Group II. A further remarkable finding in Group I was reduction in the plasma concentration of pentosidine (from 480 +/- 170 to 320 +/- 120 microg/l, P < 0.01) and decrease of proteinuria (from 3.8 +/- 2.24 to 1.6 +/- 1.0 g/24 h, P < 0.02). Plasma adiponectin rose in Group I (P < 0.01). Analysis of the lipid spectrum revealed a mild but significant decrease in total cholesterol and LPD-cholesterol (P < 0.02), more pronounced in Group I. There was also a decrease

Adsorption kinetics, isotherm, and thermodynamics studies of acetyl-11-keto-β-boswellic acids (AKBA) from Boswellia serrata extract using macroporous resin.

PubMed

Niphadkar, Sonali S; Rathod, Virendra K

2017-09-14

An acetyl-11-keto-β-boswellic acid (AKBA) is potent anti-inflammatory agent found in Boswellia serrata oleogum resin. Adsorption characteristics of AKBA from B. serrata were studied using macroporous adsorbent resin to understand separation and adsorption mechanism of targeted molecules. Different macroporous resins were screened for adsorption and desorption of AKBA and Indion 830 was screened as it showed higher adsorption capacity. The kinetic equations were studied and results showed that the adsorption of AKBA on Indion 830 was well fitted to the pseudo first-order kinetic model. The influence of two parameters such as temperature (298, 303, and 308 K) and pH (5-8) on the adsorption process was also studied. The experimental data was further investigated using Langmuir, Freundlich, and Temkin isotherm models. It was observed that Langmuir isotherm model was found to be the best fit for AKBA adsorption by Indion 830 and highest adsorption capacity (50.34 mg/g) was obtained at temperature of 303 K. The values of thermodynamic parameters such as the change of Gibbs free energy (ΔG*), entropy (ΔS*), and enthalpy (ΔH*), indicated that the process of adsorption was spontaneous, favourable, and exothermic.

Amino Acid Degradations Produced by Lipid Oxidation Products.

PubMed

Hidalgo, Francisco J; Zamora, Rosario

2016-06-10

Differently to amino acid degradations produced by carbohydrate-derived reactive carbonyls, amino acid degradations produced by lipid oxidation products are lesser known in spite of being lipid oxidation a major source of reactive carbonyls in food. This article analyzes the conversion of amino acids into Strecker aldehydes, α-keto acids, and amines produced by lipid-derived free radicals and carbonyl compounds, as well as the role of lipid oxidation products on the reactions suffered by these compounds: the formation of Strecker aldehydes and other aldehydes from α-keto acids; the formation of Strecker aldehydes and olefins from amines; the formation of shorter aldehydes from Strecker aldehydes; and the addition reactions suffered by the olefins produced from the amines. The relationships among all these reactions and the effect of reaction conditions on them are discussed. This knowledge should contribute to better control food processing in order to favor the formation of desirable beneficial compounds and to inhibit the production of compounds with deleterious properties.

Identification of 2-keto-3-deoxy-d-Gluconate Kinase and 2-keto-3-deoxy-d-Phosphogluconate Aldolase in an Alginate-Assimilating Bacterium, Flavobacterium sp. Strain UMI-01

PubMed Central

Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

2017-01-01

Recently, we identified an alginate-assimilating gene cluster in the genome of Flavobacterium sp. strain UMI-01, a member of Bacteroidetes. Alginate lyase genes and a 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH) reductase gene in the cluster have already been characterized; however, 2-keto-3-deoxy-d-gluconate (KDG) kinase and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase genes, i.e., flkin and flald, still remained uncharacterized. The amino acid sequences deduced from flkin and flald showed low identities with those of corresponding enzymes of Saccharophagus degradans 2-40T, a member of Proteobacteria (Kim et al., Process Biochem., 2016). This led us to consider that the DEH-assimilating enzymes of Bacteroidetes species are somewhat deviated from those of Proteobacteria species. Thus, in the present study, we first assessed the characteristics in the primary structures of KDG kinase and KDG aldolase of the strain UMI-01, and then investigated the enzymatic properties of recombinant enzymes, recFlKin and recFlAld, expressed by an Escherichia coli expression system. Multiple-sequence alignment among KDG kinases and KDG aldolases from several Proteobacteria and Bacteroidetes species indicated that the strain UMI-01 enzymes showed considerably low sequence identities (15%–25%) with the Proteobacteria enzymes, while they showed relatively high identities (47%–68%) with the Bacteroidetes enzymes. Phylogenetic analyses for these enzymes indicated the distant relationship between the Proteobacteria enzymes and the Bacteroidetes enzymes, i.e., they formed distinct clusters in the phylogenetic tree. recFlKin and recFlAld produced with the genes flkin and flald, respectively, were confirmed to show KDG kinase and KDPG aldolase activities. Namely, recFlKin produced 1.7 mM KDPG in a reaction mixture containing 2.5 mM KDG and 2.5 mM ATP in a 90-min reaction, while recFlAld produced 1.2 mM pyruvate in the reaction mixture containing 5 mM KDPG at the equilibrium

Aldo-keto reductase family 1 B10 affects fatty acid synthesis by regulating the stability of acetyl-CoA carboxylase-alpha in breast cancer cells.

PubMed

Ma, Jun; Yan, Ruilan; Zu, Xuyu; Cheng, Ji-Ming; Rao, Krishna; Liao, Duan-Fang; Cao, Deliang

2008-02-08

Recent studies have demonstrated that aldo-keto reductase family 1 B10 (AKR1B10), a novel protein overexpressed in human hepatocellular carcinoma and non-small cell lung carcinoma, may facilitate cancer cell growth by detoxifying intracellular reactive carbonyls. This study presents a novel function of AKR1B10 in tumorigenic mammary epithelial cells (RAO-3), regulating fatty acid synthesis. In RAO-3 cells, Sephacryl-S 300 gel filtration and DEAE-Sepharose ion exchange chromatography demonstrated that AKR1B10 exists in two distinct forms, monomers (approximately 40 kDa) bound to DEAE-Sepharose column and protein complexes (approximately 300 kDa) remaining in flow-through. Co-immunoprecipitation with AKR1B10 antibody and protein mass spectrometry analysis identified that AKR1B10 associates with acetyl-CoA carboxylase-alpha (ACCA), a rate-limiting enzyme of de novo fatty acid synthesis. This association between AKR1B10 and ACCA proteins was further confirmed by co-immunoprecipitation with ACCA antibody and pulldown assays with recombinant AKR1B10 protein. Intracellular fluorescent studies showed that AKR1B10 and ACCA proteins co-localize in the cytoplasm of RAO-3 cells. More interestingly, small interfering RNA-mediated AKR1B10 knock down increased ACCA degradation through ubiquitination-proteasome pathway and resulted in >50% decrease of fatty acid synthesis in RAO-3 cells. These data suggest that AKR1B10 is a novel regulator of the biosynthesis of fatty acid, an essential component of the cell membrane, in breast cancer cells.

9-Hydroxyprostaglandin dehydrogenase in rat kidney cortex converts prostaglandin I2 into 15-keto-13,14-dihydro 6-ketoprostaglandin E1.

PubMed

Pace-Asciak, C R; Domazet, Z

1984-11-14

15-Keto-13,14-dihydro 6-ketoprostaglandin E1 was positively identified by gas chromatography-mass spectrometry with negative-ion chemical ionisation detection from samples of rat kidney high-speed supernatant incubated with prostaglandin I2 in the presence of NAD+. A decreased formation of this product was observed when NAD+ was substituted with NADP+ and none was observed in the absence of nucleotide or substrate prostaglandin I2. Experiments with [9 beta-3H]prostaglandin I2 showed a time- and concentration-dependent loss of tritium which appeared as tritiated water, typical of reaction of [9 beta-3H]prostaglandin substrates with the enzyme, 9-hydroxyprostaglandin dehydrogenase. Time-course measurements of the appearance of tritiated water showed similar rates with 6-keto[9 beta-3H]prostaglandin F1 alpha and 15-keto-13,14-dihydro 6-keto[9 beta-3H]prostaglandin F1 alpha as substrates. These experiments suggest that the transformation of prostaglandin I2 and 6-ketoprostaglandin F1 alpha into the 15-keto-13,14-dihydro 6-ketoprostaglandin E1 catabolite occurs in this in vitro preparation via the corresponding 15-keto-13,14-dihydro catabolite of 6-ketoprostaglandin F1 alpha.

Effects of medicinal cake-separated moxibustion on plasma 6-keto-PGF1alpha and TXB2 contents in the rabbit of hyperlipemia.

PubMed

Xiaorong, Chang; Jie, Yan; Zenghui, Yue; Jing, Shen; Yaping, Lin; Shouxiang, Yi; Xiangping, Cao

2005-06-01

Hyperlipemia rabbit models established with high cholesterol and fat diet were treated with direct moxibustion and medicinal cake-separated moxibustion. The post-treatment plasma 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) and thromboxane B2 (TXB2) contents were determined by radioimmunoassay. Results indicated that the plasma 6-keto-PGF1alpha content significantly increased, the TXB2 level decreased (P < 0.05) and the TXB2 /6-keto-PGF1alpha ratio also decreased (P < 0.01) in the medicinal cake-separated moxibustion group as compared with those in the model group respectively, but there was no significant difference between the medicinal cake-separated moxibustion group and the direct moxibustion group (P > 0.05), suggesting that both the medicinal cake-separated moxibustion and direct moxibustion can regulate the plasma 6-keto-PGF1alpha and TXB2 contents, and the TXB2/6-keto-PGF1alpha ratio with similar actions, and have a certain protective action on endothelial cells of the aorta in the rabbit of hyperlipemia.

Photolysis of α-KETO Acids in Model Atmospheric Water

NASA Astrophysics Data System (ADS)

Eugene, A. J.; Guzman, M. I.

2017-12-01

Recent work has reported the potential of aqueous-phase photochemistry to promote secondary organic aerosol (SOA) formation. New aqueous photochemical SOA sources may contribute to bridging the gap between field measurements of SOA and models of SOA formation. The ubiquitous α-ketocarboxylic acids pyruvic and glyoxylic acid are known products of the atmospheric oxidation of polycyclic aromatic hydrocarbons (PAHs) as well as of biogenic volatile organic compounds (VOCs). The combination of a carbonyl chromophore (absorbing at wavelengths λ ≥ 300 nm) and hydrophilic functional groups makes these acids likely candidates for forming aqueous SOA by direct sunlight photolysis. We use a variety of analytical techniques including: 2,4-dinitrophenylhydrazine (DNPH) derivatization; ultra-high performance liquid chromatography (UHPLC) and ion chromatography (IC) coupled to mass spectrometry;1H and 13C NMR; and 13C gCOSY NMR to probe the kinetics and mechanisms of the direct photolysis of model solutions of pyruvic acid and glyoxylic acid. The results indicate that despite the structural similarity between the two acids, they each react via very different primary photochemical pathways. Pyruvic acid undergoes a proton-coupled electron transfer (PCET) mechanism with radical recombination, resulting in CO2 and 6-8 carbon organic acids. In contrast, glyoxylic acid primarily undergoes α-cleavage to generate CO, CO2, and glyoxal which is a key species in SOA formation. This work demonstrates that aqueous photolysis is a very competitive atmospheric sink for both pyruvic and glyoxylic acid, indicating that these photoreactions are capable of contributing substantially to SOA formation.

Characterisation of the broad substrate specificity 2-keto acid decarboxylase Aro10p of Saccharomyces kudriavzevii and its implication in aroma development.

PubMed

Stribny, Jiri; Romagnoli, Gabriele; Pérez-Torrado, Roberto; Daran, Jean-Marc; Querol, Amparo

2016-03-12

The yeast amino acid catabolism plays an important role in flavour generation since higher alcohols and acetate esters, amino acid catabolism end products, are key components of overall flavour and aroma in fermented products. Comparative studies have shown that other Saccharomyces species, such as S. kudriavzevii, differ during the production of aroma-active higher alcohols and their esters compared to S. cerevisiae. In this study, we performed a comparative analysis of the enzymes involved in the amino acid catabolism of S. kudriavzevii with their potential to improve the flavour production capacity of S. cerevisiae. In silico screening, based on the severity of amino acid substitutions evaluated by Grantham matrix, revealed four candidates, of which S. kudriavzevii Aro10p (SkAro10p) had the highest score. The analysis of higher alcohols and esters produced by S. cerevisiae then revealed enhanced formation of isobutanol, isoamyl alcohol and their esters when endogenous ARO10 was replaced with ARO10 from S. kudriavzevii. Also, significant differences in the aroma profile were found in fermentations of synthetic wine must. Substrate specificities of SkAro10p were compared with those of S. cerevisiae Aro10p (ScAro10p) by their expression in a 2-keto acid decarboxylase-null S. cerevisiae strain. Unlike the cell extracts with expressed ScAro10p which showed greater activity for phenylpyruvate, which suggests this phenylalanine-derivative to be the preferred substrate, the decarboxylation activities measured in the cell extracts with SkAro10p ranged with all the tested substrates at the same level. The activities of SkAro10p towards substrates (except phenylpyruvate) were higher than of those for ScAro10p. The results indicate that the amino acid variations observed between the orthologues decarboxylases encoded by SkARO10 and ScARO10 could be the reason for the distinct enzyme properties, which possibly lead to the enhanced production of several flavour compounds. The

Keto-enol tautomerism in asymmetric Schiff bases derived from p-phenylenediamine

NASA Astrophysics Data System (ADS)

Užarević, Krunoslav; Rubčić, Mirta; Stilinović, Vladimir; Kaitner, Branko; Cindrić, Marina

2010-12-01

Reaction of dehydroacetic acid and p-phenylenediamine afforded a monosubstituted Schiff base, I, with the other amino group free. In further reactions with various salicylaldehyde derivatives, I served as a precursor for synthesis of asymmetric bis-Schiff bases. The synthesized compounds are thus comprised of two subunits, dehydroacetic ( dha) and salicylidene ( sal), which are bridged by the phenylene linker. All products were investigated by means of elemental analysis, FT-IR and NMR spectroscopy, thermal methods, powder X-ray diffraction and, when possible, by single crystal X-ray crystallography. Structural and spectroscopic studies revealed that in the bis-products, the dha subunit adopts the keto-amino tautomeric form, while the sal subunit adopts the enol-imino form. Tautomeric forms were not affected if a methoxo group was introduced on the salicylidene ring. Both tautomeric subunits are stabilized by strong resonance-assisted hydrogen bonds, RAHB. The two subunits of the prepared bis-Schiff bases predominantly retain in solution the same tautomeric forms as found in the solid state.

A Keto-Mediet Approach with Coconut Substitution and Exercise May Delay the Onset of Alzheimer's Disease among Middle-Aged.

PubMed

Perng, B C; Chen, M; Perng, J C; Jambazian, P

2017-01-01

Coconut oil has been widely used to improve health because there is much information available by word of mouth, in books, and on the internet. However, researchers still continue to search for the best diets to improve the quality of life, especially for people with cognitive decline. The aim of this review is to develop a novel dietary approach, the Keto-Mediet, which may help prevent the onset of Alzheimer's disease. Evidence gained through literature review from 1982 to 2015 on gene-by-diet interaction and lipid and glucose metabolism in the brains of Alzheimer's patients is converted into the new Keto-Mediet approach. The Keto-Mediet approach combines the benefits of a Ketogenic diet and a Mediterranean diet into a pyramidal model that is rich in various types of vitamins and substitutes coconuts for saturated animal fats. Limited glucose intake is intended to delay brain degeneration. A revised adult food pyramid was created to illustrate the principles of the Keto-Mediet approach. The Keto-Mediet approach represents and interprets food groups according to the revised adult food pyramid. This approach also encourages adherence to this healthy diet and lifestyle changes including exercise for people whose age ranges from 40 to 75 years. Those who comply with this approach will significantly enhance their knowledge and adopt a healthier lifestyle, as compared to those whose modern eating patterns are typically less healthy. Therefore, the Keto-Mediet approach can be applied in hopes of preventing and decreasing Alzheimer's disease in different ethnicities and cultural groups.

Catalyst-Free Difunctionalization of Activated Alkenes in Water: Efficient Synthesis of β-Keto Sulfides and Sulfones.

PubMed

Wang, Huamin; Wang, Guangyu; Lu, Qingquan; Chiang, Chien-Wei; Peng, Pan; Zhou, Jiufu; Lei, Aiwen

2016-10-04

Difunctionalization of activated alkenes, a powerful strategy in chemical synthesis, has been accomplished for direct synthesis of a series of β-keto sulfides and β-keto sulfones. The transformation, mediated by O2 , proceeds smoothly in water and without any catalyst. Prominent advantages of this method include mild reaction conditions, purification simplicity, and gram-scale synthesis, underlining the practical utility of this methodology. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Seasonal variations of low molecular weight hydroxy-dicarboxylic acids and oxaloacetic acid in remote marine aerosols from Chichijima Island in the western North Pacific (December 2010-November 2011)

NASA Astrophysics Data System (ADS)

Gowda, Divyavani; Kawamura, Kimitaka

2018-05-01

Concentrations of homologous hydroxy-dicarboxylic acids (diacids) (hC3-hC6) and keto-diacid (oxaloacetic acid) were measured in the atmospheric aerosols collected at Chichijima Island (27.04° N, 142.13° E) in the western North Pacific from December 2010 to November 2011. The monthly averaged concentrations of hydroxy-diacids and oxaloacetic acid were significantly higher in spring followed by winter and autumn. Molecular distributions of hydroxy-diacids demonstrated that malic acid was the most abundant species in all four seasons, followed by tartronic acid in winter and spring and 3- and 2-hydroxyglutaric acids in summer and autumn. Hydroxy-diacids and keto-diacid maximized in spring and winter when air masses originated from the Asian continent with westerly winds. The concentrations of total hydroxy-diacids and oxaloacetic acid ranged from 0.1 to 27.3 ng m-3 and <0.005 to 2 ng m-3, respectively. The enhanced concentrations of diacids and their intermediates in winter and spring are associated with a long-range atmospheric transport of pollutants from East Asia to remote Chichijima Island followed by photochemical processing of organic aerosols. Seasonal molecular distribution of hydroxy-diacids and oxaloacetic acid was found to be dependent on the source strengths and plausible photochemical processing to form smaller diacids. Moderate to strong correlations among hydroxy-diacids, oxaloacetic acid and low molecular weight (LMW) diacids suggest that hydroxy-diacids and oxaloacetic acid are the intermediates in the photochemical oxidation of LMW diacid. Hence, photochemical formation of the most abundant LMW diacids, i.e., oxalic acid, could be produced from hydroxy- and keto-diacid as intermediates.

Lactate Dehydrogenase Catalysis: Roles of Keto, Hydrated, and Enol Pyruvate

ERIC Educational Resources Information Center

Meany, J. E.

2007-01-01

Many carbonyl substrates of oxidoreductase enzymes undergo hydration and enolization so that these substrate systems are partitioned between keto, hydrated (gem-diol), and enol forms in aqueous solution. Some oxidoreductase enzymes are subject to inhibition by high concentrations of substrate. For such enzymes, two questions arise pertaining to…

Detection and quantification of α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid: a metabolite of asymmetric dimethylarginine.

PubMed

Martens-Lobenhoffer, Jens; Rodionov, Roman N; Drust, Andreas; Bode-Böger, Stefanie M

2011-12-15

Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate L-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMGV. D(6)-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 μmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 μmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2. Copyright © 2011 Elsevier Inc. All rights reserved.

Branched Chain Amino Acid Oxidation in Cultured Rat Skeletal Muscle Cells

PubMed Central

Pardridge, William M.; Casanello-Ertl, Delia; Duducgian-Vartavarian, Luiza

1980-01-01

Leucine metabolism in skeletal muscle is linked to protein turnover. Since clofibrate is known both to cause myopathy and to decrease muscle protein content, the present investigations were designed to examine the effects of acute clofibrate treatment on leucine oxidation. Rat skeletal muscle cells in tissue culture were used in these studies because cultivated skeletal muscle cells, like muscle in vivo, have been shown to actively utilize branched chain amino acids and to produce alanine. The conversion of [1-14C]leucine to 14CO2 or to the [1-14C]keto-acid of leucine (α-keto-isocaproate) was linear for at least 2 h of incubation; the production of 14CO2 from [1-14C]leucine was saturable with a Km = 6.3 mM and a maximum oxidation rate (Vmax) = 31 nmol/mg protein per 120 min. Clofibric acid selectively inhibited the oxidation of [1-14C]leucine (Ki = 0.85 mM) and [U-14C]isoleucine, but had no effect on the oxidation of [U-14C]glutamate, -alanine, -lactate, or -palmitate. The inhibition of [1-14C]leucine oxidation by clofibrate was also observed in the rat quarter-diaphragm preparation. Clofibrate primarily inhibited the production of 14CO2 and had relatively little effect on the production of [1-14C]keto-acid of leucine. A physiological concentration—3.0 g/100 ml—of albumin, which actively binds clofibric acid, inhibited but did not abolish the effects of a 2-mM concentration of clofibric acid on leucine oxidation. Clofibrate treatment stimulated the net consumption of pyruvate, and inhibited the net production of alanine. The drug also increased the cytosolic NADH/NAD+ ratio as reflected by an increase in the lactate/pyruvate ratio, in association with a decrease in cell aspartate levels. The changes in pyruvate metabolism and cell redox state induced by the drug were delayed compared with the nearly immediate inhibition of leucine oxidation. These studies suggest that clofibric acid, in concentrations that approximate high therapeutic levels of the drug

Domino-Fluorination-Protodefluorination Enables Decarboxylative Cross-Coupling of α-Oxocarboxylic Acids with Styrene via Photoredox Catalysis.

PubMed

Zhang, Muliang; Xi, Junwei; Ruzi, Rehanguli; Li, Nan; Wu, Zhongkai; Li, Weipeng; Zhu, Chengjian

2017-09-15

Domino-fluorination-protodefluorination decarboxylative cross-coupling of α-keto acids with styrene has been developed via photoredox catalysis. The critical part of this strategy is the formation of the carbon-fluorine (C-F) bond by the capture of a carbon-centered radical intermediate, which will overcome side reactions during the styrene radical functionalization process. Experimental studies have provided evidence indicating a domino-fluorination-protodefluorination pathway with α-keto acid initiating the photoredox cycle. The present catalytic protocol also affords a novel approach for the construction of α,β-unsaturated ketones under mild conditions.

Improved plasma amino acids pattern following 12 months of supplemented low-protein diet in peritoneal dialysis patients.

PubMed

Jiang, Na; Qian, Jiaqi; Lin, Aiwu; Fang, Wei; Cao, Liou; Wang, Qin; Ni, Zhaohui; Lindholm, Bengt; Axelsson, Jonas; Yao, Qiang

2010-07-01

Decreased plasma essential amino acid (EAA) levels, increased nonessential amino acid (NEAA) levels, and low EAA to NEAA ratio (E/NEAA) are common in peritoneal dialysis (PD) patients and may impact uremic complications. In the present study, we investigate the impact of keto acids-supplemented low-protein (sLP) diet on plasma amino acids (AAs) patterns in stable PD patients. This is a supplemental analysis of a previously published prospective and randomized trial. Thirty-nine PD patients selected from the original population were divided to receive either low (LP: 0.6-0.8 g/kg ideal body weight [IBW]/d, n = 13), keto acids-supplemented low- (sLP: 0.6-0.8 g/kg IBW/d + 0.12 g/kg IBW/d of keto acids, n = 12), or high- (HP: 1.0-1.2 g/kg IBW/d, n = 14) protein diets and followed for 1 year. Plasma AA patterns were assessed at baseline and 12 months using high-performance liquid chromatography. Whereas there were no significant differences between the three groups at baseline, following 12 months, the E/NEAA had increased significantly in group sLP (0.58 +/- 0.16 to 0.83 +/- 0.20, p < 0.05), but was not different in either LP (0.62 +/- 0.20 to 0.72 +/- 0.13, p = ns) or HP (0.66 +/- 0.14 to 0.74 +/- 0.12, p = ns) group. This change in E/NEAA in group sLP was due to a significant decrease in NEAA concomitantly with maintained EAA levels, whereas in the other two groups, neither EAA nor NEAA changed significantly. A low-protein diet supplemented with keto acids significantly improved the pattern of plasma AA in prevalent PD patients.

The diterpenoid 7-keto-sempervirol, derived from Lycium chinense, displays anthelmintic activity against both Schistosoma mansoni and Fasciola hepatica.

PubMed

Edwards, Jennifer; Brown, Martha; Peak, Emily; Bartholomew, Barbara; Nash, Robert J; Hoffmann, Karl F

2015-03-01

Two platyhelminths of biomedical and commercial significance are Schistosoma mansoni (blood fluke) and Fasciola hepatica (liver fluke). These related trematodes are responsible for the chronic neglected tropical diseases schistosomiasis and fascioliasis, respectively. As no vaccine is currently available for anti-flukicidal immunoprophylaxis, current treatment is mediated by mono-chemical chemotherapy in the form of mass drug administration (MDA) (praziquantel for schistosomiasis) or drenching (triclabendazole for fascioliasis) programmes. This overreliance on single chemotherapeutic classes has dramatically limited the number of novel chemical entities entering anthelmintic drug discovery pipelines, raising significant concerns for the future of sustainable blood and liver fluke control. Here we demonstrate that 7-keto-sempervirol, a diterpenoid isolated from Lycium chinense, has dual anthelmintic activity against related S. mansoni and F. hepatica trematodes. Using a microtiter plate-based helminth fluorescent bioassay (HFB), this activity is specific (Therapeutic index = 4.2, when compared to HepG2 cell lines) and moderately potent (LD50 = 19.1 μM) against S. mansoni schistosomula cultured in vitro. This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening), oviposition rates and egg morphology. As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD50 = 17.7 μM). Scanning electron microscopy (SEM) evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental integrity and spine loss. 7-keto-sempervirol negatively

Enhanced Neuroprotection of Acetyl-11-Keto-β-Boswellic Acid (AKBA)-Loaded O-Carboxymethyl Chitosan Nanoparticles Through Antioxidant and Anti-Inflammatory Pathways.

PubMed

Ding, Yi; Qiao, Youbei; Wang, Min; Zhang, Huinan; Li, Liang; Zhang, Yikai; Ge, Jie; Song, Ying; Li, Yuwen; Wen, Aidong

2016-08-01

Acetyl-11-keto-β-boswellic acid (AKBA), a main active constituent from Boswellia serrata resin, is a novel candidate for therapy of cerebral ischemia-reperfusion (I/R) injury. Nevertheless, its poor solubility in aqueous solvent, bioavailability, and rapid clearance limit its curative efficacy. To enhance its potency, in our study, AKBA-loaded o-carboxymethyl chitosan nanoparticle (AKBA-NP) delivery system was synthesized. The transmission electron microscopy and transmission electron microscope images of AKBA-NPs suggested that particle size was 132 ± 18 nm, and particles were spherical in shape with smooth morphology. In pharmacokinetics study, AKBA-NPs apparently increases the area under the curve of plasma concentration-time and prolonged half-life compared with AKBA. The tissue distribution study confirmed that AKBA-NPs had a better brain delivery efficacy in comparison with AKBA. The results from our pharmacodynamic studies showed that AKBA-NPs possess better neuroprotection compared with AKBA in primary neurons with oxygen-glucose deprivation (OGD) model and in animals with middle cerebral artery occlusion (MCAO) model. Additionally, AKBA-NPs modulate antioxidant and anti-inflammatory pathways more effectively than AKBA by increasing nuclear erythroid 2-related factor 2 and heme oxygenase-1 expression, and by decreasing nuclear factor-kappa B and 5-lipoxygenase expression. Collectively, our results suggest that AKBA-NPs serve as a potent delivery vehicle for AKBA in cerebral ischemic therapy.

The synthesis of glutamic acid in the absence of enzymes: Implications for biogenesis

NASA Technical Reports Server (NTRS)

Morowitz, Harold; Peterson, Eta; Chang, Sherwood

1995-01-01

This paper reports on the non-enzymatic aqueous phase synthesis of amino acids from keto acids, ammonia and reducing agents. The facile synthesis of key metabolic intermediates, particularly in the glycolytic pathway, the citric acid cycle, and the first step of amino acid synthesis, lead to new ways of looking at the problem of biogenesis.

Synthesis of Substituted 1,4-Dioxenes through O-H Insertion and Cyclization Using Keto-Diazo Compounds.

PubMed

Davis, Owen A; Croft, Rosemary A; Bull, James A

2016-11-18

1,4-Dioxenes present interesting potential as synthetic intermediates and as unusual motifs for incorporation into biologically active compounds. Here, an efficient synthesis of functionalized 1,4-dioxenes is achieved in two steps. Using keto-diazo compounds, a ruthenium catalyzed O-H insertion with β-halohydrins followed by treatment with base results in cyclization with excellent selectivity, through O-alkylation of the keto-enolate. A variety of halohydrins and anion-stabilizing groups in the diazo-component are tolerated, affording novel functionalized dioxenes. Enantioenriched β-bromohydrins provide enantioenriched 1,4-dioxenes.

Enhanced metabolic effect of erythropoietin and keto acids in CRF patients on low-protein diet: Czech multicenter study.

PubMed

Teplan, Vladimír; Schück, Otto; Knotek, Antonín; Hajný, Jan; Horácková, Miroslava; Kvapil, Milan

2003-03-01

Our study is designed to establish whether supplementation with erythropoietin (EPO) exerts additional beneficial metabolic effects in patients with chronic renal failure (CRF) treated with keto acids (KAs) on a low-protein diet (LPD). A long-term, prospective, randomized study was designed to use three therapeutic protocols: (A) EPO plus KAs plus LPD (group I), (B) EPO plus LPD (group II), and (C) LPD (group III). One hundred eighty-six randomly selected patients (90 men, 96 women; age, 22 to 78 years) with a creatinine clearance of 22 to 36 mL/min were monitored at the beginning and at every 6 months for 3 years. During the study period, glomerular filtration rate measured as inulin clearance decreased slightly (from 26.2 +/- 3.4 to 23.4 +/- 4.1 mL/min in group I), 27.4 +/- 4.8 to 20.2 +/- 4.4 mL/min in group II, and 26.8 +/- 3.6 to 17.4 +/- 4.1 mL/min in group III; P < 0.01). Serum urea levels also declined (P < 0.01), more pronouncedly in group I (P < 0.025). In group I, there was a significant increase in levels of leucine (P < 0.01) and albumin (P < 0.01) and a decrease in proteinuria (P < 0.01). Analysis of the lipid spectrum showed a mild, yet significant, decrease in total cholesterol and low-density lipoprotein cholesterol levels (P < 0.025), more pronounced in group I. In group I, there was a decrease in plasma triglyceride levels (from 362.85 +/- 115.05 mg/dL [4.1 +/- 1.3 mmol/L] to values as low as 203.55 +/- 70.80 mg/dL [2.3 +/- 0.8 mmol/L]; P < 0.01), whereas high-density lipoprotein cholesterol levels increased (from 34.75 +/- 7.72 mg/dL [0.9 +/- 0.2 mmol/L] to 46.33 +/- 7.72 mg/dL [1.2 +/- 0.2 mmol/L]; P < 0.025). Mean arterial blood pressure was stable. EPO supplementation in patients with CRF administered KAs potentiates the beneficial effects on metabolism of proteins, amino acids, and lipids. Long-term coadministration of EPO, KA, and LPD was associated with a delay in progression of renal failure and reduction in proteinuria.

Simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and keto-doxapram in plasma using liquid chromatography - tandem mass spectrometry.

PubMed

Flint, Robert B; Bahmany, Soma; van der Nagel, Bart C H; Koch, Birgit C P

2018-05-16

A simple and specific UPLC-MS/MS method was developed and validated for simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and its active metabolite keto-doxapram. The internal standard was fentanyl-d5 for all analytes. Chromatographic separation was achieved with a reversed phase Acquity UPLC HSS T3 column with a run-time of only 5.0 minutes per injected sample. Gradient elution was performed with a mobile phase consisting of ammonium acetate, formic acid in Milli-Q ultrapure water or in methanol with a total flow rate of 0.4 mL minute -1 . A plasma volume of only 50 μL was required to achieve both adequate accuracy and precision. Calibration curves of all 5 analytes were linear. All analytes were stable for at least 48 hours in the autosampler. The method was validated according to US Food and Drug Administration guidelines. This method allows quantification of fentanyl, sufentanil, cefazolin, doxapram and keto-doxapram, which serves purposes for research, as well as therapeutic drug monitoring, if applicable. The strength of this method is the combination of a small sample volume, a short run-time, a deuterated internal standard, an easy sample preparation method and the ability to simultaneously quantify all analytes in one run. This article is protected by copyright. All rights reserved.

Combinatorial metabolic engineering of industrial Gluconobacter oxydans DSM2343 for boosting 5-keto-D-gluconic acid accumulation.

PubMed

Yuan, Jianfeng; Wu, Mianbin; Lin, Jianping; Yang, Lirong

2016-05-17

L-(+)-tartaric acid (L-TA) is an important organic acid, which is produced from the cream of tartar or stereospecific hydrolysis of the cis-epoxysuccinate. The former method is limited by the availability of raw material and the latter is dependent on the petrochemical material. Thus, new processes for the economical preparation of L-TA from carbohydrate or renewable resource would be much more attractive. Production of 5-keto-D-gluconate (5-KGA) from glucose by Gluconobacter oxydans is the first step to produce L-TA. The aim of this work is to enhance 5-KGA accumulation using combinatorial metabolic engineering strategies in G. oxydans. The sldAB gene, encoding sorbitol dehydrogenase, was overexpressed in an industrial strain G. oxydans ZJU2 under a carefully selected promoter, P0169. To enhance the efficiency of the oxidation by sldAB, the coenzyme pyrroloquinoline quinone (PQQ) and respiratory chain were engineered. Besides, the role in sldAB overexpression, coenzyme and respiratory chain engineering and their subsequent effects on 5-KGA production were investigated. An efficient, stable recombinant strain was constructed, whereas the 5-KGA production could be enhanced. By self-overexpressing the sldAB gene in G. oxydans ZJU2 under the constitutive promoter P0169, the resulting strain, G. oxydans ZJU3, produced 122.48 ± 0.41 g/L of 5-KGA. Furthermore, through the coenzyme and respiratory chain engineering, the titer and productivity of 5-KGA reached 144.52 ± 2.94 g/L and 2.26 g/(L · h), respectively, in a 15 L fermenter. It could be further improved the 5-KGA titer by 12.10 % through the fed-batch fermentation under the pH shift and dissolved oxygen tension (DOT) control condition, obtained 162 ± 2.12 g/L with the productivity of 2.53 g/(L · h) within 64 h. The 5-KGA production could be significantly enhanced with the combinatorial metabolic engineering strategy in Gluconobacter strain, including sldAB overexpression, coenzyme

Two novel mutations in the BCKDK (branched-chain keto-acid dehydrogenase kinase) gene are responsible for a neurobehavioral deficit in two pediatric unrelated patients.

PubMed

García-Cazorla, Angels; Oyarzabal, Alfonso; Fort, Joana; Robles, Concepción; Castejón, Esperanza; Ruiz-Sala, Pedro; Bodoy, Susanna; Merinero, Begoña; Lopez-Sala, Anna; Dopazo, Joaquín; Nunes, Virginia; Ugarte, Magdalena; Artuch, Rafael; Palacín, Manuel; Rodríguez-Pombo, Pilar; Alcaide, Patricia; Navarrete, Rosa; Sanz, Paloma; Font-Llitjós, Mariona; Vilaseca, Ma Antonia; Ormaizabal, Aida; Pristoupilova, Anna; Agulló, Sergi Beltran

2014-04-01

Inactivating mutations in the BCKDK gene, which codes for the kinase responsible for the negative regulation of the branched-chain α-keto acid dehydrogenase complex (BCKD), have recently been associated with a form of autism in three families. In this work, two novel exonic BCKDK mutations, c.520C>G/p.R174G and c.1166T>C/p.L389P, were identified at the homozygous state in two unrelated children with persistently reduced body fluid levels of branched-chain amino acids (BCAAs), developmental delay, microcephaly, and neurobehavioral abnormalities. Functional analysis of the mutations confirmed the missense character of the c.1166T>C change and showed a splicing defect r.[520c>g;521_543del]/p.R174Gfs1*, for c.520C>G due to the presence of a new donor splice site. Mutation p.L389P showed total loss of kinase activity. Moreover, patient-derived fibroblasts showed undetectable (p.R174Gfs1*) or barely detectable (p.L389P) levels of BCKDK protein and its phosphorylated substrate (phospho-E1α), resulting in increased BCKD activity and the very rapid BCAA catabolism manifested by the patients' clinical phenotype. Based on these results, a protein-rich diet plus oral BCAA supplementation was implemented in the patient homozygous for p.R174Gfs1*. This treatment normalized plasma BCAA levels and improved growth, developmental and behavioral variables. Our results demonstrate that BCKDK mutations can result in neurobehavioral deficits in humans and support the rationale for dietary intervention. © 2014 WILEY PERIODICALS, INC.

Predicting Keto-Enol Equilibrium from Combining UV/Visible Absorption Spectroscopy with Quantum Chemical Calculations of Vibronic Structures for Many Excited States. A Case Study on Salicylideneanilines.

PubMed

Zutterman, Freddy; Louant, Orian; Mercier, Gabriel; Leyssens, Tom; Champagne, Benoît

2018-06-21

Salicylideneanilines are characterized by a tautomer equilibrium, between an enol and a keto form of different colors, at the origin of their remarkable thermochromic, solvatochromic, and photochromic properties. The enol form is usually the most stable but appropriate choice of substituents and conditions (solvent, crystal, host compound) can displace the equilibrium toward the keto form so that there is a need for fast prediction of the keto:enol abundance ratio. Here we demonstrate the reliability of a combined theoretical-experimental method, based on comparing simulated and measured UV/visible absorption spectra, to determine this keto/enol ratio. The calculations of the excitation energies, oscillator strengths, and vibronic structures of both enol and keto forms are performed for all excited states absorbing in the relevant (visible and near-UV) wavelength range at the time-dependent density functional theory level by accounting for solvent effects using the polarizable continuum model. This approach is illustrated for two salicylideneaniline derivatives, which are present, in solution, under the form of keto-enol mixtures. The results are compared to those of chemometric analysis as well as ab initio predictions of the reaction free enthalpies.

Effect of 3-keto-1,5-bisphosphonates on obese-liver's rats.

PubMed

Lahbib, Karima; Touil, Soufiane

2016-10-01

Obesity is associated with an oxidative stress status, which is defined by an excess of reactive oxygen species (ROS) vs. the antioxidant defense system. We report in this present work, the link between fat deposition and oxidative stress markers using a High Fat Diet-(HFD) induced rat obesity and liver-oxidative stress. We further determined the impact of chronic administration of 3-keto-1, 5-BPs 1 (a & b) (40μg/kg/8 weeks/i.p.) on liver's level. In fact, exposure of rats to HFD during 16 weeks induced body and liver weight gain and metabolic disruption with an increase on liver Alanine amino transférase (ALAT) and Aspartate aminotransférase (ASAT) concentration. HFD increased liver calcium level as well as free iron, whereas, it provoked a decrease on liver lipase activity. HFD also induced liver-oxidative stress status vocalized by an increase in reactive oxygen species (ROS) as superoxide radical (O 2 ), hydroxyl radical (OH) and Hydrogen peroxide (H 2 O 2 ). Consequently, different deleterious damages as an increase on Malon Dialdehyde MDA, Carbonyl protein PC levels with a decrease in non-protein sulfhydryls NPSH concentrations, have been detected. Interestingly, our results demonstrate a decrease in antioxidant enzymes activities such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx) and peroxidases (POD). Importantly, 3-keto-1,5-bisphosphonates treatment corrected the majority of the deleterious effects caused by HFD, but it failed to correct some liver's disruptions as mineral profile, oxidative damages (PC and NPSH levels) as well as SOD and lipase activities. Our investigation point that 3-keto-1,5-bisphosphonates could be considered as safe antioxidant agents on the hepatic level that should also find other potential biological applications. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-L-gulonic acid from D-sorbitol.

PubMed

Gao, Lili; Hu, Yudong; Liu, Jie; Du, Guocheng; Zhou, Jingwen; Chen, Jian

2014-07-01

2-Keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C, is currently produced by a two-step fermentation route from D-sorbitol. However, this route involves three bacteria, making the mix-culture system complicated and redundant. Thus, replacement of the conventional two-step fermentation process with a one-step process could be revolutionary in vitamin C industry. In this study, different combinations of five L-sorbose dehydrogenases (SDH) and two L-sorbosone dehydrogenases (SNDH) from Ketogulonicigenium vulgare WSH-001 were introduced into Gluconobacter oxydans WSH-003, an industrial strain used for the conversion of d-sorbitol to L-sorbose. The optimum combination produced 4.9g/L of 2-KLG. In addition, 10 different linker peptides were used for the fusion expression of SDH and SNDH in G. oxydans. The best recombinant strain (G. oxydans/pGUC-k0203-GS-k0095) produced 32.4g/L of 2-KLG after 168h. Furthermore, biosynthesis of pyrroloquinoline quinine (PQQ), a cofactor of those dehydrogenases, was enhanced to improve 2-KLG production. With the stepwise metabolic engineering of G. oxydans, the final 2-KLG production was improved to 39.2g/L, which was 8.0-fold higher than that obtained using independent expression of the dehydrogenases. These results bring us closer to the final one-step industrial-scale production of vitamin C. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Indium-catalyzed synthesis of keto esters from cyclic 1,3-diketones and alcohols and application to the synthesis of seratrodast.

PubMed

Kuninobu, Yoichiro; Kawata, Atsushi; Noborio, Taihei; Yamamoto, Syun-Ichi; Matsuki, Takashi; Takata, Kazumi; Takai, Kazuhiko

2010-04-01

Esterification reactions from cyclic 1,3-diketones and alcohols are carried out in the presence of several Lewis acids. In particular, indium(III) triflate, In(OTf)(3), iron(III) triflate, Fe(OTf)(3), copper(II) triflate, Cu(OTf)(2), and silver(I) triflate, AgOTf, show high catalytic activities. These reactions proceed through the carbon-carbon bond cleavage by a retro-aldol reaction and were found to be highly regioselective even in the presence of other functional groups. This type of reaction can also be applied to the preparation of the keto esters during the synthesis of seratrodast, which is an antiasthmatic and eicosanoid antagonist.

Metformin inhibits Branched Chain Amino Acid (BCAA) derived ketoacidosis and promotes metabolic homeostasis in MSUD.

PubMed

S Sonnet, Davis; N O'Leary, Monique; A Gutierrez, Mark; M Nguyen, Steven; Mateen, Samiha; Hsu, Yuehmei; P Mitchell, Kylie; J Lopez, Antonio; Vockley, Jerry; K Kennedy, Brian; Ramanathan, Arvind

2016-07-04

Maple Syrup Urine Disease (MSUD) is an inherited disorder caused by the dysfunction in the branched chain keto-acid dehydrogenase (BCKDH) enzyme. This leads to buildup of branched-chain keto-acids (BCKA) and branched-chain amino acids (BCAA) in body fluids (e.g. keto-isocaproic acid from the BCAA leucine), leading to numerous clinical features including a less understood skeletal muscle dysfunction in patients. KIC is an inhibitor of mitochondrial function at disease relevant concentrations. A murine model of intermediate MSUD (iMSUD) shows significant skeletal muscle dysfunction as by judged decreased muscle fiber diameter. MSUD is an orphan disease with a need for novel drug interventions. Here using a 96-well plate (liquid chromatography- mass spectrometry (LC-MS) based drug-screening platform we show that Metformin, a widely used anti-diabetic drug, reduces levels of KIC in patient-derived fibroblasts by 20-50%. This Metformin-mediated effect was conserved in vivo; Metformin-treatment significantly reduced levels of KIC in the muscle (by 69%) and serum (by 56%) isolated from iMSUD mice, and restored levels of mitochondrial metabolites (e.g. AMP and other TCA). The drug also decreased the expression of mitochondrial branched chain amino transferase (BCAT) which produces KIC in skeletal muscle. This suggests that Metformin can restore skeletal muscle homeostasis in MSUD by decreasing mitochondrial KIC production.

The aldo-keto reductase superfamily homepage.

PubMed

Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

2003-02-01

The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

Computational Study of the Malonic Acid Tautomerization Products in Highly Concentrated Particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dick-Pérez, Marilú; Windus, Theresa L.

Knowing the tautomeric form of malonic acid (MA) in concentrated particles is critical to understanding its effect on the atmosphere. Energies and vibrational modes of hydrated MA particles were calculated using density functional theory (DFT) at the B3LYP/6-31G(d,p) level and the effective fragment potential (EFP) method. Visualization of the keto and enol isomer vibrational modes enabled the assignment of keto isomer peaks in the 1710–1750 cm –1 range, and previously unidentified experimental IR peaks in the 1690–1710 cm –1 can now be attributed to the enol isomer. Furthermore, a comparison of calculated spectra of pure hydrated enol or keto isomersmore » confirm recent experimental evidence, of a shift in the keto–enol tautomer equilibrium when MA exists as concentrated particles.« less

Computational Study of the Malonic Acid Tautomerization Products in Highly Concentrated Particles

DOE PAGES

Dick-Pérez, Marilú; Windus, Theresa L.

2017-03-09

Knowing the tautomeric form of malonic acid (MA) in concentrated particles is critical to understanding its effect on the atmosphere. Energies and vibrational modes of hydrated MA particles were calculated using density functional theory (DFT) at the B3LYP/6-31G(d,p) level and the effective fragment potential (EFP) method. Visualization of the keto and enol isomer vibrational modes enabled the assignment of keto isomer peaks in the 1710–1750 cm –1 range, and previously unidentified experimental IR peaks in the 1690–1710 cm –1 can now be attributed to the enol isomer. Furthermore, a comparison of calculated spectra of pure hydrated enol or keto isomersmore » confirm recent experimental evidence, of a shift in the keto–enol tautomer equilibrium when MA exists as concentrated particles.« less

Deposition Form and Bioaccessibility of Keto-carotenoids from Mamey Sapote (Pouteria sapota), Red Bell Pepper (Capsicum annuum), and Sockeye Salmon (Oncorhynchus nerka) Filet.

PubMed

Chacón-Ordóñez, Tania; Esquivel, Patricia; Jiménez, Víctor M; Carle, Reinhold; Schweiggert, Ralf M

2016-03-09

The ultrastructure and carotenoid-bearing structures of mamey sapote (Pouteria sapota) chromoplasts were elucidated using light and transmission electron microscopy and compared to carotenoid deposition forms in red bell pepper (Capsicum annuum) and sockeye salmon (Oncorhynchus nerka). Globular-tubular chromoplasts of sapote contained numerous lipid globules and tubules embodying unique provitamin A keto-carotenoids in a lipid-dissolved and presumably liquid-crystalline form, respectively. Bioaccessibility of sapotexanthin and cryptocapsin was compared to that of structurally related keto-carotenoids from red bell pepper and salmon. Capsanthin from bell pepper was the most bioaccessible pigment, followed by sapotexanthin and cryptocapsin esters from mamey sapote. In contrast, astaxanthin from salmon was the least bioaccessible keto-carotenoid. Thermal treatment and fat addition consistently enhanced bioaccessibility, except for astaxanthin from naturally lipid-rich salmon, which remained unaffected. Although the provitamin A keto-carotenoids from sapote were highly bioaccessible, their qualitative and quantitative in vivo bioavailability and their conversion to vitamin A remains to be confirmed.

In vivo assessment of the mitochondrial response to caloric restriction in obese women by the 2-keto[1-C]isocaproate breath test.

PubMed

Parra, Dolores; González, Alvaro; Martínez, J Alfredo; Labayen, Idoia; Díez, Nieves

2003-04-01

The 2-keto[1-(13)C]isocaproate breath test has been proposed as a tool to detect mitochondrial dysfunction in alcoholic liver disease. The aim of this study was to evaluate if the 2-keto[1-(13)C]isocaproate breath test could detect in vivo dynamic changes on mitochondrial activity due to caloric restriction in obese women. Fifteen obese women (body mass index [BMI] > 30 kg/m(2)) participated in the study at baseline. Ten of these women agreed to participate on a diet program to induce body weight loss. Fifteen lean women (BMI < 25 kg/m(2)) were included as a control group. The breath test was performed by the oral administration of the tracer measuring (13)CO(2) enrichment in breath before and after ingestion using isotope ratio mass spectrometry. Body composition, resting energy expenditure, and plasma levels of insulin and leptin were measured. There were no relationships observed between the 2-keto[1-(13)C]isocaproate breath test and the plasma insulin (before diet: P =.863; after diet: P =.879), or leptin (before diet: P =.500; after diet: P =.637). In obese women before treatment, kilograms of fat free mass (P =.108), resting energy expenditure adjusted for body composition (P =.312), and the 2-keto[1-(13)C]isocaproate breath test (P =.205) were similar in comparison to lean women. However, 2-keto[1-(13)C]isocaproate oxidation tended to increase after dieting and was significantly higher than in controls (P =.015). These data suggest that the 2-keto[1-(13)C]isocaproate breath test reflected the adaptive modifications in mitochondrial oxidation in response to caloric restriction in obese women. Copyright 2003 Elsevier, Inc. All rights reserved.

Keto-supplemented Low Protein Diet: A Valid Therapeutic Approach for Patients with Steroid-resistant Proteinuria during Early-stage Chronic Kidney Disease.

PubMed

Zhang, J; Xie, H; Fang, M; Wang, K; Chen, J; Sun, W; Yang, L; Lin, H

2016-04-01

Low protein diets supplemented with keto acid (sLPD) are recommended for patients with stage 3-5 chronic kidney disease (CKD). This study assessed whether sLPD is beneficial for patients with steroid-resistant proteinuria during early-stage CKD. A 1-year randomized controlled trial was conducted from 2010 to 2012. In this study, 108 proteinuric patients who were steroid-resistant were assigned to a sLPD group (0.6 g/kg/d with 0.09 g/kg/d keto acids) or a normal protein diet group (NPD, 1.0 g/kg/d). Estimated dietary protein intake, urinary protein excretion, remission rate, renal function, nutritional status, and blood pressure were measured. Baseline characteristics were comparable between the sLPD group (47 patients) and the NPD group (49 patients). Urinary protein excretion significantly decreased in sLPD compared to NPD in months 6, 9, and 12 (P<0.05). Proteinuria reduction was higher in sLPD than in NPD (P<0.001) at the end of the study. Complete remission and partial remission rates were higher in sLPD than in NPD. Serum albumin and pre-albumin levels were higher in sLPD than in NPD in months 9 and 12 (P<0.05). Serum total cholesterol and triglyceride levels declined more significantly in sLPD than in NPD (P<0.01) at the end of the study. There were no differences in nutritional status, renal function, hemoglobin, or blood pressure between the two groups. sLPD is both nutritionally safe and beneficial, providing nephroprotective effects for early-stage CKD patients with steroid-resistant proteinuria.

Thermophilic enzymes and their applications in biocatalysis: a robust aldo-keto reductase.

PubMed

Willies, Simon; Isupov, Misha; Littlechild, Jennifer

2010-09-01

Extremophiles are providing a good source of novel robust enzymes for use in biocatalysis for the synthesis of new drugs. This is particularly true for the enzymes from thermophilic organisms which are more robust than their mesophilic counterparts to the conditions required for industrial bio-processes. This paper describes a new aldo-keto reductase enzyme from a thermophilic eubacteria, Thermotoga maritima which can be used for the production of primary alcohols. The enzyme has been cloned and over-expressed in Escherichia coli and has been purified and subjected to full biochemical characterization. The aldo-keto reductase can be used for production of primary alcohols using substrates including benzaldehyde, 1,2,3,6-tetrahydrobenzaldehyde and para-anisaldehyde. It is stable up to 80 degrees C, retaining over 60% activity for 5 hours at this temperature. The enzyme at pH 6.5 showed a preference for the forward, carbonyl reduction. The enzyme showed moderate stability with organic solvents, and retained 70% activity in 20% (v/v) isopropanol or DMSO. These properties are favourable for its potential industrial applications.

Structure and mechanism of human UDP-xylose synthase: evidence for a promoting role of sugar ring distortion in a three-step catalytic conversion of UDP-glucuronic acid.

PubMed

Eixelsberger, Thomas; Sykora, Sabine; Egger, Sigrid; Brunsteiner, Michael; Kavanagh, Kathryn L; Oppermann, Udo; Brecker, Lothar; Nidetzky, Bernd

2012-09-07

UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-D-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with NAD(+) and UDP reveals a homodimeric short-chain dehydrogenase/reductase (SDR), belonging to the NDP-sugar epimerases/dehydratases subclass. We show that enzymatic reaction proceeds in three chemical steps via UDP-4-keto-D-glucuronic acid and UDP-4-keto-pentose intermediates. Molecular dynamics simulations reveal that the D-glucuronyl ring accommodated by UXS features a marked (4)C(1) chair to B(O,3) boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C(4) (step 1) by aligning the enzymatic base Tyr(147) with the reactive substrate hydroxyl and it brings the carboxylate group at C(5) into an almost fully axial position, ideal for decarboxylation of UDP-4-keto-D-glucuronic acid in the second chemical step. The protonated side chain of Tyr(147) stabilizes the enolate of decarboxylated C(4) keto species ((2)H(1) half-chair) that is then protonated from the Si face at C(5), involving water coordinated by Glu(120). Arg(277), which is positioned by a salt-link interaction with Glu(120), closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C(4) keto group by NADH, assisted by Tyr(147) as catalytic proton donor, yields UDP-xylose adopting the relaxed (4)C(1) chair conformation (step 3).

Arachidonic acid and prostaglandin endoperoxide metabolism in isolated rabbit and coronary microvessels and isolated and cultivated coronary microvessel endothelial cells.

PubMed Central

Gerritsen, M E; Cheli, C D

1983-01-01

Isolated microvessels and isolated and cultured microvessel endothelial cells were prepared from rabbit cardiac muscle. Pathways of arachidonic acid metabolism were determined by measurement of exogenous substrate utilization [( 1-14C]arachidonic acid incorporation and release from intact tissue and cells; [1-14C]prostaglandin H2 (PGH2) metabolism by broken cell preparations) and by quantification of endogenous products (immunoreactive 6-keto-prostaglandin F1 alpha (PGF1 alpha) and prostaglandin E (PGE) release) by selective radioimmunoassay. Rabbit coronary microvessels and derived microvascular endothelial cells (RCME cells) synthesized two major products of the cyclooxygenase pathway: 6-keto-PGF1 alpha (hydrolytic product of prostaglandin I2) and PGE2. A reduced glutathione requiring PGH-E isomerase was demonstrated in coronary microvessels and RCME cells, but not in rabbit circumflex coronary artery or aorta. In addition, a minor amount of a compound exhibiting similar characteristics to 6-keto-PGE1 was found to be produced by microvessels and RCME cells. Measurement of endogenously released prostaglandins indicated that under basal and stimulated conditions, PGE release exceeded that of 6-keto-PGF1 alpha. Microvessels and microvessel endothelial cells derived from cardiac muscle of rabbit exhibit pathways of arachidonate metabolism that are different from those of many large blood vessels and derived endothelial cells. Images PMID:6415116

Divergent shifts in lipid mediator profile following supplementation with n-3 docosapentaenoic acid and eicosapentaenoic acid.

PubMed

Markworth, James F; Kaur, Gunveen; Miller, Eliza G; Larsen, Amy E; Sinclair, Andrew J; Maddipati, Krishna Rao; Cameron-Smith, David

2016-11-01

In contrast to the well-characterized effects of specialized proresolving lipid mediators (SPMs) derived from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), little is known about the metabolic fate of the intermediary long-chain (LC) n-3 polyunsaturated fatty acid (PUFA) docosapentaenoic acid (DPA). In this double blind crossover study, shifts in circulating levels of n-3 and n-6 PUFA-derived bioactive lipid mediators were quantified by an unbiased liquid chromatography-tandem mass spectrometry lipidomic approach. Plasma was obtained from human subjects before and after 7 d of supplementation with pure n-3 DPA, n-3 EPA or placebo (olive oil). DPA supplementation increased the SPM resolvin D5 n -3DPA (RvD5 n -3DPA ) and maresin (MaR)-1, the DHA vicinal diol 19,20-dihydroxy-DPA and n-6 PUFA derived 15-keto-PG E 2 (15-keto-PGE 2 ). EPA supplementation had no effect on any plasma DPA or DHA derived mediators, but markedly elevated monohydroxy-eicosapentaenoic acids (HEPEs), including the e-series resolvin (RvE) precursor 18-HEPE; effects not observed with DPA supplementation. These data show that dietary n-3 DPA and EPA have highly divergent effects on human lipid mediator profile, with no overlap in PUFA metabolites formed. The recently uncovered biologic activity of n-3 DPA docosanoids and their marked modulation by dietary DPA intake reveals a unique and specific role of n-3 DPA in human physiology.-Markworth, J. F., Kaur, G., Miller, E. G., Larsen, A. E., Sinclair, A. J., Maddipati, K. R., Cameron-Smith, D. Divergent shifts in lipid mediator profile following supplementation with n-3 docosapentaenoic acid and eicosapentaenoic acid. © FASEB.

Unique occurrence of unusual fatty acids and their industrial utilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hosamani, K.M.

1996-01-01

Ebenaceae plant family consists of 7 genera and more than 320 species. The present investigation describes the unique occurrence of hitherto unknown 9-keto-cis-13-octadecenoic acid (29.0%) and the cyclopropenoid fatty acids (malvalic acid 12.7% and sterculic acid 8.7%) in the seed oil of Diospyros melanoxylon as well as in the Ebenaceae plant family. Other normal fatty acids are also detected. The identification and characterization were based on FTIR, {sup 1}H NMR, {sup 13}C NMR, MS, and GLC techniques and chemical degradations.

Domain analysis of 3 Keto Acyl-CoA synthase for structural variations in Vitis vinifera and Oryza brachyantha using comparative modelling.

PubMed

Sagar, Mamta; Pandey, Neetesh; Qamar, Naseha; Singh, Brijendra; Shukla, Akanksha

2015-03-01

The long chain fatty acids incorporated into plant lipids are derived from the iterative addition of C2 units which is provided by malonyl-CoA to an acyl-CoA after interactions with 3-ketoacyl-CoA synthase (KCS), found in several plants. This study provides functional characterization of three 3 ketoacyl CoA synthase like proteins in Vitis vinifera (one) and Oryza brachyantha (two proteins). Sequence analysis reveals that protein of Oryza brachyantha shows 96% similarity to a hypothetical protein in Sorghum bicolor; total 11 homologs were predicted in Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type III polyketide synthase-like protein, Thiolase-like, subgroup; Thiolase-like and 3-Oxoacyl-ACP synthase III, C-terminal and chalcone synthase like domain but very long chain 3-keto acyl CoA domain is absent. All three proteins were found to have Chalcone and stilbene synthases C terminal domain which is similar to domain of thiolase and β keto acyl synthase. Its N terminal domain is absent in J3M9Z7 protein of Oryza brachyantha and F6HH63 protein of Vitis vinifera. Differences in N-terminal domain is responsible for distinguish activity. The J3MF16 protein of Oryza brachyantha contains N terminal domain and C terminal domain and characterized using annotation of these domains. Domains Gcs (streptomyces coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase (Gerbera hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in three proteins. This similarity points toward anthocyanin biosynthetic process. Similarity to chalcone synthase 2 reveals its possible role in Naringenine and Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza brachyantha. Active site residues C-240, H-407, N-447 are present in J3MF16 protein that are common in these three protein at different positions. Structural variations among dimer interface, product binding site, malonyl-CoA binding sites, were predicted in

Pharmacologic activation of peroxisome proliferator-activating receptor-α accelerates hepatic fatty acid oxidation in neonatal pigs

PubMed Central

Shim, Kwanseob; Jacobi, Sheila; Odle, Jack; Lin, Xi

2018-01-01

Up-regulation of peroxisome proliferator-activating receptor-α (PPARα) and increasing fatty acid oxidation are important for reducing pre-weaning mortality of pigs. We examined the time-dependent regulatory effects of PPARα activation via oral postnatal clofibrate administration (75 mg/(kg-BW·d) for up to 7 days) on mitochondrial and peroxisomal fatty acid oxidation in pigs, a species with limited hepatic fatty acid oxidative capacity due to low ketogenesis. Hepatic oxidation was increased by 44-147% (depending on fatty acid chain-length) and was attained after only 4 days of clofibrate treatment. Acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase I (CPTI) activities accelerated in parallel. The increase in CPTI activity was accompanied by a rapid reduction in the sensitivity of CPTI to malonyl-CoA inhibition. The mRNA abundance of CPTI and ACO, as well as peroxisomal keto-acyl-CoA thiolase (KetoACoA) and mitochondrial malonyl-CoA decarboxylase (MCD), also were augmented greatly. However, the increase in ACO activity and MCD expression were different from CPTI, and significant interactions were observed between postnatal age and clofibrate administration. Furthermore, the expression of acetyl-CoA carboxylase β (ACCβ) decreased with postnatal age and clofibrate had no effect on its expression. Collectively these results demonstrate that the expression of PPARα target genes and the increase in fatty acid oxidation induced by clofibrate are time- and age-dependent in the liver of neonatal pigs. Although the induction patterns of CPTI, MCD, ACO, KetoACoA, and ACCβ are different during the early postnatal period, 4 days of exposure to clofibrate were sufficient to robustly accelerate fatty acid oxidation.

Keto analogues and amino acids supplementation induces a decrease of white blood cell counts and a reduction of muscle damage during intense exercise under thermoneutral conditions.

PubMed

Lima, R C P; Camerino, S R A S; França, T C L; Rodrigues, D S A; Gouveia, M G S; Ximenes-da-Silva, A; Bassini, A; Prado, E S; Cameron, L C

2017-04-19

This study evaluated the acute effect of keto analogue and amino acid (AA-KAAA) supplementation on both white blood cell counts and the established biomarkers of muscle damage during exercise under thermoneutral conditions. Sixteen male cyclists received a ketogenic diet for two days and were divided into two equal groups: a group taking AA-KAAA (KA) or a control group (PL). The athletes performed a two hour cycling session followed by a maximum incremental test until voluntary exhaustion (VExh). Blood samples were obtained at rest and during exercise for further hematological and biochemical analyses. Exercise-induced ammonemia increased in the PL group at VExh (75%) but remained unchanged in the KA group. Both groups exhibited a significant increase in leukocyte and neutrophil counts of ∼85% (∼13 × 10 9 L -1 ), but the shape of the lymphocytes and the eosinophil counts suggest that AA-KAAA supplementation helps prevent lymphocytosis. AA-KAAA supplementation induced a decrease in creatine kinase and aspartate aminotransferase levels at VExh while showing a significant decrease in lactate dehydrogenase at 120 min. We found that AA-KAAA supplementation decreases both the lymphocyte count response in blood and the established biomarkers of muscle damage after intense exercise under a low heat stress environment.

Stereocontrolled reduction of alpha- and beta-keto esters with micro green algae, Chlorella strains.

PubMed

Ishihara, K; Yamaguchi, H; Adachi, N; Hamada, H; Nakajima, N

2000-10-01

The stereocontrolled reduction of alpha- and beta-keto esters using micro green algae was accomplished by a combination of the cultivation method and the introduction of an additive. The reduction of ethyl pyruvate and ethyl benzoylformate by the photoautotrophically cultivated Chlorella sorokiniana gave the corresponding alcohol in high e.e. (>99% e.e. (S) and >99% e.e. (R), respectively). In the presence of glucose as an additive, the reduction of ethyl 3-methyl-2-oxobutanoate by the heterotrophically cultivated C. sorokiniana afforded the corresponding (R)-alcohol. On the other hand, the reduction in the presence of ethyl propionate gave the (S)-alcohol. Ethyl 2-methyl-3-oxobutanoate was reduced in the presence of glycerol by the photoautotrophically cultivated C. sorokiniana or the heterotrophically cultivated C. sorokiniana to the corresponding syn-(2R,3S)-hydroxy ester with high diastereo- and enantiomeric excess (e.e.). Some additives altered the stereochemical course in the reduction of alpha- and beta-keto esters.

A novel aldo-keto reductase from Escherichia coli can increase resistance to methylglyoxal toxicity.

PubMed

Grant, Anne W; Steel, Gavin; Waugh, Hugh; Ellis, Elizabeth M

2003-01-21

A novel aldo-keto reductase (AKR) from Escherichia coli has been cloned, expressed and purified. This protein, YghZ, is distantly related (<40%) to mammalian aflatoxin dialdehyde reductases of the aldo-keto reductase AKR7 family and to potassium channel beta-subunits in the AKR6 family. The enzyme has been placed in a new AKR family (AKR14), with the designation AKR14A1. Sequences encoding putative homologues of this enzyme exist in many other bacteria. The enzyme can reduce several aldehyde and diketone substrates, including the toxic metabolite methylglyoxal. The K(m) for the model substrate 4-nitrobenzaldehyde is 1.06 mM and for the endogenous dicarbonyl methylglyoxal it is 3.4 mM. Overexpression of the recombinant enzyme in E. coli leads to increased resistance to methylglyoxal. It is possible that this enzyme plays a role in the metabolism of methylglyoxal, and can influence its levels in vivo.

Dimerization of the keto tautomer of acetohydroxamic acid—infrared matrix isolation and theoretical study

NASA Astrophysics Data System (ADS)

Sałdyka, Magdalena; Mielke, Zofia

2005-05-01

Dimerization of the keto tautomer of acetohydroxamic acid has been studied using FTIR matrix isolation spectroscopy and DFT(B3LYP)/6-31+G(d,p) calculations. Analysis of CH 3CONHOH/Ar matrix spectra indicates formation of two dimers in which two intramolecular CO···H sbnd ON bonds within two interacting acetohydroxamic acid molecules are retained. A chain dimer I is stabilized by the intermolecular CO···H sbnd N hydrogen bond, whereas the cyclic dimer II is stabilized by two intermolecular N sbnd H···O(H)N bonds. Twelve vibrations were identified for dimer I and six vibrations for dimer II; the observed frequency shifts show a good agreement with the calculated ones for the structures I and II. Both dimers have comparable binding energies ( ΔEZPECPI, II = -7.02, -6.34 kcal mol -1) being less stable than calculated structures III and IV ( ΔEZPECPIII, IV = -9.50, -8.87 kcal mol -1) in which one or two intramolecular hydrogen bonds are disrupted. In the most stable 10-membered cyclic dimer III, two intermolecular CO···H sbnd ON hydrogen bonds are formed at expense of intramolecular hydrogen bonds of the same type. The formation of the less stable (AHA) 2 dimers in the studied matrixes indicates that the formation of (AHA) 2 is kinetically and not thermodynamically controlled.

SOLVENT-FREE FACILE SYNTHESIS OF NOVEL α-TOSYLOXY β-KETO SULFONES USING [HYDROXY(TOSYLOXY)IODO]BENZENE

EPA Science Inventory

A facile, general and high yielding protocol for the synthesis of novel α-tosyloxy β-keto sulfones is described utilizing relatively non-toxic, [hydroxy(tosyloxy)iodo]benzene, under solvent-free conditions at room temperature.

Gold-catalyzed synthesis of benzil derivatives and α-keto imides via oxidation of alkynes.

PubMed

Xu, Cheng-Fu; Xu, Mei; Jia, Yi-Xia; Li, Chuan-Ying

2011-03-18

An efficient process based on the gold-catalyzed redox reaction has been developed to oxidize 1,2-diarylacetylene or ynamide to 1,2-diaryldiketone or α-keto imide respectively. This process can tolerate a variety of functional groups and affords 1,2-dicarbonyl compounds in excellent yields under mild reaction conditions.

β-Keto esters from ketones and ethyl chloroformate: a rapid, general, efficient synthesis of pyrazolones and their antimicrobial, in silico and in vitro cytotoxicity studies

PubMed Central

2013-01-01

Background Pyrazolones are traditionally synthesized by the reaction of β-keto esters with hydrazine and its derivatives. There are methods to synthesize β-keto esters from esters and aldehydes, but these methods have main limitation in varying the substituents. Often, there are a number of methods such as acylation of enolates in which a chelating effect has been employed to lock the enolate anion using lithium and magnesium salts; however, these methods suffer from inconsistent yields in the case of aliphatic acylation. There are methods to synthesize β-keto esters from ketones like caboxylation of ketone enolates using carbon dioxide and carbon monoxide sources in the presence of palladium or transition metal catalysts. Currently, the most general and simple method to synthesize β-keto ester is the reaction of dimethyl or ethyl carbonate with ketone in the presence of strong bases which also requires long reaction time, use of excessive amount of reagent and inconsistent yield. These factors lead us to develop a simple method to synthesize β-keto esters by changing the base and reagent. Results A series of β-keto esters were synthesized from ketones and ethyl chloroformate in the presence of base which in turn are converted to pyrazolones and then subjected to cytotoxicity studies towards various cancer cell lines and antimicrobial activity studies towards various bacterial and fungal strains. Conclusion The β-keto esters from ethyl chloroformate was successfully attempted, and the developed method is simple, fast and applicable to the ketones having the alkyl halogens, protecting groups like Boc and Cbz that were tolerated and proved to be useful in the synthesis of fused bicyclic and tricyclic pyrazolones efficiently using cyclic ketones. Since this method is successful for different ketones, it can be useful for the synthesis of pharmaceutically important pyrazolones also. The synthesized pyrazolones were subjected to antimicrobial, docking and

[Influence of exogenous gamma-aminobutyric acid (GABA) on GABA metabolism and amino acid contents in roots of melon seedling under hypoxia stress].

PubMed

Wang, Chun-Yan; Li, Jing-Rui; Xia, Qing-Ping; Wu, Xiao-Lei; Gao, Hong-Bo

2014-07-01

This paper investigated the influence of gamma-aminobutyric acid (GABA) on GABA metabolism and amino acid content under hypoxia stress by accurately controlling the level of dissolved oxygen in hydroponics, using the roots of melon 'Xiyu 1' seedlings as the test material. The results showed that compared with the control, the growth of roots was inhibited seriously under hypoxia stress. Meanwhile, the hypoxia-treated roots had significantly higher activities of glutamate decarboxylase (GAD), glutamate dehydrogenase (GDH), glutamate synthase (GOGAT), glutamine synthetase (GS), alanine aminotransferase (ALT), aspartate aminotransferase (AST) as well as the contents of GABA, pyruvic acid, alanine (Ala) and aspartic acid (Asp). But the contents of glutamic acid (Glu) and alpha-keto glutaric acid in roots under hypoxia stress was obviously lower than those of the control. Exogenous treatment with GABA alleviated the inhibition effect of hypoxia stress on root growth, which was accompanied by an increase in the contents of endogenous GABA, Glu, alpha-keto glutaric acid and Asp. Furthermore, under hypoxia stress, the activities of GAD, GDH, GOGAT, GS, ALT, AST as well as the contents of pyruvic acid and Ala significantly decreased in roots treated with GABA. However, adding GABA and viny-gamma-aminobutyric acid (VGB) reduced the alleviation effect of GABA on melon seedlings under hypoxia stress. The results suggested that absorption of GABA by roots could alleviate the injury of hypoxia stress to melon seedlings. This meant that GABA treatment allows the normal physiological metabolism under hypoxia by inhibiting the GAD activity through feedback and maintaining higher Glu content as well as the bal- ance of carbon and nitrogen.

A FACILE ONE-POT SYNTHESIS OF β-KETO SULFONES FROM KETONES UNDER SOLVENT-FREE CONDITIONS

EPA Science Inventory

An easy solvent-free method is described for the conversion of ketones into β-keto sulfones in high yields that involves in situ generation of α-tosyloxyketones followed by nucleophilic substitution with sodium arene sulfinate in presence of tetra-butylammonium bromide at ...

3-Keto-1,5-bisphosphonates Alleviate Serum-Oxidative Stress in the High-fat Diet Induced Obesity in Rats.

PubMed

Lahbib, Karima; Aouani, Iyadh; Cavalier, Jean-François; Touil, Soufiane

2015-09-01

Obesity has become a leading global health problem owing to its strong association with a high incidence of oxidative stress. Many epidemiologic studies showed that an antioxidant supplementation decreases the state of oxidative stress. In the present work, a HFD-induced rat obesity and oxidative stress were used to investigate the link between fat deposition and serum-oxidative stress markers. We also studied the effect of a chronic administration of 3-keto-1,5-bisphosphonates 1 (a & b) (40 μg/kg/8 weeks/i.p.). Exposure of rats to HFD during 16 weeks induced fat deposition, weight gain and metabolic disruption characterized by an increase in cholesterol, triglyceride and glycemia levels, and a decrease in ionizable calcium and free iron concentrations. HFD also induced serum-oxidative stress status vocalized by an increase in ROS (H2 O2 ), MDA and PC levels, with a decrease in antioxidant enzyme activity (CAT, GPx, SOD). Importantly, 3-keto-1,5-bisphosphonates corrected all the deleterious effects of HFD treatment in vivo, but it failed to inhibit lipases in vitro and in vivo. These studies suggest that 3-keto-1,5-bisphosphonates 1 could be considered as safe antioxidant agents that should also find other potential biological applications. © 2014 John Wiley & Sons A/S.

Preparation of porous carbon spheres from 2-keto-l-gulonic acid mother liquor by oxidation and activation for electric double-layer capacitor application.

PubMed

Hao, Zhi-Qiang; Cao, Jing-Pei; Zhao, Xiao-Yan; Wu, Yan; Zhu, Jun-Sheng; Dang, Ya-Li; Zhuang, Qi-Qi; Wei, Xian-Yong

2018-03-01

A novel strategy is proposed for the increase of specific surface area (SSA) of porous carbon sphere (PCS) by oxidation and activation. 2-keto-l-gulonic acid mother liquor (GAML) as a high-pollution waste has a relatively high value of reutilization. For its high value-added utilization, GAML is used as the precursor for preparation of PCS as carbon-based electrode materials for electric double-layer capacitor. PCS is prepared by hydrothermal carbonization, carbonization and KOH activation, and Fe(NO 3 ) 3 9H 2 O is used as an oxidizing agent during carbonization. The as-prepared PCS has excellent porosity and high SSA of 2478 m 2  g -1 . Meanwhile, the pore structure of PCS can be controlled by the adjustment of carbonization parameters (carbonization temperature and the loading of Fe(NO 3 ) 3 9H 2 O). Besides, the SSA and specific capacitance of PCS can be increased remarkably when Fe(NO 3 ) 3 9H 2 O is added in carbonization. The specific capacitance of PCS can reach 303.7 F g -1 at 40 mA g -1 . PCSs as electrode material have superior electrochemical stability. After 8000 cycles, the capacitance retention is 98.3% at 2 A g -1 . The electric double-layer capacitance of PCS is improved when CS is carbonized with Fe(NO 3 ) 3 9H 2 O, and the economic and environmental benefits are achieved by the effective recycle of GAML. Copyright © 2017 Elsevier Inc. All rights reserved.

A new member of the aldo-keto reductase family from the plant pathogen Xylella fastidiosa.

PubMed

Rosselli, Luciana K; Oliveira, Cristiano L P; Azzoni, Adriano R; Tada, Susely F S; Catani, Cleide F; Saraiva, Antonio M; Soares, José Sérgio M; Medrano, Francisco J; Torriani, Iris L; Souza, Anete P

2006-09-15

The Xylella fastidiosa genome program generated a large number of gene sequences that belong to pathogenicity, virulence and adaptation categories from this important plant pathogen. One of these genes (XF1729) encodes a protein similar to a superfamily of aldo-keto reductase together with a number of structurally and functionally related NADPH-dependent oxidoreductases. In this work, the similar sequence XF1729 from X. fastidiosa was cloned onto the pET32Xa/LIC vector in order to overexpress a recombinant His-tag fusion protein in Escherichia coli BL21(DE3). The expressed protein in the soluble fraction was purified by immobilized metal affinity chromatography (agarose-IDA-Ni resin). Secondary structure contents were verified by circular dichroism spectroscopy. Small angle X-ray scattering (SAXS) measurements furnish general structural parameters and provide a strong indication that the protein has a monomeric form in solution. Also, ab initio calculations show that the protein has some similarities with a previously crystallized aldo-keto reductase protein. The recombinant XF1729 purified to homogeneity catalyzed the reduction of dl-glyceraldehyde (K(cat) 2.26s(-1), Km 8.20+/-0.98 mM) and 2-nitrobenzaldehyde (K(cat) 11.74 s(-1), Km 0.14+/-0.04 mM) in the presence of NADPH. The amino acid sequence deduced from XF1729 showed the highest identity (40% or higher) with several functional unknown proteins. Among the identified AKRs, we found approximately 29% of identity with YakC (AKR13), 30 and 28% with AKR11A and AKR11B, respectively. The results establish XF1729 as the new member of AKR family, AKR13B1. Finally, the first characterization by gel filtration chromatography assays indicates that the protein has an elongated shape, which generates an apparent higher molecular weight. The study of this protein is an effort to fight X. fastidiosa, which causes tremendous losses in many economically important plants.

Aldo-keto reductase enzymes detoxify glyphosate and improve herbicide resistance in plants.

PubMed

Vemanna, Ramu S; Vennapusa, Amaranatha Reddy; Easwaran, Murugesh; Chandrashekar, Babitha K; Rao, Hanumantha; Ghanti, Kirankumar; Sudhakar, Chinta; Mysore, Kirankumar S; Makarla, Udayakumar

2017-07-01

In recent years, concerns about the use of glyphosate-resistant crops have increased because of glyphosate residual levels in plants and development of herbicide-resistant weeds. In spite of identifying glyphosate-detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an aldo-keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologues in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate-mediated cucumber seedling growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1- or OsAKRI-expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Modified chiral triazolium salts for enantioselective benzoin cyclization of enolizable keto-aldehydes: synthesis of (+)-sappanone B.

PubMed

Takikawa, Hiroshi; Suzuki, Keisuke

2007-07-05

Asymmetric synthesis of (+)-sappanone B (1), a natural product with a 3-hydroxy chromanone structure, was achieved via enantioselective benzoin cyclization by using a modified Rovis catalyst and triethylamine. This catalyst enabled the successful benzoin cyclization of readily enolizable keto-aldehydes.

One-pot synthesis of keto thioethers by palladium/gold-catalyzed click and pinacol reactions.

PubMed

Cadu, Alban; Watile, Rahul A; Biswas, Srijit; Orthaber, Andreas; Sjöberg, Per J R; Samec, Joseph S M

2014-11-07

An atom-efficient synthesis of keto thioethers was devised via tandem gold/palladium catalysis. The reaction proceeds through a regioselective thiol attack at the β-position of the alcohol, followed by an alkyl, aryl, or benzyl 1,2-shift. Both acyclic and cyclic systems were studied, in the latter case leading to the ring expansion of cyclic substrates.

Effect of restricted protein diet supplemented with keto analogues in end-stage renal disease: a systematic review and meta-analysis.

PubMed

Jiang, Zheng; Tang, Yi; Yang, Lichuan; Mi, Xuhua; Qin, Wei

2018-04-01

To evaluate the efficacy and safety of the restricted protein diet supplemented with keto analogues when applied in end-stage renal disease (ESRD). The Cochrane Library, PubMed, Embase, CBM and CENTRAL databases were searched and reviewed up to January 2017. Clinical trials were analyzed using RevMan 5.3 software. Five randomized controlled trials were selected and included in this study according to our inclusion and exclusion criteria. Changes in serum albumin, PTH, triglyceride, cholesterol, calcium, phosphorus, hemoglobin, Kt/v and CRP before and after treatment were analyzed. Meta-analysis results indicated that, compared with normal protein diet, low-protein diet (LPD) supplemented with keto analogues (sLPD) could improve serum albumin (P < 0.00001), hyperparathyroidism (P < 0.00001) and hyperphosphatemia (P = 0.008). No differences in triglyceride, cholesterol, hemoglobin, Kt/v and CRP were observed between different protein intake groups. Restricted protein diet supplemented with keto analogues (sLPD) may improve nutritional status and prevent hyperparathyroidism in ESRD patients. The current data were mainly obtained from short-term, single-center trails with small sample sizes and limited nutritional status indexes, indicating a need for further study.

The keto-enol equilibrium and thermal conversion kinetics of 2- and 4-hydroxyacetophenone in the gas phase: a DFT study

NASA Astrophysics Data System (ADS)

Monascal, Yeljair; Gallardo, Eliana; Cartaya, Loriett; Maldonado, Alexis; Bentarcurt, Yenner; Chuchani, Gabriel

2018-01-01

Keto-enol tautomeric equilibrium and the mechanism of thermal conversion of 2- and 4-hydroxyacetophenone in gas phase have been studied by means of electronic structure calculations using density functional theory (DFT). A topological analysis of electron density evidence that the structure of keto and enol forms of 2-hydroxyacetophenone are stabilised by a relatively strong intramolecular hydrogen bond. 2- and 4-hydroxyacetophenone undergo deacetylation reactions yielding phenol and ketene. Two possible mechanisms are considered for these eliminations: the process takes place from the keto form (mechanism A), or occurs from the enolic form of the substrate (mechanism B). Quantum chemical calculations support the mechanism B, being found a good agreement with the experimental activation parameters. These results suggest that the rate-limiting step is the reaction of the enol through a concerted, non-synchronous, semi-polar, four-membered cyclic transition state (TS). The most advanced reaction coordinate in the TS is the rupture of O1...H1 bond, with an evolution in the order of 79.7%-80.9%. Theoretical results also suggest a three-step mechanism for the phenyl acetate formation from 2-hydroxyacetophenone.

Inferring High-Confidence Human Protein-Protein Interactions

DTIC Science & Technology

2012-01-01

comprised proteins that had the same specific func- tion or were subunits of the same protein complex, such as branched chain keto acid E1 alpha (BCKDHA...and branched chain keto acid E1 beta (BCKDHB) [3,29], and dynein cytoplasmic 2 intermediate chain 1 (D2LIC) and dynein cytoplasmic 2 heavy chain 1...474.3 28.0 1337.0 BCKDHA 5 Branched chain keto acid dehydro. E1, alpha BCKDHB 4 Branched chain keto acid dehydro. E1, beta 4 471.4 29.0 1337.5 ARTN 2

Effect of restricted protein diet supplemented with keto analogues in chronic kidney disease: a systematic review and meta-analysis.

PubMed

Jiang, Zheng; Zhang, Xiaoyan; Yang, Lichuan; Li, Zi; Qin, Wei

2016-03-01

To evaluate the efficacy and safety of the restricted protein diet (low or very low protein diet) supplemented with keto analogues in the treatment of chronic kidney disease (CKD). The Cochrane library, PubMed, Embase, CBM and CENTRAL databases were searched and reviewed up to April 2015. Clinical trials were analyzed using RevMan 5.3 software. Seven random control trials, one cross-over trial and one non-randomized concurrent control trial were selected and included in this study according to our inclusion and exclusion criteria. The changes of eGFR, BUN, Scr, albumin, PTH, triglyceride, cholesterol, calcium, phosphorus and nutrition indexes (BMI, lean body mass and mid-arm muscular circumference) before and after treatment were analyzed. The meta-analysis results indicated that, comparing with normal protein diet, low protein diet (LPD) or very low protein diet (vLPD) supplemented with keto analogues (s(v)LPD) could significantly prevent the deterioration of eGFR (P < 0.001), hyperparathyroidism (P = 0.04), hypertension (P < 0.01) and hyperphosphatemia (P < 0.001). No differences in BUN, Scr, Albumin, triglyceride, cholesterol, hemoglobin, calcium and nutrition indexes were observed between different protein intake groups. Restricted protein diet supplemented with keto analogues (s(v)LPD) could delay the progression of CKD effectively without causing malnutrition.

Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Eun-Jung; Son, Hyeoncheol Francis; Kim, Sangwoo

Highlights: • We determined a crystal structure of β-keto thiolase from Ralstonia eutropha H16 (ReBktB). • Distinct substrate binding mode ReBktB was elucidated. • Enzymatic kinetic parameters of ReBktB were revealed. - Abstract: ReBktB is a β-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3 Å, and the overall structure has a similar fold to that of type II biosynthetic thiolases, suchmore » as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBktB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that K{sub m}, V{sub max}, and K{sub cat} values of 11.58 μM, 1.5 μmol/min, and 102.18 s{sup −1}, respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.« less

Kinetic and Mechanistic Studies of the Deuterium Exchange in Classical Keto-Enol Tautomeric Equilibrium Reactions

ERIC Educational Resources Information Center

Nichols, Michael A.; Waner, Mark J.

2010-01-01

An extension of the classic keto-enol tautomerization of beta-dicarbonyl compounds into a kinetic analysis of deuterium exchange is presented. It is shown that acetylacetone and ethyl acetoacetate undergo nearly complete deuterium exchange of the alpha-methylene carbon when dissolved in methanol-d[subscript 4]. The extent of deuteration may be…

Urinary 11-dehydro-thromboxane B₂ and 2,3-dinor-6-keto-prostaglandin-F₁α in healthy post-menopausal and pre-menopausal women receiving aspirin 100 mg.

PubMed

Hartanto, Marcia Dewi; Arieselia, Zita; Setiabudy, Rianto; Setiawati, Arini; Baziad, Ali

2012-07-01

The prevalence of cardiovascular diseases in women increases sharply after menopause. In postmenopausal women, thromboxane production increases while prostacyclin decreases. Low dose aspirin reduces the production of both thromboxane and prostacyclin. The present study was an open-label clinical trial with two parallel groups of 15 premenopausal women and 15 postmenopausal women. Twenty-four hours urine was collected from each subject before and after aspirin 100 mg daily for 7 days. The concentration of thromboxane and prostacyclin was measured as their metabolites (11-dehydro-thromboxane B(2) and 2,3-dinor-6-keto-prostaglandin-F(1α)) in urine using enzyme immunoassay methods. This study showed that aspirin significantly reduced thromboxane in both groups with significantly larger percentage reduction in postmenopausal women compared to premenopausal women (73.32 vs. 61.13%, p = 0.021). This study also showed that aspirin reduced prostacyclin significantly in both groups, but the percentage reduction between the groups was not significantly different. The decrease in the ratio of 11-dTXB(2)/2,3-dinor-6-keto-PGF(1α) should be compared to assess aspirin efficacy as an antithrombotic. Calculation of the ratio of 11-dTXB(2)/2,3-dinor-6-keto-PGF(1α) before aspirin consumption was higher in postmenopausal women than in premenopausal women. The decrease in 11-dTXB(2)/2,3-dinor-6-keto-PGF(1α) ratio by aspirin was greater in postmenopausal women than in premenopausal women (1.91 vs. 0.17; p = 0.022). It was concluded that aspirin reduced thromboxane and prostacyclin significantly in each group with significant 11-dTXB(2) percentage reduction between groups and non-significant 2,3-dinor-6-keto-PGF(1α) percentage reduction between groups, but reduced the 11-dTXB(2)/2,3-dinor-6-keto-PGF(1α) ratio much larger in postmenopausal women compared to that in premenopausal women.

Pyrimidine Nucleosides with a Reactive (β-Chlorovinyl)sulfone or (β-Keto)sulfone Group at the C5 Position, Their Reactions with Nucleophiles and Electrophiles, and Their Polymerase-Catalyzed Incorporation into DNA

PubMed Central

2018-01-01

Transition-metal-catalyzed chlorosulfonylation of 5-ethynylpyrimidine nucleosides provided (E)-5-(β-chlorovinyl)sulfones A, which undergo nucleophilic substitution with amines or thiols affording B. The treatment of vinyl sulfones A with ammonia followed by acid-catalyzed hydrolysis of the intermediary β-sulfonylvinylamines gave 5-(β-keto)sulfones C. The latter reacts with electrophiles, yielding α-carbon-alkylated or -sulfanylated analogues D. The 5′-triphosphates of A and C were incorporated into double-stranded DNA, using open and one-nucleotide gap substrates, by human or Escherichia coli DNA-polymerase-catalyzed reactions. PMID:29732453

Effects of glucogenic and ketogenic feeding strategies on splanchnic glucose and amino acid metabolism in postpartum transition Holstein cows.

PubMed

Larsen, M; Kristensen, N B

2012-10-01

Nine periparturient Holstein cows catheterized in major splanchnic vessels were used in a complete randomized design with repeated measurements to investigate effects of glucogenic and ketogenic feeding strategies on splanchnic metabolism of glucose and amino acids. At parturition, cows were assigned to 1 of 3 feeding strategies: a glucogenic diet (GLCG) based on sodium hydroxide treated wheat grain (56.5% of diet dry matter); a ketogenic diet (KETO) based on fodder beets (40.5% of diet dry matter); or an alfalfa-glucogenic strategy (ALF-GLCG) supplying 100% alfalfa (Medicago sativa L.) haylage at the day of parturition, followed by a 6-d linear shift to the GLCG diet. Samples were obtained 14 d before expected parturition as well as at 4, 15, and 29 d in milk (DIM). The net portal release of glucose was greatest with GLCG, reflecting the higher intake of ruminal escape starch with GLCG, as compared with a lower starch intake with KETO. Postpartum, the portal recovery of feed starch was greater (28 ± 3%, mean ± SEM) with KETO as compared with GLCG (15 ± 4%). At 4 DIM, the net hepatic release of glucose was greatest with KETO and least with ALF-GLCG, whereafter it increased as lactation progressed with ALF-GLCG and GLCG, but not with KETO. The high alfalfa haylage allowance at 4 DIM with the ALF-GLCG treatment induced the lowest net release of nutrients from the splanchnic tissues at 4 DIM. The hepatic removal of lactate as percent of total influx (mean ± SEM) increased from 27 ± 3% prepartum to 56 ± 3% at 4 DIM. The hepatic removal of lactate as percent of net portal release increased from 144 ± 10% prepartum to 329 ± 17% at 4 DIM with ALF-GLCG and KETO as compared with 242 ± 20% in GLCG. No clear evidence for an amino acid sparing effect in splanchnic tissues from increasing small intestinal glucose absorption was observed. In conclusion, the glucogenic feeding strategy induced the highest glucogenic status among the tested feeding strategies due to

Enantioselective copper catalysed intramolecular C-H insertion reactions of α-diazo-β-keto sulfones, α-diazo-β-keto phosphine oxides and 2-diazo-1,3-diketones; the influence of the carbene substituent.

PubMed

Shiely, Amy E; Slattery, Catherine N; Ford, Alan; Eccles, Kevin S; Lawrence, Simon E; Maguire, Anita R

2017-03-22

Enantioselectivities in C-H insertion reactions, employing the copper-bis(oxazoline)-NaBARF catalyst system, leading to cyclopentanones are highest with sulfonyl substituents on the carbene carbon, and furthermore, the impact is enhanced by increased steric demand on the sulfonyl substituent (up to 91%ee). Enantioselective intramolecular C-H insertion reactions of α-diazo-β-keto phosphine oxides and 2-diazo-1,3-diketones are reported for the first time.

Theoretical and vibrational spectroscopic approach to keto-enol tautomerism in methyl-2-(4-methoxybenzoyl)-3-(4-methoxyphenyl)-3-oxopropanoylcarbamate

NASA Astrophysics Data System (ADS)

Arı, Hatice; Özpozan, Talat; Büyükmumcu, Zeki; Kabacalı, Yiğit; Saçmaci, Mustafa

2016-10-01

A carbamate compound having tricarbonyl groups, methyl-2-(4-methoxybenzoyl)-3-(4-methoxyphenyl)-3-oxopropanoylcarbamate (BPOC) was investigated from theoretical and vibrational spectroscopic point of view employing quantum chemical methods. Hybrid Density Functionals (B3LYP, X3LYP and B3PW91) with 6-311 G(d,p) basis set were used for the calculations. Rotational barrier and conformational analyses were performed to find the most stable conformers of keto and enol forms of the molecule. Three transition states for keto-enol tautomerism in gas phase were determined. The results of the calculations show that enol-1 form of BPOC is more stable than keto and enol-2 forms. Hydrogen bonding investigation including Natural bond orbital analysis (NBO) for all the tautomeric structures was employed to compare intra-molecular interactions. The energies of HOMO and LUMO molecular orbitals for all tautomeric forms of BPOC were predicted. Normal Coordinate Analysis (NCA) was carried out for the enol-1 to assign vibrational bands of IR and Raman spectra. The scaling factors were calculated as 0.9721, 0.9697 and 0.9685 for B3LYP, X3LYP and B3PW91 methods, respectively. The correlation graphs of experimental versus calculated vibrational wavenumbers were plotted and X3LYP method gave better frequency agreement than the others.

Radical-cationic gaseous amino acids: a theoretical study.

PubMed

Sutherland, Kailee N; Mineau, Philippe C; Orlova, Galina

2007-08-16

Three major forms of gaseous radical-cationic amino acids (RCAAs), keto (COOH), enolic (C(OH)OH), and zwitterionic (COO(-)), as well as their tautomers, are examined for aliphatic Ala(.+), Pro(.+), and Ser(.+), sulfur-containing Cys(.+), aromatic Trp(.+), Tyr(.+), and Phe(.+), and basic His(.+). The hybrid B3LYP exchange-correlation functional with various basis sets along with the highly correlated CCSD(T) method is used. For all RCAAs considered, the main stabilizing factor is spin delocalization; for His(.+), protonation of the basic side chain is equally important. Minor stabilizing factors are hydrogen bonding and 3e-2c interactions. An efficient spin delocalization along the N-C(alpha)-C(O-)O moiety occurs upon H-transfer from C(alpha) to the carboxylic group to yield the captodative enolic form, which is the lowest-energy isomer for Ala(.+), Pro(.+), Ser(.+), Cys(.+), Tyr(.+), and Phe(.+). This H-transfer occurs in a single step as a 1,3-shift through the sigma-system. For His(.+), the lowest-energy isomer is formed upon H-transfer from C(alpha) to the basic side chain, which results in a keto form, with spin delocalized along the N-C(alpha)-C=O fragment. Trp(.+) is the only RCAA that favors spin delocalization over an aromatic system given the low ionization energy of indole. The lowest-energy isomer of Trp(.+) is a keto form, with no H-transfer.

[Roles of sialic acids in sperm maturation and capacitation and sperm-egg recognition].

PubMed

Feng, Ying; Wang, Lin; Wu, Yi-Lun; Liu, Hong-Hua; Ma, Fang

2016-10-01

Sialic acids are a subset of nine-carbon alpha-keto aldonic acids involved in various biological functions. Sialic acid on the sperm surface is closely related to sperm maturation and capacitation and sperm-egg recognition, which makes sperm negatively charged to avoid accumulation and covers some antigenic determinants there to increase the survival rate of sperm in the female reproductive tract. The loss of sialic acids is an important factor mediating sperm capacitation. Moreover, the sialic acid at the extremity of the protein polymer is involved in signal identification in sperm-egg recognition. Here, we review the current understanding of sialic acids in sperm maturation and capacitation and sperm-egg recognition.

Comprehensive analysis of mycolic acid subclass and molecular species composition of Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105).

PubMed

Uenishi, Yuko; Fujita, Yukiko; Kusunose, Naoto; Yano, Ikuya; Sunagawa, Makoto

2008-02-01

The mycobacterial cell envelope consists of a characteristic cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidoglycan complex, and related hydrophobic components that contribute to the cell surface properties. Since mycolic acids have recently been reported to play crucial roles in host immune response, detailed molecular characterization of mycolic acid subclasses and sub-subclasses of CWS from Mycobacterium bovis BCG Tokyo 172 (SMP-105) was performed. Mycolic acids were liberated by alkali hydrolysis from SMP-105, and their methyl esters were separated by silica gel TLC into three subclasses: alpha-, methoxy-, and keto-mycolates. Each mycolate subclass was further separated by silver nitrate (AgNO(3))-coated silica gel TLC into sub-subclasses. Molecular weights of individual mycolic acid were determined by MALDI-TOF mass spectrometry. alpha-Mycolates were sub-grouped into cis, cis-dicyclopropanoic (alpha1), and cis-monocyclopropanoic-cis-monoenoic (alpha2) series; methoxy-mycolates were sub-grouped into cis-monocyclopropanoic (m1), trans-monocyclopropanoic (m2), trans-monoenoic (m3), cis-monocyclopropanoic-trans-monoenoic (m4), cis-monoenoic (m5), and cis-monocyclopropanoic-cis-monoenoic (m6) series; and keto-mycolates were sub-grouped into cis-monocyclopropanoic (k1), trans-monocyclopropanoic (k2), trans-monoenoic (k3), cis-monoenoic (k4), and cis-monocyclopropanoic-cis-monoenoic (k5) series. The position of each functional group, including cyclopropane rings and methoxy and keto groups, was determined by analysis of the meromycolates with fast atom bombardment (FAB) mass spectrometry and FAB mass-mass spectrometry, and the cis/trans ratio of cyclopropane rings and double bonds were determined by NMR analysis of methyl mycolates. Mycolic acid subclass and molecular species composition of SMP-105 showed characteristic features including newly-identified cis-monocyclopropanoic-trans-monoenoic mycolic acid (m4).

Bismuth(III) trifluoromethanesulfonate catalyzed ring opening reaction of mono epoxy oleochemicals to form keto and diketo derivatives

USDA-ARS?s Scientific Manuscript database

Using a catalytic system, methyl oleate is transformed into long chain keto and diketo derivatives via an epoxide route. Methyl 9(10)-oxooctadecanoate and methyl 9,10-dioxooctadecanoate were made by a ring opening reaction of epoxidized methyl oleate using bismuth triflate catalyst. Lower reaction t...

Mechanistic Insights into the Catalytic Oxidation of Carboxylic Acids on Au/TiO 2: Partial Oxidation of Propionic and Butyric Acid to Gold Ketenylidene through Unsaturated Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

McEntee, Monica; Tang, Wenjie; Neurock, Matthew

Here, the partial oxidation of model C 2–C 4 (acetic, propionic, and butyric) carboxylic acids on Au/TiO 2 catalysts consisting of Au particles ~3 nm in size was investigated using transmission infrared spectroscopy and density functional theory. All three acids readily undergo oxidative dehydrogenation on Au/TiO 2. Propionic and butyric acid dehydrogenate at the C2–C3 positions, whereas acetic acid dehydrogenates at the C1–C2 position. The resulting acrylate and crotonate intermediates are subsequently oxidized to form β-keto acids that decarboxylate. All three acids form a gold ketenylidene intermediate, Au 2C=C=O, along the way to their full oxidation to form CO 2.more » Infrared measurements of Au 2C=C=O formation as a function of time provides a surface spectroscopic probe of the kinetics for the activation and oxidative dehydrogenation of the alkyl groups in the carboxylate intermediates that form.« less

Mechanistic Insights into the Catalytic Oxidation of Carboxylic Acids on Au/TiO 2: Partial Oxidation of Propionic and Butyric Acid to Gold Ketenylidene through Unsaturated Acids

DOE PAGES

McEntee, Monica; Tang, Wenjie; Neurock, Matthew; ...

2014-12-12

Here, the partial oxidation of model C 2–C 4 (acetic, propionic, and butyric) carboxylic acids on Au/TiO 2 catalysts consisting of Au particles ~3 nm in size was investigated using transmission infrared spectroscopy and density functional theory. All three acids readily undergo oxidative dehydrogenation on Au/TiO 2. Propionic and butyric acid dehydrogenate at the C2–C3 positions, whereas acetic acid dehydrogenates at the C1–C2 position. The resulting acrylate and crotonate intermediates are subsequently oxidized to form β-keto acids that decarboxylate. All three acids form a gold ketenylidene intermediate, Au 2C=C=O, along the way to their full oxidation to form CO 2.more » Infrared measurements of Au 2C=C=O formation as a function of time provides a surface spectroscopic probe of the kinetics for the activation and oxidative dehydrogenation of the alkyl groups in the carboxylate intermediates that form.« less

Solvent Dependency of the UV-Vis Spectrum of Indenoisoquinolines: Role of Keto-Oxygens as Polarity Interaction Probes

PubMed Central

Coletta, Andrea; Castelli, Silvia; Chillemi, Giovanni; Sanna, Nico; Cushman, Mark; Pommier, Yves; Desideri, Alessandro

2013-01-01

Indenoisoquinolines are the most promising non-campthotecins topoisomerase IB inhibitors. We present an integrated experimental/computational investigation of the UV-Vis spectra of the IQNs parental compound (NSC314622) and two of its derivatives (NSC724998 and NSC725776) currently undergoing Phase I clinical trials. In all the three compounds a similar dependence of the relative absorption intensities at 270 nm and 290 nm on solvent polarity is found. The keto-oxygens in positions 5 and 11 of the molecular scaffold of the molecule are the principal chromophores involved in this dependence. Protic interactions on these sites are also found to give rise to absorptions at wavelength <250 nm observed in water solution, due to the stabilization of highly polarized tautomers of the molecule. These results suggest that the keto-oxygens are important polarizable groups that can act as useful interactors with the molecular receptor, providing at the same time an useful fingerprint for the monitoring of the drug binding to topoisomerase IB. PMID:24086299

Copper-Catalyzed Oxidative Reaction of β-Keto Sulfones with Alcohols via C-S Bond Cleavage: Reaction Development and Mechanism Study.

PubMed

Du, Bingnan; Wang, Wenmin; Wang, Yang; Qi, Zhenghang; Tian, Jiaqi; Zhou, Jie; Wang, Xiaochen; Han, Jianlin; Ma, Jing; Pan, Yi

2018-02-16

A Cu-catalyzed cascade oxidative radical process of β-keto sulfones with alcohols has been achieved by using oxygen as an oxidant. In this reaction, β-keto sulfones were converted into sulfinate esters under the oxidative conditions via cleavage of C-S bond. Experimental and computational studies demonstrate that a new pathway is involved in this reaction, which proceeds through the formation of the key four-coordinated Cu II intermediate, O-O bond homolysis induced C-S bond cleavage and Cu-catalyzed esterification to form the final products. This reaction provides a new strategy to sulfonate esters and enriches the research content of C-S bond cleavage and transformations. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acetyl-11-Keto-β-Boswellic Acid Promotes Osteoblast Differentiation by Inhibiting Tumor Necrosis Factor-α and Nuclear Factor-κB Activity.

PubMed

Bai, Fan; Chen, Xuewu; Yang, Hui; Xu, Hong-Guang

2018-06-20

Tumor necrosis factor (TNF) -α plays a crucial role in rheumatoid arthritis (RA)-related bone loss disease. The main mechanism of action of RA induced bone loss is the significant inhibitory effect of TNF-α on osteoblast differentiation. TNF-α inhibits osteoblast differentiation mainly by activating nuclear factor (NF) -κB signaling pathway. Owing to the crucial role of TNF-α and NF-κB in the inhibition of osteoblast differentiation, they are considered as targets for the development of therapeutic drugs. In the present study, we evaluated the NF-κB inhibitor Boswellic acid (BA) and its derivatives in the regulation of osteoblast differentiation and the molecular mechanism. Based on the cell model of TNF-α induced inhibition of osteoblast differentiation of MC3T3-E1, the regulatory role of BAs was studied. The result of MTT assay indicated that bone morphogenetic protein (BMP) -2, TNF-α, or acetyl-11-keto-β-BA (AKBA) impact no significant effect for cell viability of MC3T3-E1. The results of alkaline phosphatase (ALP activity assay and real-time polymerase chain reaction indicated that AKBA blocked TNF-α-induced inhibition of the expression of osteoblast markers, suggesting that AKBA rescued osteoblast differentiation from TNF-α-induced inhibition. Additionally, AKBA stimulated the BMP-2-induced expression of osteoblast markers, suggesting that AKBA promotes osteoblast differentiation directly. The results of western blotting and luciferase assay indicated that N-κB signaling was activated by TNF-α. The overexpression of NF-κB component p65 in MC3T3-E1 was found to attenuate the positive effect of AKBA in osteoblast differentiation, suggesting that AKBA potentiates osteoblast differentiation by inhibiting NF-κB signaling. Collectively, AKBA promotes osteoblast differentiation by inhibiting TNF-α and NF-κB. Our study revealed a new discovery of AKBA in regulating osteoblast differentiation, and demonstrated that AKBA may be a potential anabolic

A novel bacteriophage KSL-1 of 2-Keto-gluconic acid producer Pseudomonas fluorescens K1005: isolation, characterization and its remedial action

PubMed Central

2012-01-01

Background Bacteriophages have the destructive damage on the industrial bioprocess. 2-Keto-gluconic acid (2KGA) producing bacteria had also been attacked and lysed by bacteriophages which lowered the glucose consumption and 2KGA yield and even stopped the fermentation process. In this study, we presented the characteristics of a novel virulent bacteriophage specifically infecting Pseudomonas fluorescens K1005 and proposed an efficient remedial action for this phage infection to reduce the production loss. Results The phage KSL-1 of Pseudomonas fluorescens K1005 was isolated from abnormal 2KGA fermentation broth. It belonged to the Siphoviridae family with a hexagonal head diameter of about 99 nm and a non-contractile tail of about 103 nm × 39 nm. The genome size of phage KSL-1 was estimated to be approximately 53 kbp. Its optimal MOI to infect P. fluorescens K1005 was about 0.001. One-step growth curve gave its latent and burst periods of 90 min and 75 min with a burst size of 52 phage particles per infected cell. This phage was stable with a pH range of 7.0–10.0, and sensitive to thermal treatment. Finally, a simple remedial action was proposed by feeding fresh seed culture. Compared with the infected 2KGA fermentation, the remedial experiments restored 2KGA fermentation performance by increasing the produced 2KGA concentration to 159.89 g/L and shortening the total fermentation time of 80 h with the productivity and yield of 2.0 g/L.h and 0.89 g/g. The obtained data proved that this method was effective to combat the phage infections problems during the 2KGA fermentation. Conclusion The phage KSL-1 was a novel bacteriophage specifically infecting Pseudomonas fluorescens K1005. The remedial action of feeding fresh seed culture to the infected broth was an easily-operating and effective method to maintain a high 2KGA yield and avoid the draft of infected broth. PMID:22747634

Transition metal-free one-pot cascade synthesis of 7-oxa-2-azatricyclo[7.4.0.0(2,6)]trideca-1(9),10,12-trien-3-ones from biomass-derived levulinic acid under mild conditions.

PubMed

Jha, Amitabh; Naidu, Ajaya B; Abdelkhalik, Ashraf M

2013-11-21

An efficient, environmentally benign, transition-metal free, tandem C-N, C-O bond formation reaction is developed for the synthesis of tricyclic 7-oxa-2-azatricyclo[7.4.0.0(2,6)]trideca-1(9),10,12-trien-3-ones and their homologs from easily available starting materials, including renewable levulinic acid, a keto acid. The reaction of keto acids with methyl chloroformate and variously substituted o-aminobenzyl alcohols using triethylamine as a base in toluene at room temperature gave good to excellent yields. This newly developed protocol was successfully utilized for the synthesis of a variety of polycyclic 7-oxa-2-azatricyclo[7.4.0.0(2,6)]trideca-1(9),10,12-trien-3-ones and related compounds.

In Vitro Enzymatic Synthesis of New Penicillins Containing Keto Acids as Side Chains

PubMed Central

Ferrero, Miguel A.; Reglero, Angel; Martínez-Blanco, Honorina; Fernández-Valverde, Martiniano; Luengo, Jose M.

1991-01-01

Seven different penicillins containing α-ketobutyric, β-ketobutyric, γ-ketovaleric, α-ketohexanoic, δ-ketohexanoic, ε-ketoheptanoic, and α-ketooctanoic acids as side chains have been synthesized in vitro by incubating the enzymes phenylacetyl coenzyme A (CoA) ligase from Pseudomonas putida and acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum with CoA, ATP, Mg2+, dithiothreitol, 6-aminopenicillanic acid, and the corresponding side chain precursor. PMID:1952871

Rapid solid-phase immunoassay for 6-keto prostaglandin F1 alpha on microplates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schramm, W.; Smith, R.H.; Jackson, T.M.

1990-03-01

We describe, for the measurement of 6-keto prostaglandin F1 alpha in biological media, a solid-phase immunoassay with immobilized antibodies that requires a total processing time of less than 2 h with hands-on time less than 30 min for 40 samples. The method combines the convenience of the microplate format with the sensitivity of radiolabeled prostaglandin derivatives as tracers in a competitive immunoassay. The intra- and interassay variations at 50% displacement of the radiolabeled prostaglandin derivative as tracer were 9.0% and 11.8%, respectively. At 50% displacement of the radiolabeled tracer, the sensitivity is about 20 pg per well. Optimal incubation timemore » is between 60 and 90 min. Nonspecific binding was less than 1% if about 8 pg of tracer (approximately 25,000 counts/min per well) was used. Inhibition curves of samples in different dilutions were parallel to standard curves. The variation of bound radiolabeled prostaglandin derivative within the wells of one microplate (n = 96) was less than 3%. Human plasma samples and medium from tissue culture assayed for 6-keto prostaglandin F1 alpha correlated well with results obtained with a solid-phase assay based on use of magnetic particles (r = 0.99, n = 24) for culture-medium samples; r = 0.99; n = 26 for plasma samples.« less

A systems approach for discovering linoleic acid derivatives that potentially mediate pain and itch

PubMed Central

Ramsden, Christopher E.; Domenichiello, Anthony F.; Yuan, Zhi-Xin; Sapio, Matthew R.; Keyes, Gregory S.; Mishra, Santosh K.; Gross, Jacklyn R.; Majchrzak-Hong, Sharon; Zamora, Daisy; Horowitz, Mark S.; Davis, John M.; Sorokin, Alexander V.; Dey, Amit; LaPaglia, Danielle M.; Wheeler, Joshua J.; Vasko, Michael R.; Mehta, Nehal N.; Mannes, Andrew J.; Iadarola, Michael J.

2018-01-01

Chronic pain and itch are common hypersensitivity syndromes that are affected by endogenous mediators. We applied a systems-based, translational approach to predict, discover, and characterize mediators of pain and itch that are regulated by diet and inflammation. Profiling of tissue-specific precursor abundance and biosynthetic gene expression predicted that inflamed skin would be abundant in four previously unknown 11-hydroxy-epoxy-or 11-keto-epoxy-octadecenoate linoleic acid derivatives and four previously identified 9- or 13-hydroxy-epoxy- or 9- or 13-keto-epoxy-octadecenoate linoleic acid derivatives. All of these mediators were confirmed to be abundant in rat and human skin by mass spectrometry. However, only the two 11-hydroxy-epoxy-octadecenoates sensitized rat dorsal root ganglion neurons to release more calcitonin gene–related peptide (CGRP), which is involved in pain transmission, in response to low pH (which mimics an inflammatory state) or capsaicin (which activates ion channels involved in nociception). The two 11-hydroxy-epoxy-octadecenoates share a 3-hydroxy-Z-pentenyl-E-epoxide moiety, thus suggesting that this substructure could mediate nociceptor sensitization. In rats, intradermal hind paw injection of 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate elicited C-fiber–mediated sensitivity to thermal pain. In a randomized trial testing adjunctive strategies to manage refractory chronic headaches, reducing the dietary intake of linoleic acid was associated with decreases in plasma 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate, which correlated with clinical pain reduction. Human psoriatic skin had 30-fold higher 9-keto-12,13-trans-epoxy-(10E)-octadecenoate compared to control skin, and intradermal injection of this compound induced itch-related scratching behavior in mice. Collectively, these findings define a family of endogenous mediators with potential roles in pain and itch. PMID:28831021

The Baker's Yeast Reduction of Keto-Esters in Organic Solvents: A One Week Research Project for Undergraduate Students

NASA Astrophysics Data System (ADS)

North, Michael

1998-05-01

An experiment has been designed which allows final year undergraduate students to carry out a mini-research project in one week and thus get a flavour of the joys and tribulations of conducting chemical research before they undertake a major research project. The experiment is an investigation into the reduction of alpha- or beta-keto esters using non-fermenting Baker's yeast in petroleum ether. There are a number of advantages to this method of using Baker's yeast, including a reduction in the amount of organic solvent used, and a much simplified purification procedure. During the course of the mini-project, the substrate specificity of the yeast is investigated, and the conditions for the optimisation of a particular keto ester are determined. Each product is analysed by a variety of analytical techniques including polarimetry, IR, NMR, and GC. In addition, the use of correct stereochemical nomenclature to describe prochiral, and chiral compounds as well as chemical reactions are discussed.

Aqueous-Phase Acetic Acid Ketonization over Monoclinic Zirconia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai, Qiuxia; Lopez-Ruiz, Juan A.; Cooper, Alan R.

The effect of aqueous phase on the acetic acid ketonization over monoclinic zirconia has been investigated using first-principles based density functional theory (DFT) calculations. To capture the aqueous phase chemistry over the solid zirconia catalyst surface, the aqueous phase is represented by 111 explicit water molecules with a liquid water density of 0.93 g/cm3 and the monoclinic zirconia is modeled by the most stable surface structure . The dynamic nature of aqueous phase/ interface was studied using ab initio molecular dynamics simulation, indicating that nearly half of the surface Zr sites are occupied by either adsorbed water molecules or hydroxylmore » groups at 550 K. DFT calculations show that the adsorption process of acetic acid from the liquid water phase to the surface is nearly thermodynamically neutral with a Gibbs free energy of -2.3 kJ/mol although the adsorption strength of acetic acid on the surface in aqueous phase is much stronger than in vapor phase. Therefore it is expected that the adsorption of acetic acid will dramatically affects aqueous phase ketonization reactivity over the monoclinic zirconia catalyst. Using the same ketonization mechanism via the β-keto acid intermediate, we have compared acetic acid ketonization to acetone in both vapor and aqueous phases. Our DFT calculation results show although the rate-determining step of the β-keto acid formation via the C-C coupling is not pronouncedly affected, the presence of liquid water molecules will dramatically affect dehydrogenation and hydrogenation steps via proton transfer mechanism. This work was financially supported by the United States Department of Energy (DOE)’s Bioenergy Technologies Office (BETO) and performed at the Pacific Northwest National Laboratory (PNNL). PNNL is a multi-program national laboratory operated for DOE by Battelle Memorial Institute. Computing time and advanced catalyst characterization use was granted by a user proposal at the William R. Wiley

Advances in the Biology and Chemistry of Sialic Acids

PubMed Central

Chen, Xi; Varki, Ajit

2010-01-01

Sialic acids are a subset of nonulosonic acids, which are nine-carbon alpha-keto aldonic acids. Natural existing sialic acid-containing structures are presented in different sialic acid forms, various sialyl linkages, and on diverse underlying glycans. They play important roles in biological, pathological, and immunological processes. Sialobiology has been a challenging and yet attractive research area. Recent advances in chemical and chemoenzymatic synthesis as well as large-scale E. coli cell-based production have provided a large library of sialoside standards and derivatives in amounts sufficient for structure-activity relationship studies. Sialoglycan microarrays provide an efficient platform for quick identification of preferred ligands for sialic acid-binding proteins. Future research on sialic acid will continue to be at the interface of chemistry and biology. Research efforts will not only lead to a better understanding of the biological and pathological importance of sialic acids and their diversity, but could also lead to the development of therapeutics. PMID:20020717

Macrocyclic lactones: A versatile source for omega radiohalogenated fatty acid analogs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dougan, A.H.; Lyster, D.M.; Robertson, K.A.

For each omega halogenated fatty acid there exists a potential omega hydroxy fatty acid and the corresponding macrocyclic lactone. The authors have utilized such lactones as starting materials for omega /sup 123/I fatty acid analogs intended for myocardial imaging. Macrocyclic musk lactones are industrially available; 120 analogs are described in the literature. The preparation requires saponification, tosylation, and radio-iodide substitution. Iodo-fatty acids are readily separated from tosylate fatty acids on TLC. While providing a secure source of 16-iodo-hexadecanoic acid and 17-iodo-heptadecanoic acid, the scheme allows ready access to a large number of untried fatty acid analogs. Examples presented are 16-iodo-hexadecanoicmore » acid, 16-iodo-7-hexadecanoic acid, 16-iodo-12-oxa-hexadecanoic acid, 15-iodo-pentadecanoic acid, and 15-iodo-12-keto-pentadecanoic acid. Metabolic studies are in progress in mice and dogs to assess the utility of these analogs for myocardial imaging.« less

A comparison of dehydroepiandrosterone and 7-keto dehydroepiandrosterone with other drugs that modulate ethanol intake in rats responding under a multiple schedule

PubMed Central

Amato, Russell J.; Hulin, Mary W.; Winsauer, Peter J.

2012-01-01

Dehydroepiandrosterone (DHEA), 7-keto DHEA, and several comparison drugs (ethanol, chlordiazepoxide, rauwolscine, and RO15-4513) were administered to male rats responding under a multiple schedule of food and ethanol presentation to determine their selectively for decreasing ethanol-maintained responding. DHEA and 7-keto DHEA significantly decreased both ethanol- and food-maintained responding, compared to control, while also decreasing blood ethanol concentration (BEC). Acute ethanol administration also decreased responding for both food and ethanol; however, ethanol-maintained responding was more potently decreased than food-maintained responding. BEC remained relatively stable after increasing ethanol doses. Among the other drugs tested, RO15-4513 was the most selective for decreasing ethanol-maintained responding compared to food-maintained responding, and it decreased BECs as ethanol-maintained responding decreased. The largest dose of rauwolscine significantly decreased responding for food, while not affecting ethanol-maintained responding compared to control. Low to intermediate doses of rauwolscine produced small, non-significant increases in ethanol-maintained responding and BECs. Chlordiazepoxide produced significant decreases in food-maintained responding and the dose of ethanol presented, but only at the highest dose tested. Although DHEA and 7-keto DHEA did not decrease ethanol-maintained responding as selectively as ethanol or RO15-4513 under the multiple schedule, these neurosteroids may be valuable pharmacological tools in the development of new treatments for alcohol abuse and dependence. PMID:22473025

Human aldo-keto reductases 1B1 and 1B10: a comparative study on their enzyme activity toward electrophilic carbonyl compounds.

PubMed

Shen, Yi; Zhong, Linlin; Johnson, Stephen; Cao, Deliang

2011-05-30

Aldo-keto reductase family 1 member B1 (AKR1B1, 1B1 in brief) and aldo-keto reductase family 1 member B10 (AKR1B10, 1B10 in brief) are two proteins with high similarities in their amino acid sequences, stereo structures, and substrate specificity. However, these two proteins exhibit distinct tissue distributions; 1B10 is primarily expressed in the gastrointestinal tract and adrenal gland, whereas 1B1 is ubiquitously present in all tissues/organs, suggesting their difference in biological functions. This study evaluated in parallel the enzyme activity of 1B1 and 1B10 toward alpha, beta-unsaturated carbonyl compounds with cellular and dietary origins, including acrolein, crotonaldehyde, 4-hydroxynonenal, trans-2-hexenal, and trans-2,4-hexadienal. Our results showed that 1B10 had much better enzyme activity and turnover rates toward these chemicals than 1B1. By detecting the enzymatic products using high-performance liquid chromatography, we measured their activity to carbonyl compounds at low concentrations. Our data showed that 1B10 efficiently reduced the tested carbonyl compounds at physiological levels, but 1B1 was less effective. Ectopically expressed 1B10 in 293T cells effectively eliminated 4-hydroxynonenal at 5 μM by reducing to 1,4-dihydroxynonene, whereas endogenously expressed 1B1 did not. The 1B1 and 1B10 both showed enzyme activity to glutathione-conjugated carbonyl compounds, but 1B1 appeared more active in general. Together our data suggests that 1B10 is more effectual in eliminating free electrophilic carbonyl compounds, but 1B1 seems more important in the further detoxification of glutathione-conjugated carbonyl compounds. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Electrophilic fluorinating reagent mediated synthesis of fluorinated alpha-keto Ethers, benzil, and 6,6'-dialkoxy-2,2'-bipyridines.

PubMed

Manandhar, Sudha; Singh, Rajendra P; Eggers, Gary V; Shreeve, Jean'ne M

2002-09-06

Interactions of various fluorinated and nonfluorinated alcohols with trans-stilbene in the presence of electrophilic reagents were studied. Under neat conditions, reactions of trans-stilbene (1) with fluorinated alcohols, R(f)OH (R(f) = CF3CH2-, CFH2CH2-, CF3CF2CH2-, CF2H(CF2)3CH2-, (CF3)2CH-, (CF3)3C- (2a-f) in the presence of an electrophilic reagent, 1-(chloromethyl)-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (Selectfluor) or N,N-difluoro-2,2'-bipyridinium bis(tetrafluoroborate) (MEC-31), gave alpha-keto ethers (3a-f) and benzil (4) in good to moderate yields. alpha-Keto ether and benzil formation was very much dependent on the reaction time, the degree of fluorination of the alcohols, and whether a solvent such as CH3CN, DMF or DMA was utilized. In solution, alpha-keto ethers and benzil did not form. Interestingly, under neat conditions, nonfluorinated alcohols, ROH (R = CH3-, CH3CH2-, CH3CH2CH2-, CH3CH2CH2CH2-, CH3CH2CH2CH2CH2CH2-) (5g-k) did not react with trans-stilbene in the presence of MEC-31 but gave 6,6'-dialkoxy-2,2'-bipyridines (6g-k), regioselectively, in excellent isolated yields. On the other hand, fluorinated alcohols did not react with MEC-31. Reaction of MEC-31 with an excess of diethylene glycol (7) gave the bipyridine derivative (8) in 88% isolated yield. Reaction of 8 either with diethylaminosulfur trifluoride (DAST) or bis(2-methoxyethyl)aminosulfur trifluoride (Deoxofluor) readily produced the corresponding difluoro derivative (9) in 85% isolated yield. All new compounds have been characterized by spectroscopic and elemental analysis.

Conversion of leucine to β‐hydroxy‐β‐methylbutyrate by α‐keto isocaproate dioxygenase is required for a potent stimulation of protein synthesis in L6 rat myotubes

PubMed Central

Vílchez, José D.; Salto, Rafael; Manzano, Manuel; Sevillano, Natalia; Campos, Nefertiti; Argilés, Josep M.; Rueda, Ricardo; López‐Pedrosa, José M.

2015-01-01

Abstract Background L‐Leu and its metabolite β‐hydroxy‐β‐methylbutyrate (HMB) stimulate muscle protein synthesis enhancing the phosphorylation of proteins that regulate anabolic signalling pathways. Alterations in these pathways are observed in many catabolic diseases, and HMB and L‐Leu have proven their anabolic effects in in vivo and in vitro models. The aim of this study was to compare the anabolic effects of L‐Leu and HMB in myotubes grown in the absence of any catabolic stimuli. Methods Studies were conducted in vitro using rat L6 myotubes under normal growth conditions (non‐involving L‐Leu‐deprived conditions). Protein synthesis and mechanistic target of rapamycin signalling pathway were determined. Results Only HMB was able to increase protein synthesis through a mechanism that involves the phosphorylation of the mechanistic target of rapamycin as well as its downstream elements, pS6 kinase, 4E binding protein‐1, and eIF4E. HMB was significantly more effective than L‐Leu in promoting these effects through an activation of protein kinase B/Akt. Because the conversion of L‐Leu to HMB is limited in muscle, L6 cells were transfected with a plasmid that codes for α‐keto isocaproate dioxygenase, the key enzyme involved in the catabolic conversion of α‐keto isocaproate into HMB. In these transfected cells, L‐Leu was able to promote protein synthesis and mechanistic target of rapamycin regulated pathway activation equally to HMB. Additionally, these effects of leucine were reverted to a normal state by mesotrione, a specific inhibitor of α‐keto isocaproate dioxygenase. Conclusion Our results suggest that HMB is an active L‐Leu metabolite able to maximize protein synthesis in skeletal muscle under conditions, in which no amino acid deprivation occurred. It may be proposed that supplementation with HMB may be very useful to stimulate protein synthesis in wasting conditions associated with chronic diseases, such as cancer or

7alpha- and 12alpha-Hydroxysteroid dehydrogenases from Acinetobacter calcoaceticus lwoffii: a new integrated chemo-enzymatic route to ursodeoxycholic acid.

PubMed

Giovannini, Pier Paolo; Grandini, Alessandro; Perrone, Daniela; Pedrini, Paola; Fantin, Giancarlo; Fogagnolo, Marco

2008-12-22

We report the very efficient biotransformation of cholic acid to 7-keto- and 7,12-diketocholic acids with Acinetobacter calcoaceticus lwoffii. The enzymes responsible of the biotransformation (i.e. 7alpha- and 12alpha-hydroxysteroid dehydrogenases) are partially purified and employed in a new chemo-enzymatic synthesis of ursodeoxycholic acid starting from cholic acid. The first step is the 12alpha-HSDH-mediated total oxidation of sodium cholate followed by the Wolf-Kishner reduction of the carbonyl group to chenodeoxycholic acid. This acid is then quantitatively oxidized with 7alpha-HSDH to 7-ketochenodeoxycholic acid, that was chemically reduced to ursodeoxycholic acid (70% overall yield).

Three new fatty acids from the roots of Boehmeria nivea (L.) Gaudich and their antifungal activities.

PubMed

Xu, Qiong-Ming; Liu, Yan-Li; Li, Xiao-Ran; Li, Xia; Yang, Shi-Lin

2011-03-01

Three new unsaturated fatty acids, (Z)-9,10,11-trihydroxy-12-octadecenoic acid (1), (Z)-7,8,9-trihydroxy-10-hexadecenoic acid (2) and (Z)-12-keto-7,8,9-trihydroxy-10-hexadecenoic acid (3) were isolated from the roots of Boehmeria nivea (L.) Gaudich, along with a known unsaturated fatty acid, (E)-8,11,12-trihydroxy-9-octadecenoic acid (4). The structures of the new compounds were established by HR ESI-MS, (1)H, (13)C, 2D ((1)H-(1)H COSY, HSQC, HMBC) NMR experiments. The known compound was identified by a comparison of its spectral data with published references. The three new compounds showed some antifungal activities by agar assay.

Antiplasmodial activity of novel keto-enamine chalcone-chloroquine based hybrid pharmacophores.

PubMed

Sashidhara, Koneni V; Kumar, Manoj; Modukuri, Ram K; Srivastava, Rajeev Kumar; Soni, Awakash; Srivastava, Kumkum; Singh, Shiv Vardan; Saxena, J K; Gauniyal, Harsh M; Puri, Sunil K

2012-05-01

A series of novel keto-enamine chalcone-chloroquine based hybrids were synthesized following new methodology developed in our laboratory. The synthesized compounds were screened against chloroquine sensitive strain (3D7) of Plasmodium falciparum in an in vitro model. Some of the compounds were showing comparable antimalarial activity at par with chloroquine. Compounds with significant in vitro antimalarial activity were then evaluated for their in vivo efficacy in Swiss mice against Plasmodium yoelii (chloroquine resistant N-67 strain), wherein compounds 25 and 27 each showed an in vivo suppression of 99.9% parasitaemia on day 4. Biochemical studies reveal that inhibition of hemozoin formation is the primary mechanism of action of these analogues. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Aldo-Keto Reductase Superfamily and its Role in Drug Metabolism and Detoxification

PubMed Central

Barski, Oleg A.; Tipparaju, Srinivas M.; Bhatnagar, Aruni

2008-01-01

The Aldo-Keto Reductase (AKR) superfamily comprises of several enzymes that catalyze redox transformations involved in biosynthesis, intermediary metabolism and detoxification. Substrates of the family include glucose, steroids, glycosylation end products, lipid peroxidation products, and environmental pollutants. These proteins adopt a (β/α)8 barrel structural motif interrupted by a number of extraneous loops and helixes that vary between proteins and bring structural identity to individual families. The human AKR family differs from the rodent families. Due to their broad substrate specificity, AKRs play an important role in the Phase II detoxification of a large number of pharmaceuticals, drugs, and xenobiotics. PMID:18949601

Microbial catabolism of sterols: focus on the enzymes that transform the sterol 3β-hydroxy-5-en into 3-keto-4-en.

PubMed

Kreit, Joseph

2017-02-01

An overview on the microbial sterol catabolism is described with a focus on the catabolic step of the 3β-hydroxy-5-en structure. Cholesterol oxidase transforms this structure into the corresponding 3-keto-4-en feature, and thus initiates the sterol molecule catabolism. The oxidase has been found in a large number of microorganisms, especially in Actinobacteria as species of Rhodococcus and Streptomyces. Other Actinobacteria as species of Mycobacterium and Nocardia possess NAD(P)-dependent dehydrogenase for this catabolic step. In Rhodococcus jostii, oxidation of the C26 of the sterol side chain is the initiating step. The resulting stenone or sterol-C26-oic acid is then catabolized according to two subpathways: cleavage of the sterol side chain and degradation of the steroid nucleus. Divergent items concerned with the enzymes that transform the sterol 3β-hydroxy-5-en are discussed. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Modeling of chemical reactions in micelle: water-mediated keto-enol interconversion as a case study.

PubMed

Marracino, Paolo; Amadei, Andrea; Apollonio, Francesca; d'Inzeo, Guglielmo; Liberti, Micaela; di Crescenzo, Antonello; Fontana, Antonella; Zappacosta, Romina; Aschi, Massimiliano

2011-06-30

The effect of a zwitterionic micelle environment on the efficiency of the keto-enol interconversion of 2-phenylacetylthiophene has been investigated by means of a joint application of experimental and theoretical/computational approaches. Results have revealed a reduction of the reaction rate constant if compared with bulk water essentially because of the different solvation conditions experienced by the reactant species, including water molecules, in the micelle environment. The slight inhibiting effect due to the application of a static electric field has also been theoretically investigated and presented.

Effects of dietary valine:lysine ratio on the performance, amino acid composition of tissues and mRNA expression of genes involved in branched-chain amino acid metabolism of weaned piglets

PubMed Central

Xu, Ye Tong; Ma, Xiao Kang; Wang, Chun Lin; Yuan, Ming Feng; Piao, Xiang Shu

2018-01-01

Objective The goal of this study was to investigate the effects of dietary standard ileal digestible (SID) valine:lysine ratios on performance, intestinal morphology, amino acids of liver and muscle, plasma indices and mRNA expression of branched-chain amino acid (BCAA) metabolism enzymes. Methods A total of 144 crossbred pigs (Duroc×Landrace×Large White) weaned at 28±4 days of age (8.79±0.02 kg body weight) were randomly allotted to 1 of 4 diets formulated to provide SID valine:lysine ratios of 50%, 60%, 70%, or 80%. Each diet was fed to 6 pens of pigs with 6 pigs per pen (3 gilts and 3 barrows) for 28 days. Results Average daily gain increased quadratically (p<0.05), the villous height of the duodenum, jejunum and ileum increased linearly (p<0.05) as the SID valine:lysine ratio increased. The concentrations of plasma α-keto isovaleric and valine increased linearly (p<0.05), plasma aspartate, asparagine and cysteine decreased (p<0.05) as the SID valine:lysine ratio increased. An increase in SID lysine:valine levels increased mRNA expression levels of mitochondrial BCAA transaminase and branched-chain α-keto acid dehydrogenase in the longissimus dorsi muscle (p<0.05). Conclusion Using a quadratic model, a SID valine:lysine ratio of 68% was shown to maximize the growth of weaned pigs which is slightly higher than the level recommended by the National Research Council [6]. PMID:28728397

Branched-chain amino acid (BCAA) supplementation enhances adaptability to exercise training of mice with a muscle-specific defect in the control of BCAA catabolism.

PubMed

Xu, Minjun; Kitaura, Yasuyuki; Shindo, Daichi; Shimomura, Yoshiharu

2018-03-01

Branched-chain α-keto acid dehydrogenase (BCKDH) kinase (BDK) suppresses the branched-chain amino acid (BCAA) catabolism by inactivation of the BCKDH complex. The muscle-specific BDK-deficient (BDK-mKO) mice showed accelerated BCAA oxidation in muscle and decreased endurance capacity after training (Xu et al. PLoS One. 12 (2017) e0180989). We here report that BCAA supplementation overcompensated endurance capacity in BDK-mKO mice after training.

Branched-chain amino acid supplementation: impact on signaling and relevance to critical illness.

PubMed

Mattick, John S A; Kamisoglu, Kubra; Ierapetritou, Marianthi G; Androulakis, Ioannis P; Berthiaume, Francois

2013-01-01

The changes that occur in mammalian systems following trauma and sepsis, termed systemic inflammatory response syndrome, elicit major changes in carbohydrate, protein, and energy metabolism. When these events persist for too long they result in a severe depletion of lean body mass, multiple organ dysfunction, and eventually death. Nutritional supplementation has been investigated to offset the severe loss of protein, and recent evidence suggests that diets enriched in branched-chain amino acids (BCAAs) may be especially beneficial. BCAAs are metabolized in two major steps that are differentially expressed in muscle and liver. In muscle, BCAAs are reversibly transaminated to the corresponding α-keto acids. For the complete degradation of BCAAs, the α-keto acids must travel to the liver to undergo oxidation. The liver, in contrast to muscle, does not significantly express the branched-chain aminotransferase. Thus, BCAA degradation is under the joint control of both liver and muscle. Recent evidence suggests that in liver, BCAAs may perform signaling functions, more specifically via activation of mTOR (mammalian target of rapamycin) signaling pathway, influencing a wide variety of metabolic and synthetic functions, including protein translation, insulin signaling, and oxidative stress following severe injury and infection. However, understanding of the system-wide effects of BCAAs that integrate both metabolic and signaling aspects is currently lacking. Further investigation in this respect will help rationalize the design and optimization of nutritional supplements containing BCAAs for critically ill patients. Copyright © 2013 Wiley Periodicals, Inc.

The effect of trinitrobenzene sulfonic acid (TNB) on colonocyte arachidonic acid metabolism.

PubMed

Stratton, M D; Sexe, R; Peterson, B; Kaminski, D L; Li, A P; Longo, W E

1996-02-01

In previous studies we found that luminal perfusion of the isolated left colon of the rabbit with the hapten, trinitrobenzene, resulted in the production of an acute inflammatory process associated with alterations in eicosanoid metabolism. As the colitis was attenuated by cyclooxygenase inhibitors it is possible that the inflammation was mediated by arachidonic acid metabolites. In the present study it was intended to evaluate the effect of trinitrobenzene on eicosanoid metabolism in transformed human colonic cells by exposing Caco-2++ cells to various doses of trinitrobenzene. Cell injury was evaluated by measuring lactate dehydrogenase levels and cyclooxygenase and lipoxygenase activity was evaluated by measuring prostanoid and leukotriene production. In separate experiments resting and trinitrobenzene stimulated cells were treated with indomethacin and dexamethasone. Trinitrobenzene produced increased prostaglandin E2 and 6-keto prostaglandin F1alpha++ and increased lactate dehydrogenase levels. Leukotriene B4 was significantly increased compared to control values at the highest TNB concentration administered. Indomethacin inhibited the lactate dehydrogenase and prostanoid changes, suggesting that the inflammatory changes produced were mediated by the prostanoids. Dexamethasone administered for 1 hr prior to trinitrobenzene decreased the 6-keto prostaglandin F1alpha but did not alter trinitrobenzene produced changes in lactate dehydrogenase concentrations. Exposure of Caco-2 cells to dexamethasone for 24 hr decreased the trinitrobenzene produced lactate dehydrogenase and eicosanoid changes. The results suggest that trinitrobenzene produces an acute injury to Caco-2 cells that may be mediated by the cyclooxygenase enzymes.

Overproduction of a rice aldo-keto reductase increases oxidative and heat stress tolerance by malondialdehyde and methylglyoxal detoxification.

PubMed

Turóczy, Zoltán; Kis, Petra; Török, Katalin; Cserháti, Mátyás; Lendvai, Agnes; Dudits, Dénes; Horváth, Gábor V

2011-03-01

The accumulation of toxic compounds generated by the interaction between reactive oxygen species and polyunsaturated fatty acids of membrane lipids can significantly damage plant cells. A plethora of enzymes act on these reactive carbonyls, reducing their toxicity. Based on the chromosomal localization and on their homology with other stress-induced aldo-keto reductases (AKRs) we have selected three rice AKR genes. The transcription level of OsAKR1 was greatly induced by abscisic acid and various stress treatments; the other two AKR genes tested were moderately stress-inducible. The OsAKR1 recombinant protein exhibited a high nicotinamide adenine dinucleotide phosphate-dependent catalytic activity to reduce toxic aldehydes including glycolysis-derived methylglyoxal (MG) and lipid peroxidation-originated malondialdehyde (MDA). The function of this enzyme in MG detoxification was demonstrated in vivo in E. coli and in transgenic plants overproducing the OsAKR1 protein. Heterologous synthesis of the OsAKR1 enzyme in transgenic tobacco plants resulted in increased tolerance against oxidative stress generated by methylviologen (MV) and improved resistance to high temperature. In these plants lower levels of MDA were detected both following MV and heat treatment due to the activity of the OsAKR1 enzyme. The transgenic tobaccos also exhibited higher AKR activity and accumulated less MG in their leaves than the wild type plants; both in the presence and absence of heat stress. These results support the positive role of OsAKR1 in abiotic stress-related reactive aldehyde detoxification pathways and its use for improvement of stress tolerance in plants.

Effect of keto-acidosis on seizure occurrence in diabetic patients.

PubMed

Gao, X; Wee, A S; Nick, T G

2005-05-01

It has been observed anecdotally that diabetics are usually non-ketotic at the time of their seizure presentation. In order to establish some validity on this observation, we reviewed the medical records of patients with diagnoses of diabetes and seizure. Study subjects were diabetics presenting with seizures. Control subjects were random sampling of all diabetics. In 51 diabetics presenting with seizures, 38 were nonketotic and 13 were ketotic. In the control group of 119 diabetics, 63 were non-ketotic and 56 were ketotic. There were no significant differences in the serum levels of glucose, sodium, potassium, chloride, and calcium between the seizure and control groups. Multiple regression analysis showed that non-ketotic patients were at risk for developing seizures with an odds ratio of 4.03 (p=0.001). Male sex and type 1 diabetes were also risk factors while age was not a risk factor. Keto-acidosis may play a role in preventing epileptic seizures from occurring in diabetic patients.

A novel regulatory defect in the branched-chain α-keto acid dehydrogenase complex due to a mutation in the PPM1K gene causes a mild variant phenotype of maple syrup urine disease.

PubMed

Oyarzabal, Alfonso; Martínez-Pardo, Mercedes; Merinero, Begoña; Navarrete, Rosa; Desviat, Lourdes R; Ugarte, Magdalena; Rodríguez-Pombo, Pilar

2013-02-01

This article describes a hitherto unreported involvement of the phosphatase PP2Cm, a recently described member of the branched-chain α-keto acid dehydrogenase (BCKDH) complex, in maple syrup urine disease (MSUD). The disease-causing mutation was identified in a patient with a mild variant phenotype, involving a gene not previously associated with MSUD. SNP array-based genotyping showed a copy-neutral homozygous pattern for chromosome 4 compatible with uniparental isodisomy. Mutation analysis of the candidate gene, PPM1K, revealed a homozygous c.417_418delTA change predicted to result in a truncated, unstable protein. No PP2Cm mutant protein was detected in immunocytochemical or Western blot expression analyses. The transient expression of wild-type PPM1K in PP2Cm-deficient fibroblasts recovered 35% of normal BCKDH activity. As PP2Cm has been described essential for cell survival, apoptosis and metabolism, the impact of its deficiency on specific metabolic stress variables was evaluated in PP2Cm-deficient fibroblasts. Increases were seen in ROS levels along with the activation of specific stress-signaling MAP kinases. Similar to that described for the pyruvate dehydrogenase complex, a defect in the regulation of BCKDH caused the aberrant metabolism of its substrate, contributing to the patient's MSUD phenotype--and perhaps others. © 2012 WILEY PERIODICALS, INC.

[Imbalance of system of glutamin - glutamic acid in the placenta and amniotic fluid at placental insufficiency].

PubMed

Pogorelova, T N; Gunko, V O; Linde, V A

2014-01-01

Metabolism of glutamine and glutamic acid has been investigated in the placenta and amniotic fluid under conditions of placental insufficiency. The development of placental insufficiency is characterized by the increased content of glutamic acid and a decrease of glutamine in both placenta and amniotic fluid. These changes changes were accompanied by changes in the activity of enzymes involved in the metabolism of these amino acids. There was a decrease in glutamate dehydrogenase activity and an increase in glutaminase activity with the simultaneous decrease of glutamine synthetase activity. The compensatory decrease in the activity of glutamine keto acid aminotransferase did not prevent a decrease in the glutamine level. The impairments in the system glutamic acid-glutamine were more pronounced during the development of premature labor.

Industrial production of L-ascorbic Acid (vitamin C) and D-isoascorbic acid.

PubMed

Pappenberger, Günter; Hohmann, Hans-Peter

2014-01-01

L-ascorbic acid (vitamin C) was first isolated in 1928 and subsequently identified as the long-sought antiscorbutic factor. Industrially produced L-ascorbic acid is widely used in the feed, food, and pharmaceutical sector as nutritional supplement and preservative, making use of its antioxidative properties. Until recently, the Reichstein-Grüssner process, designed in 1933, was the main industrial route. Here, D-sorbitol is converted to L-ascorbic acid via 2-keto-L-gulonic acid (2KGA) as key intermediate, using a bio-oxidation with Gluconobacter oxydans and several chemical steps. Today, industrial production processes use additional bio-oxidation steps with Ketogulonicigenium vulgare as biocatalyst to convert D-sorbitol to the intermediate 2KGA without chemical steps. The enzymes involved are characterized by a broad substrate range, but remarkable regiospecificity. This puzzling specificity pattern can be understood from the preferences of these enyzmes for certain of the many isomeric structures which the carbohydrate substrates adopt in aqueous solution. Recently, novel enzymes were identified that generate L-ascorbic acid directly via oxidation of L-sorbosone, an intermediate of the bio-oxidation of D-sorbitol to 2KGA. This opens the possibility for a direct route from D-sorbitol to L-ascorbic acid, obviating the need for chemical rearrangement of 2KGA. Similar concepts for industrial processes apply for the production of D-isoascorbic acid, the C5 epimer of L-ascorbic acid. D-isoascorbic acid has the same conformation at C5 as D-glucose and can be derived more directly than L-ascorbic acid from this common carbohydrate feed stock.

Self-Controlled Synthesis of Hyperbranched Poly(etherketone)s from A2 + B3 Approach in Poly(phosphoric acid)

DTIC Science & Technology

2009-01-01

aromatic keto -band arisen from carboxylic acids, which could be part of terminal groups of HPEKs, ranged from 1708 to 1719 cm1. The carbonyl bands from...1999, 143 , 1–34; (d) Inoue, K. Prog Polym Sci 2000, 25, 453–571; (e) Voit, B. J Polym Sci Part A: Polym Chem 2000, 36, 2505–2525; (f) Hult, A

A human fatty acid synthase inhibitor binds β-ketoacyl reductase in the keto-substrate site.

PubMed

Hardwicke, Mary Ann; Rendina, Alan R; Williams, Shawn P; Moore, Michael L; Wang, Liping; Krueger, Julie A; Plant, Ramona N; Totoritis, Rachel D; Zhang, Guofeng; Briand, Jacques; Burkhart, William A; Brown, Kristin K; Parrish, Cynthia A

2014-09-01

Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the β-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.

DNA from uncultured organisms as a source of 2,5-diketo-L-gluconic acid reductases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eschenfeldt, W. H.; Stols, L.; Rosenbaum, H.

2001-09-01

Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-D-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressedmore » in Escherichia coli. The purified gene products catalyzed the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid. Compared to previously described DKGRs isolated from Corynebacterium spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higher k{sub cat}/K{sub m} values than those previously determined, primarily as a result of better binding of substrate. The K{sub m} values for the two new reductases were 57 and 67 {mu}M, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.« less

Determination of Solvent Effects on Keto-Enol Equilibria of 1,3-Dicarbonyl Compounds Using NMR: Revisiting a Classic Physical Chemistry Experiment

ERIC Educational Resources Information Center

Cook, A. Gilbert; Feltman, Paul M.

2007-01-01

The use of proton NMR to determine the equilibrium position of tautomeric 1,3-dicarbonyl compounds in various solvents has been a classic physical chemistry experiment. We are presenting an expansion of the excellent description of this experiment by Garland, Shoemaker, and Nibler. Often the assumption is made that the keto tautomer is always the…

Design, optimization, and biological evaluation of novel keto-benzimidazoles as potent and selective inhibitors of phosphodiesterase 10A (PDE10A).

PubMed

Hu, Essa; Kunz, Roxanne K; Chen, Ning; Rumfelt, Shannon; Siegmund, Aaron; Andrews, Kristin; Chmait, Samer; Zhao, Sharon; Davis, Carl; Chen, Hang; Lester-Zeiner, Dianna; Ma, Ji; Biorn, Christopher; Shi, Jianxia; Porter, Amy; Treanor, James; Allen, Jennifer R

2013-11-14

Our development of PDE10A inhibitors began with an HTS screening hit (1) that exhibited both high p-glycoprotein (P-gp) efflux ratios in rat and human and poor metabolic stability. On the basis of cocrystal structure of 1 in human PDE10A enzyme, we designed a novel keto-benzimidazole 26 with comparable PDE10A potency devoid of efflux liabilities. On target in vivo coverage of PDE10A in rat brain was assessed using our previously reported LC-MS/MS receptor occupancy (RO) technology. Compound 26 achieved 55% RO of PDE10A at 30 mg/kg po and covered PDE10A receptors in rat brain in a dose-dependent manner. Cocrystal structure of 26 in PDE10A confirmed the binding mode of the novel scaffold. Further optimization resulted in the identification of keto-benzimidazole 34, which showed an increased in vivo efficacy of 57% RO in rats at 10 mg/kg po and an improved in vivo rat clearance and oral bioavailability.

Regulation of vascular prostaglandin synthesis by metabolites of arachidonic acid in perfused rabbit aorta.

PubMed Central

Kent, R S; Diedrich, S L; Whorton, A R

1983-01-01

To address the hypothesis that metabolites of arachidonic acid are important regulators of prostaglandin (PG) synthesis in intact vascular tissue, we studied arachidonate metabolism in rabbit aortas in response to a continuous infusion of arachidonic acid, 10 micrograms/ml. Prostacyclin (PGI2; measured as 6-keto-PGF1 alpha) production rate accelerated during the first 2 min, reached peak velocity at 2 min, and then progressively decelerated. The velocity profile of PGI2 production was similar to that previously reported for cyclooxygenase holoenzyme assayed in vitro, and was consistent with progressive inactivation of the enzymes leading to PGI2 synthesis. We determined the specific inhibition of cyclooxygenase and prostacyclin synthetase by measuring PGI2 and PGE2 production rates and by infusing cyclic endoperoxides. Our results indicate preferential inactivation of cyclooxygenase during arachidonate metabolism, most likely due to cyclooxygenase-derived oxidative intermediates. This was a dose-dependent response and resulted in a progressive decrease in the 6-keto-PGF1 alpha/PGE2 ratio. Exogenously added 15-hydroperoxy eicosatetraenoic acid, on the other hand, actually stimulated cyclooxygenase activity at low doses, while markedly inhibiting prostacyclin synthetase. This finding, along with the accelerating nature of arachidonate metabolism, is consistent with the concept of "peroxide tone" as a mediator of cyclooxygenase activity in this system. These results demonstrate that arachidonate metabolites regulate PG synthesis in intact blood vessels. The progressive enzymatic inhibition intrinsic to arachidonate metabolism may be a model for similar changes occurring in states of enhanced lipid peroxidation. These metabolic alterations might greatly influence the numerous vascular functions known to involve arachidonic acid metabolism. PMID:6409932

Evolution of the biosynthesis of the branched-chain amino acids

NASA Technical Reports Server (NTRS)

Keefe, Anthony D.; Lazcano, Antonio; Miller, Stanley L.

1995-01-01

The origins of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threomine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from alpha-ketoisovalerc acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use fo the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

Amino acid metabolism during exercise in trained rats: the potential role of carnitine in the metabolic fate of branched-chain amino acids.

PubMed

Ji, L L; Miller, R H; Nagle, F J; Lardy, H A; Stratman, F W

1987-08-01

The influence of endurance training and an acute bout of exercise on plasma concentrations of free amino acids and the intermediates of branched-chain amino acid (BCAA) metabolism were investigated in the rat. Training did not affect the plasma amino acid levels in the resting state. Plasma concentrations of alanine (Ala), aspartic acid (Asp), asparagine (Asn), arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val) were significantly lower, whereas glutamate (Glu), glycine (Gly), ornithine (Orn), tryptophan (Trp), tyrosine (Tyr), creatinine, urea, and ammonia levels were unchanged, after one hour of treadmill running in the trained rats. Plasma concentration of glutamine (Glu), the branched-chain keto acids (BCKA) and short-chain acyl carnitines were elevated with exercise. Ratios of plasma BCAA/BCKA were dramatically lowered by exercise in the trained rats. A decrease in plasma-free carnitine levels was also observed. These data suggest that amino acid metabolism is enhanced by exercise even in the trained state. BCAA may only be partially metabolized within muscle and some of their carbon skeletons are released into the circulation in forms of BCKA and short-chain acyl carnitines.

Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes

PubMed Central

Spite, Matthew; Baba, Shahid P.; Ahmed, Yonis; Barski, Oleg A.; Nijhawan, Kanchan; Petrash, J. Mark; Bhatnagar, Aruni; Srivastava, Sanjay

2007-01-01

Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone (‘core’ aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte–endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C16:0-20:4 phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C16:0-20:4 phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are

Effects of carnitine and its congeners on eicosanoid discharge from rat cells: implications for release of TNFα

PubMed Central

Elliott, Graham R.; Pruimboom, Wanda M.; Zijlstra, Freek J.; Bonta, Iván L.

1993-01-01

THE acyl carrier coenzyme A (CoA) is involved in fatty acid metabolism. The carnitine/CoA ratio is of particular importance in regulating the transport of long-chain fatty acids into mitochondria for oxidation. Also CoA has a role in the formation and breakdown of products from both the cyclooxygenase and lipoxygenase pathways of the precursor arachidonic acid. In the present study the effect of 4 days feeding of 300 mg/kg/day of L-carnitine, acetyl Lcarnitine and propionyl L-carnitine on the basal and calcium ionophore (A23187) stimulated release of arachidonic acid metabolites from rat carrageenin elicited peritoneal cells was investigated. There were two series of experiments carried out. In the first, the harvested peritoneal cell population consisted of less than 90% macrophages and additional polymorphonuclear (PMN) leucocytes. The basal release of prostaglandin E2 (PGE2), 6-ketoprostaglandin F1α (6-keto-PGF1α) and leukotriene B4 (LTB4) was stimulated by all treatments. The A23187 stimulated release of 6-keto-PGF1α and LTB4 was increased by all three compounds. The 6-keto-PGF1α:TxB2 and 6-keto-PGF1α:LTB4 ratios were increased by carnitine treatment. These results suggested that carnitine could modify the macrophage component of an inflammatory site in vivo. In the second series of experiments the harvested cell population was highly purified (>95% macrophages) and none of the compounds fed to the rats caused a change of either eicosanoid or TNFα formation. Moreover the 6-keto-PGF1α:TxB2 and 6-keto-PGF1α:LTB4 ratios were not enhanced by any of the compounds tested. It is conceivable that in the first series the increased ratios 6-keto-PGF1α:TxB2 and 6-keto-PGF1α:LTB4 reflected the effect of carnitine or its congeners on PMN leucocytes rather than on macrophages. PMID:18475573

Production and Recovery of Pyruvic Acid: Recent Advances

NASA Astrophysics Data System (ADS)

Pal, Dharm; Keshav, Amit; Mazumdar, Bidyut; Kumar, Awanish; Uslu, Hasan

2017-12-01

Pyruvic acid is an important keto-carboxylic acid and can be manufactured by both chemical synthesis and biotechnological routes. In the present paper an overview of recent developments and challenges in various existing technique for the production and recovery of pyruvic acid from fermentation broth or from waste streams has been presented. The main obstacle in biotechnological production of pyruvic acid is development of suitable microorganism which can provide high yield and selectivity. On the other hand, technical limitation in recovery of pyruvic acid from fermentation broth is that, it could not be separated as other carboxylic acid in the form of salts by addition of alkali. Besides, pyruvic acid cannot be crystallized. Commercial separation by distillation is very expensive because pyruvic acid decomposes at higher temperature. It is also chemically reactive due to its peculiar molecular structure and has tendency to polymerize. Thus, at high concentration the various type of reaction leads to lower yield of the product, and hence, conventional methods are not favorable. Alternate separation technologies viable to both synthetic and biological routes are the current research areas. Latest techniques such as reactive extraction is new to the field of recovery of pyruvic acid. Recent development and future prospects in downstream processing of biochemically produced pyruvic acids has been discussed in this review article.

The oxidation of amino acids by ferrate(V). A pre-mix pulse radiolysis study.

PubMed

Rush, J D; Bielski, B H

1995-06-01

The forms of ferrate(V) which are derived from the one-electron reduction of potassium ferrate (K2FeO4) by ethanol radicals react with representative amino acids (glycine, methionine, phenylalanine and serine) at rates that are greater than 10(5)M-1s-1 near pH 10. The predominant interaction in the alkaline pH range is between the protonated ferrate(V) species, HFeO4(2-), and the amino acid anion. Fe(V) + amino acid-->Fe(III) + NH3 + alpha-keto acid The rate-determining process is the two electron reduction of ferrate(V) to iron(III) with oxidation and subsequent deamination of the amino acid. The reaction appears to involve an entry of the amino acid into the inner coordination sphere of ferrate(V). In all cases, ferrate(V) exhibits preferred attack on the amino group in contrast to the OH radical which attacks the thioether site of methionine and the phenyl ring of phenylalanine.

Synthesis of positively charged CdTe quantum dots and detection for uric acid

NASA Astrophysics Data System (ADS)

Zhang, Tiliang; Sun, Xiangying; Liu, Bin

2011-09-01

The CdTe dots (QDs) coated with 2-Mercaptoethylamine was prepared in aqueous solution and characterized with fluorescence spectroscopy, UV-Vis absorption spectra, high-resolution transmission electron microscopy and infrared spectroscopy. When the λex = 350 nm, the fluorescence peak of positively charged CdTe quantum dots is at 592 nm. The uric acid is able to quench their fluorescence. Under optimum conditions, the change of fluorescence intensity is linearly proportional to the concentration of uric acid in the range 0.4000-3.600 μmol L -1, and the limit of detection calculated according to IUPAC definitions is 0.1030 μmol L -1. Compared with routine method, the present method determines uric acid in human serum with satisfactory results. The mechanism of this strategy is due to the interaction of the tautomeric keto/hydroxyl group of uric acid and the amino group coated at the CdTe QDs.

Investigating Metabolic Control of Persister Formation in Biofilms

DTIC Science & Technology

2013-10-01

References Albe, K.R., Butler, M.H., and Wright, B.E. (1990). Cellular concentrations of enzymes and their substrates. Journal of theoretical biology 143 ...0.00 1,2- Propanediol 0.00±0.00 Tween 40 0.25±0.04 α- Keto - Glutaric Acid 0.26±0.07 α- Keto -Butyric Acid 0.18±0.03 α-Methyl-D

Stereochemical analysis of the elimination reaction catalyzed by D-amino-acid oxidase.

PubMed

Cheung, Y F; Walsh, C

1976-06-01

The stereochemistry of the intramolecular proton transfer catalyzed by the flavoenzyme, D-amino-acid oxidase, during the elimination reaction of beta-chloro-alpha-amino acid substrates (Walsh et al. (1973), J. Biol. Chem. 248, 1964) has been established. Both D-erythro- and D-threo-2-amino-3-chloro(2-3H) butyrate have been shown to yield (3R)-2-keto (3-3H)-2- butyrate predominantly. Tritium kinetic isotope effects on the rate of the reaction (4.7 for the D-erythro, and 3.8 for the D-threo compound) and percentages of intramolecular triton transfer (7.2% for the D-erythro- and 2.6% for the D-threo compound) have been measured. Their implications on the mechanism of this unusual elimination reaction are discussed.

Solvent effects on the absorption spectrum and first hyperpolarizability of keto-enol tautomeric forms of anil derivatives: A Monte Carlo/quantum mechanics study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adriano Junior, L.; Fonseca, T. L.; Castro, M. A.

2016-06-21

Theoretical results for the absorption spectrum and electric properties of the enol and keto tautomeric forms of anil derivatives in the gas-phase and in solution are presented. The electronic properties in chloroform, acetonitrile, methanol, and water were determined by carrying out sequential Monte Carlo simulations and quantum mechanics calculations based on the time dependent density functional theory and on the second-order Møller–Plesset perturbation theory method. The results illustrate the role played by electrostatic interactions in the electronic properties of anil derivatives in a liquid environment. There is a significant increase of the dipole moment in solution (20%-100%) relative to themore » gas-phase value. Solvent effects are mild for the absorption spectrum and linear polarizability but they can be particularly important for first hyperpolarizability. A large first hyperpolarizability contrast between the enol and keto forms is observed when absorption spectra present intense lowest-energy absorption bands. Dynamic results for the first hyperpolarizability are in qualitative agreement with the available experimental results.« less

Effect of phospholipid-based formulations of Boswellia serrata extract on the solubility, permeability, and absorption of the individual boswellic acid constituents present.

PubMed

Hüsch, Jan; Gerbeth, Kathleen; Fricker, Gert; Setzer, Constanze; Zirkel, Jürgen; Rebmann, Herbert; Schubert-Zsilavecz, Manfred; Abdel-Tawab, Mona

2012-10-26

Boswellia serrata gum resin extracts are used widely for the treatment of inflammatory diseases. However, very low concentrations in the plasma and brain were observed for the boswellic acids (1-6, the active constituents of B. serrata). The present study investigated the effect of phospholipids alone and in combination with common co-surfactants (e.g., Tween 80, vitamin E-TPGS, pluronic f127) on the solubility of 1-6 in physiologically relevant media and on the permeability in the Caco-2 cell model. Because of the high lipophilicity of 1-6, the permeability experiments were adapted to physiological conditions using modified fasted state simulated intestinal fluid as apical (donor) medium and 4% bovine serum albumin in the basolateral (receiver) compartment. A formulation composed of extract/phospholipid/pluronic f127 (1:1:1 w/w/w) increased the solubility of 1-6 up to 54 times compared with the nonformulated extract and exhibited the highest mass net flux in the permeability tests. The oral administration of this formulation to rats (240 mg/kg) resulted in 26 and 14 times higher plasma levels for 11-keto-β-boswellic acid (1) and acetyl-11-keto-β-boswellic acid (2), respectively. In the brain, five times higher levels for 2 compared to the nonformulated extract were determined 8 h after oral administration.

Nutrition, phosphorus, and keto-analogues in hemodialysis patients: a Chinese perspective.

PubMed

Chen, Jing

2013-05-01

The optimal dietary protein requirements in maintenance hemodialysis (HD) patients and how to balance the treatments between the nutritional intervention and other approaches are still controversies among nephrologists. In China, excessive dietary intake, low dose of dialysis, and lack of non-calcium-containing phosphorus binders are the main causes of hyperphosphatemia among HD patients. If the daily protein intake reached the recommended dose of 1.2 g/kg body weight per the Kidney Disease Outcomes Quality Initiative guidelines, the net accumulation of phosphorus in patients receiving conventional thrice-weekly low-flux HD may reach 1550 mg per week on the basis of our studies on the assessment of phosphorus removal by HD and residual renal function. In fact, a relatively low-protein diet supplemented with keto-analogues could maintain the stable nutritional status in dialysis patients and provide additional beneficial effects. An individualized nutritional intervention is worth trying to treat hyperphosphatemia in HD patients. Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

Inactivation of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii by 2 prime -chloro-2 prime -deoxyuridine 5 prime -triphosphate: A 3 prime -2 prime hydrogen transfer during the formation of 3 prime -keto-2 prime -deoxyuridine 5 prime -triphosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ashley, G.W.; Harris, G.; Stubbe, J.

1988-10-04

The ribonucleoside triphosphate reductase of Lactobacillus leichmannii converts the substrate analogue 2{prime}-chloro-2{prime}-deoxyuridine 5{prime}-triphosphate (C1UTP) into a mixture of 2{prime}-deoxyuridine triphosphate (dUTP) and the unstable product 3{prime}-keto-2{prime}-deoxyuridine triphosphate (3{prime}-keto-dUTP). This ketone can be trapped by reduction with NaBH{sub 4}, producing a 4:1 mixture of xylo-dUTP and dUTP. When (3{prime}-{sup 3}H)C1UTP is treated with enzyme in the presence of NaBH{sub 4}, the isomeric deoxyuridines isolated after alkaline phosphatase treatment retained 15% of the {sup 3}H in C1UTP. Degradation of these isomeric nucleosides has established the location of the {sup 3}H in 3{prime}-keto-dUTP as predominantly 2{prime}(S). The xylo-dU had 98.6% of its labelmore » at the 2{prime}(S) position and 1.5% at 2{prime}(R). The isolated dU had 89.6% of its label at 2{prime}(S) and 1.4% at 2{prime}(R), with the remaining 9% label inferred to be at the 3{prime}-carbon, this resulting from the direct enzymic production of dUTP. These results are consistent with enzymic production of a 1:1,000 mixture of dUTP and 3{prime}-keto-dUTP, where the 3{prime}-hydrogen of C1UTP is retained at 3{prime} during production of dUTP and is transferred to 2{prime}(S) during production of 3{prime}-keto-dUTP. The implications of these results and the unique role of the cofactor adenosylcobalamin are discussed in terms of reductase being a model for the B{sub 12}-dependent rearrangement reactions.« less

Apoptotic signaling pathways induced by acute administration of branched-chain amino acids in an animal model of maple syrup urine disease.

PubMed

Vilela, Thais C; Scaini, Giselli; Furlanetto, Camila B; Pasquali, Matheus A B; Santos, João Paulo A; Gelain, Daniel P; Moreira, José Cláudio F; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

2017-02-01

Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of the branched-chain α-keto acid dehydrogenase complex activity. This blockage leads to accumulation of the branched-chain amino acids leucine, isoleucine and valine, as well as their corresponding α-keto acids and α-hydroxy acids. The affected patients present severe neurological symptoms, such as coma and seizures, as well as edema and cerebral atrophy. Considering that the mechanisms of the neurological symptoms presented by MSUD patients are still poorly understood, in this study, protein levels of apoptotic factors are measured, such as Bcl-2, Bcl-xL, Bax, caspase-3 and -8 in hippocampus and cerebral cortex of rats submitted to acute administration of branched-chain amino acids during their development. The results in this study demonstrated that BCAA acute exposure during the early postnatal period did not significantly change Bcl-2, Bcl-xL, Bax and caspase-8 protein levels. However, the Bax/Bcl-2 ratio and procaspase-3 protein levels were decreased in hippocampus. On the other hand, acute administration of BCAA in 30-day-old rats increase in Bax/Bcl-2 ratio followed by an increased caspase-3 activity in cerebral cortex, whereas BCAA induces apoptosis in hippocampus through activation and cleavage of caspase-3 and -8 without changing the Bax/Bcl-2 ratio. In conclusion, the results suggest that apoptosis could be of pivotal importance in the developmental neurotoxic effects of BCAA. In addition, the current studies also suggest that multiple mechanisms may be involved in BCAA-induced apoptosis in the cerebral cortex and hippocampus.

Molecular characterization of an aldo-keto reductase from Marivirga tractuosa that converts retinal to retinol.

PubMed

Hong, Seung-Hye; Nam, Hyun-Koo; Kim, Kyoung-Rok; Kim, Seon-Won; Oh, Deok-Kun

2014-01-01

A recombinant aldo-keto reductase (AKR) from Marivirga tractuosa was purified with a specific activity of 0.32unitml(-1) for all-trans-retinal with a 72kDa dimer. The enzyme had substrate specificity for aldehydes but not for alcohols, carbonyls, or monosaccharides. The enzyme turnover was the highest for benzaldehyde (kcat=446min(-1)), whereas the affinity and catalytic efficiency were the highest for all-trans-retinal (Km=48μM, kcat/Km=427mM(-1)min(-1)) among the tested substrates. The optimal reaction conditions for the production of all-trans-retinol from all-trans-retinal by M. tractuosa AKR were pH 7.5, 30°C, 5% (v/v) methanol, 1% (w/v) hydroquinone, 10mM NADPH, 1710mgl(-1) all-trans-retinal, and 3unitml(-1) enzyme. Under these optimized conditions, the enzyme produced 1090mgml(-1) all-trans-retinol, with a conversion yield of 64% (w/w) and a volumetric productivity of 818mgl(-1)h(-1). AKR from M. tractuosa showed no activity for all-trans-retinol using NADP(+) as a cofactor, whereas human AKR exhibited activity. When the cofactor-binding residues (Ala158, Lys212, and Gln270) of M. tractuosa AKR were changed to the corresponding residues of human AKR (Ser160, Pro212, and Glu272), the A158S and Q270E variants exhibited activity for all-trans-retinol. Thus, amino acids at positions 158 and 270 of M. tractuosa AKR are determinant residues of the activity for all-trans-retinol. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

A laboratory study of the persistence of carbofuran and its 3-hydroxy- and 3 keto-metabolites in sterile and natural mineral and organic soils.

PubMed

Miles, J R; Tu, C M; Harris, C R

1981-01-01

In a laboratory study, the persistence of carbofuran and its 3-hydroxy- and 3-keto-metabolites was examined separately over 16 wk in sterile and natural organic (muck) and mineral (loam) soils. Carbofuran was relatively persistent in sterile soils; at 8 wk 77% remained in the sterile muck and about 50% remained in the sterile loam. In the natural muck 25% of initial carbofuran remained at wk whereas in the natural loam carbofuran had completely disappeared by that time. The 3-ketocarbofuran was very short-lived even in the sterile muck where only 50% remained at 1 wk. The 3-hydroxycarbofuran degraded appreciably on zero day in the natural soils (with conversion to 3-keto-carbofuran) and about 90% had disappeared in 1 wk. A more detailed study of the persistence of 3-hydroxycarbofuran in the natural soils showed complete disappearance in 2 days in loam and in 3 days in muck. The 3-ketocarbofuran produced from the 3-hydroxy-carbofuran reached a maximum concentration in 1 day and then disappeared within 4 days in loam and about 1 wk in muck.

Impaired adiponectin signaling contributes to disturbed catabolism of branched-chain amino acids in diabetic mice.

PubMed

Lian, Kun; Du, Chaosheng; Liu, Yi; Zhu, Di; Yan, Wenjun; Zhang, Haifeng; Hong, Zhibo; Liu, Peilin; Zhang, Lijian; Pei, Haifeng; Zhang, Jinglong; Gao, Chao; Xin, Chao; Cheng, Hexiang; Xiong, Lize; Tao, Ling

2015-01-01

The branched-chain amino acids (BCAA) accumulated in type 2 diabetes are independent contributors to insulin resistance. The activity of branched-chain α-keto acid dehydrogenase (BCKD) complex, rate-limiting enzyme in BCAA catabolism, is reduced in diabetic states, which contributes to elevated BCAA concentrations. However, the mechanisms underlying decreased BCKD activity remain poorly understood. Here, we demonstrate that mitochondrial phosphatase 2C (PP2Cm), a newly identified BCKD phosphatase that increases BCKD activity, was significantly downregulated in ob/ob and type 2 diabetic mice. Interestingly, in adiponectin (APN) knockout (APN(-/-)) mice fed with a high-fat diet (HD), PP2Cm expression and BCKD activity were significantly decreased, whereas BCKD kinase (BDK), which inhibits BCKD activity, was markedly increased. Concurrently, plasma BCAA and branched-chain α-keto acids (BCKA) were significantly elevated. APN treatment markedly reverted PP2Cm, BDK, BCKD activity, and BCAA and BCKA levels in HD-fed APN(-/-) and diabetic animals. Additionally, increased BCKD activity caused by APN administration was partially but significantly inhibited in PP2Cm knockout mice. Finally, APN-mediated upregulation of PP2Cm expression and BCKD activity were abolished when AMPK was inhibited. Collectively, we have provided the first direct evidence that APN is a novel regulator of PP2Cm and systematic BCAA levels, suggesting that targeting APN may be a pharmacological approach to ameliorating BCAA catabolism in the diabetic state. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Tautomerism in N-(2-hydroxy-1-naphthylidene)amino acids and the search for an answer to the difficult question about where the proton belongs

NASA Astrophysics Data System (ADS)

Warncke, Gisela; Fels, Sabine; Brendler, Erica; Böhme, Uwe

2016-08-01

N-(2-hydroxy-1-naphthylidene)-L-valine 1, N-(2-hydroxy-1-naphthylidene)-L-phenylalanine 2, and N-(2-hydroxy-1-naphthylidene)-L-threonine 3 were prepared and characterized with spectroscopic methods, elemental analyses, and values of optical rotation. Compound 1 undergoes a solid state order-disorder phase transition at 231 K. The X-ray structures of the high and low temperature phase of 1 have been determined. Single crystal X-ray structures of 2 and 3 have been determined as well. The tautomerism of N-(2-hydroxy-1-naphthylidene)amino acid derivatives is discussed controversial in the literature. A bond lengths statistical analysis shows that all three compounds exist uniformly in the keto-amine form in the solid state. Quantum chemical calculations, NMR, and UV-Vis spectroscopy were used to obtain further insight into the existence of phenol-imine and keto-amine structures in this class of compounds.

Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Taeho; Flick, Robert; Brunzelle, Joseph

The nonnatural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases (AKRs). This pathway requires an AKR selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one-pot synthesis. In this work, we screened more than 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 fromPseudomonas aeruginosashowed the highest activity and was selected for comparativemore » studies with STM2406 fromSalmonella entericaserovar Typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as a cofactor, reduced a broad range of aldehydes, and showed low activities toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for the activity against 3-HB and aromatic aldehydes, which include the residues of the substrate-binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced the activity and the affinity of this protein toward 3-HB, resulting in a 7-fold increase ink cat/K m. Our work provides further insights into the molecular mechanisms of the substrate selectivity of AKRs and for the rational design of these enzymes toward new substrates. IMPORTANCEIn this study, we identified several aldo-keto reductases with significant activity in reducing 3-hydroxybutanal to 1,3-butanediol (1,3-BDO), an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate-binding residues, including the two

PHYSIOLOGY OF THE AMINO ACIDS.

PubMed

VAN Slyke, D D

1942-03-13

We have followed the amino acids from their entrance into the alimentary tract in the form of food proteins through the successive steps of digestion, absorption into the blood stream and passage from the blood stream into the tissues, where they are concentrated by some unknown mechanism to many times their concentration in the blood plasma. We have seen something of the way in which certain of the amino acids can be transformed into one another in the body or synthesized from ammonia and keto acids. However, we have had to admit that our bodies can form in such ways only about half of the different amino acids that are required, and that the other half must be made for us by plants, bacteria or other organisms which have greater synthetic powers than we. And finally we have seen something of the manifold fates of the amino acids after they have entered our tissues; how they may be destroyed and their nitrogenous parts turned into urea in the liver before it is possible to put them to their more specialized uses, how their carbon fractions can be used to form glucose, how they may sacrifice themselves to protect us from toxic products, how they can serve as source material for certain vitamins, hormones and other compounds with physiological functions still to be identified, and how finally those amino acids which are not deflected to these various fates may enter into the proteins of the tissues and become for a time parts of our living structures.

The aldo-keto reductase AKR1B7 coexpresses with renin without influencing renin production and secretion.

PubMed

Machura, Katharina; Iankilevitch, Elina; Neubauer, Björn; Theuring, Franz; Kurtz, Armin

2013-03-01

On the basis of evidence that within the adult kidney, the aldo-keto reductase AKR1B7 (aldo-keto reductase family 1, member 7, also known as mouse vas deferens protein, MVDP) is selectively expressed in renin-producing cells, we aimed to define a possible role of AKR1B7 for the regulation and function of renin cells in the kidney. We could confirm colocalization and corecruitment of renin and of AKR1B7 in wild-type kidneys. Renin cells in AKR1B7-deficient kidneys showed normal morphology, numbers, and intrarenal distribution. Plasma renin concentration (PRC) and renin mRNA levels of AKR1B7-deficient mice were normal at standard chow and were lowered by a high-salt diet directly comparable to wild-type mice. Treatment with a low-salt diet in combination with an angiotensin-converting enzyme inhibitor strongly increased PRC and renin mRNA in a similar fashion both in AKR1B7-deficient and wild-type mice. Under this condition, we also observed a strong retrograde recruitment of renin-expressing cell along the preglomerular vessels, however, without a difference between AKR1B7-deficient and wild-type mice. The isolated perfused mouse kidney model was used to study the acute regulation of renin secretion by ANG II and by perfusion pressure. Regarding these parameters, no differences were observed between AKR1B7-deficient and wild-type kidneys. In summary, our data suggest that AKR1B7 is not of major relevance for the regulation of renin production and secretion in spite of its striking coregulation with renin expression.

Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli

PubMed Central

Howard, Thomas P.; Middelhaufe, Sabine; Moore, Karen; Edner, Christoph; Kolak, Dagmara M.; Taylor, George N.; Parker, David A.; Lee, Rob; Smirnoff, Nicholas; Aves, Stephen J.; Love, John

2013-01-01

Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e., aliphatic n- and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (Cn) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane Cn and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules. PMID:23610415

Pretreated Starch Solutions for Low Environmental Impact Aircraft Deicing

DTIC Science & Technology

2010-09-01

modified monomeric organic molecules, have been the focus of recent study. Several organic acids and keto acids, such as succinate and acetate, have...1992), but the authors noted low economic feasibility without additional recovered products. Ganjyal et al. (2007) produced levulinate, a keto acid...534 -23.4 25 -22.7 52 569 -25.3 5 -25.8 104 585 -28.0 Sigma Aldrich DARCO 12-20 Mesh 0 -28.0 143 634 -28.0 50 -26.2 100 626 -27.4 Calgon F-300 9

Lignases and aldo-keto reductases for conversion of lignin-containing materials to fermentable products

DOEpatents

Scharf, Michael; Sethi, Amit

2016-09-13

Termites have specialized digestive systems that overcome the lignin barrier in wood to release fermentable simple sugars. Using the termite Reticulitermes flavipes and its gut symbionts, high-throughput titanium pyrosequencing and proteomics approaches experimentally compared the effects of lignin-containing diets on host-symbiont digestome composition. Proteomic investigations and functional digestive studies with recombinant lignocellulases conducted in parallel provided strong evidence of congruence at the transcription and translational levels and provide enzymatic strategies for overcoming recalcitrant lignin barriers in biofuel feedstocks. Briefly described, therefore, the disclosure provides a system for generating a fermentable product from a lignified plant material, the system comprising a cooperating series of at least two catalytically active polypeptides, where said catalytically active polypeptides are selected from the group consisting of: cellulase Cell-1, .beta.-glu cellulase, an aldo-keto-reductase, a catalase, a laccase, and an endo-xylanase.

Let the substrate flow, not the enzyme: Practical immobilization of d-amino acid oxidase in a glass microreactor for effective biocatalytic conversions.

PubMed

Bolivar, Juan M; Tribulato, Marco A; Petrasek, Zdenek; Nidetzky, Bernd

2016-11-01

Exploiting enzymes for chemical synthesis in flow microreactors necessitates their reuse for multiple rounds of conversion. To achieve this goal, immobilizing the enzymes on microchannel walls is a promising approach, but practical methods for it are lacking. Using fusion to a silica-binding module to engineer enzyme adsorption to glass surfaces, we show convenient immobilization of d-amino acid oxidase on borosilicate microchannel plates. In confocal laser scanning microscopy, channel walls appeared uniformly coated with target protein. The immobilized enzyme activity was in the range expected for monolayer coverage of the plain surface with oxidase (2.37 × 10(-5)  nmol/mm(2) ). Surface attachment of the enzyme was completely stable under flow. The operational half-life of the immobilized oxidase (25°C, pH 8.0; soluble catalase added) was 40 h. Enzymatic oxidation of d-Met into α-keto-γ-(methylthio)butyric acid was characterized in single-pass and recycle reactor configurations, employing in-line measurement of dissolved O2 , and off-line determination of the keto-acid product. Reaction-diffusion time-scale analysis for different flow conditions showed that the heterogeneously catalyzed reaction was always slower than diffusion of O2 to the solid surface (DaII  ≤ 0.3). Potential of the microreactor for intensifying O2 -dependent biotransformations restricted by mass transfer in conventional reactors is thus revealed. Biotechnol. Bioeng. 2016;113: 2342-2349. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Molecular and Enantiomeric Analysis of Organic Compounds in Carbonaceous Meteorites

NASA Technical Reports Server (NTRS)

Cooper, George

2003-01-01

Carbonaceous meteorites are relatively enriched in carbon. Much of this carbon is in the form of soluble organic compounds. The Murchison and Murray meteorites are the best-characterized carbonaceous meteorites with respect to organic chemistry. Their content of organic compounds has led to an initial understanding of early solar system organic chemistry as well as what compounds may have played a role in the origin of life (Cronin and Chang, 1993). Reported compounds include: amino acids, amides, carboxylic acids, sulfonic acids, and polyols. This talk will focus on the molecular and enantiomeric analysis of individual meteoritic compounds: polyol acids; and a newly identified class of meteorite compounds, keto acids, i.e., acetoacetic acid, levulinic acid, etc. Keto acids (including pyruvic) are critically important in all contemporary organisms. They are key intermediates in metabolism and processes such as the citric acid cycle. Using gas chromatography-mass spectrometry we identified individual meteoritic keto acids after derivatization to one or more of the following forms: isopropyl ester (ISP), trimethyIsiIy1 (TMS), tert-butyldimethylsilyl (BDMS). Ongoing analyses will determine if, in addition to certain amino acids from Murchison (Cronin and Pizzarello, 1997), other potentially important prebiotic compounds also contain enantiomeric excesses, i.e., excesses that could have contributed to the current homochirality of life.

The tautomerization between keto- to phenol-hydrazone induced by anions in the solution

NASA Astrophysics Data System (ADS)

Shang, Xuefang; Yuan, Jianmei; Wang, Yingling; Zhang, Jinlian; Xu, Xiufang

2012-02-01

Two simple anion receptors, 2-[(2-hydroxy-5-nitrophenyl)methylene]hydrazone (1) and 2-[(3,5-dibromo-2-hydroxyphenyl)methylene]hydrazone (2) with -OH binding sites, were synthesized and characterized. The anion binding ability of receptors 1 and 2 with halide anions (F-, Cl-, Br- and I-), AcO- and HPO4- was investigated using visual (naked-eye), UV-vis titration experiments in dry DMSO together with DFT theoretical calculation. The addition of F-, AcO- and HPO4- to the host solution resulted in a red shift of the charge-transfer absorbance band accompanied by a color change from yellow to orange in the naked-eye experiments. Receptor 1 containing a nitro group at the para position and receptor 2 containing two bromine groups at the ortho and para positions both showed strong binding ability for HPO4- ion in the form of phenol-hydrazone. Moreover, receptor 1, induced by anion species in the solution, converted to the form of phenol-hydrazone from keto-hydrazone.

Secondary Structures in a Freeze-Dried Lignite Humic Acid Fraction Caused by Hydrogen-Bonding of Acidic Protons with Aromatic Rings.

PubMed

Cao, Xiaoyan; Drosos, Marios; Leenheer, Jerry A; Mao, Jingdong

2016-02-16

A lignite humic acid (HA) was separated from inorganic and non-HA impurities (i.e., aluminosilicates, metals) and fractionated by a combination of dialysis and XAD-8 resin. Fractionation revealed a more homogeneous structure of lignite HA. New and more specific structural information on the main lignite HA fraction is obtained by solid-state nuclear magnetic resonance (NMR) spectroscopy. Quantitative (13)C multiple cross-polarization (multiCP) NMR indicated oxidized phenyl propane structures derived from lignin. MultiCP experiments, conducted on potassium HA salts titrated to pH 10 and pH 12, revealed shifts consistent with carboxylate and phenolate formation, but structural changes associated with enolate formation from aromatic beta keto acids were not detected. Two-dimensional (1)H-(13)C heteronuclear correlation (2D HETCOR) NMR indicated aryl-aliphatic ketones, aliphatic and aromatic carboxyl groups, phenol, and methoxy phenyl ethers. Acidic protons from carboxyl groups in both the lignite HA fraction and a synthetic HA-like polycondensate were found to be hydrogen-bonded with electron-rich aromatic rings. Our results coupled with published infrared spectra provide evidence for the preferential hydrogen bonding of acidic hydrogens with electron-rich aromatic rings rather than adjacent carbonyl groups. These hydrogen-bonding interactions likely result from stereochemical arrangements in primary structures and folding.

Aldo-Keto Reductases 1B in Adrenal Cortex Physiology

PubMed Central

Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A. Marie

2016-01-01

Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions. PMID:27499746

Pd(II) and Pt(II) complexes of α-keto stabilized sulfur ylide: Synthesis, structural, theoretical and catalytic activity studies

NASA Astrophysics Data System (ADS)

Sabounchei, Seyyed Javad; Hashemi, Ali; Sedghi, Asieh; Bayat, Mehdi; Akhlaghi Bagherjeri, Fateme; Gable, Robert W.

2017-05-01

Reaction of dimethyl sulfide with 2, 3‧-dibromoacetophenone led to formation of sulfonium salt [Me2SCH2C(O)C6H4-m-Br]Br (1). The resulted sulfonium salt was treated with NaOH and gave the α-keto stabilized sulfur ylide Me2SC(H)C(O)C6H4-m-Br (2). This ligand was reacted with [MCl2(cod)] (M = Pd, Pt; cod = 1,5-cyclooctadiene) to form the new cis- and trans-[MCl2(ylide)2] (M = Pd (cis- and trans-3), Pt (cis- and trans-4)) complexes. Characterization of the obtained compounds was performed by elemental analysis, IR, 1H and 13C NMR. Recrystallization of dichlorobis(ylide) palladium(II) and platinum(II) complexes from DMSO solution yielded the crystalline products, which X-ray diffraction data revealed that the both compounds were crystallized as cis-[MCl2(ylide)(DMSO)] (M = Pd (5), Pt (6)) complexes. Also, a theoretical study on structure and nature of the Msbnd C bonding between the Y ligand (ylide) and [MCl2·DMSO] fragments in [YMCl2·DMSO] (M = Pd, Pt) complexes has been reported via NBO and energy-decomposition analysis (EDA). Furthermore, the palladium catalyzed Suzuki-Miyaura reaction of various aryl chlorides with arylboronic acids was performed. The results showed that the Pd(II) complexes cis- and trans-3 catalyzed efficiently coupling reactions at low catalyst loading and short reaction time.

Asymmetric reduction of ketones and β-keto esters by (S)-1-phenylethanol dehydrogenase from denitrifying bacterium Aromatoleum aromaticum.

PubMed

Dudzik, A; Snoch, W; Borowiecki, P; Opalinska-Piskorz, J; Witko, M; Heider, J; Szaleniec, M

2015-06-01

Enzyme-catalyzed enantioselective reductions of ketones and keto esters have become popular for the production of homochiral building blocks which are valuable synthons for the preparation of biologically active compounds at industrial scale. Among many kinds of biocatalysts, dehydrogenases/reductases from various microorganisms have been used to prepare optically pure enantiomers from carbonyl compounds. (S)-1-phenylethanol dehydrogenase (PEDH) was found in the denitrifying bacterium Aromatoleum aromaticum (strain EbN1) and belongs to the short-chain dehydrogenase/reductase family. It catalyzes the stereospecific oxidation of (S)-1-phenylethanol to acetophenone during anaerobic ethylbenzene mineralization, but also the reverse reaction, i.e., NADH-dependent enantioselective reduction of acetophenone to (S)-1-phenylethanol. In this work, we present the application of PEDH for asymmetric reduction of 42 prochiral ketones and 11 β-keto esters to enantiopure secondary alcohols. The high enantioselectivity of the reaction is explained by docking experiments and analysis of the interaction and binding energies of the theoretical enzyme-substrate complexes leading to the respective (S)- or (R)-alcohols. The conversions were carried out in a batch reactor using Escherichia coli cells with heterologously produced PEDH as whole-cell catalysts and isopropanol as reaction solvent and cosubstrate for NADH recovery. Ketones were converted to the respective secondary alcohols with excellent enantiomeric excesses and high productivities. Moreover, the progress of product formation was studied for nine para-substituted acetophenone derivatives and described by neural network models, which allow to predict reactor behavior and provides insight on enzyme reactivity. Finally, equilibrium constants for conversion of these substrates were derived from the progress curves of the reactions. The obtained values matched very well with theoretical predictions.

omega-Amino acid:pyruvate transaminase from Alcaligenes denitrificans Y2k-2: a new catalyst for kinetic resolution of beta-amino acids and amines.

PubMed

Yun, Hyungdon; Lim, Seongyop; Cho, Byung-Kwan; Kim, Byung-Gee

2004-04-01

Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed omega-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic beta-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for L-beta-amino-n-butyric acid (L-beta-ABA). The enzyme converts various beta-amino acids and amines to the corresponding beta-keto acids and ketones by using pyruvate as an amine acceptor. The apparent K(m) and V(max) for L-beta-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM L-beta-ABA, the apparent K(m) and V(max) for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM D,L-beta-ABA, producing optically pure D-beta-ABA (99% enantiomeric excess) with 53% conversion.

ω-Amino Acid:Pyruvate Transaminase from Alcaligenes denitrificans Y2k-2: a New Catalyst for Kinetic Resolution of β-Amino Acids and Amines

PubMed Central

Yun, Hyungdon; Lim, Seongyop; Cho, Byung-Kwan; Kim, Byung-Gee

2004-01-01

Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed ω-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic β-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for l-β-amino-n-butyric acid (l-β-ABA). The enzyme converts various β-amino acids and amines to the corresponding β-keto acids and ketones by using pyruvate as an amine acceptor. The apparent Km and Vmax for l-β-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM l-β-ABA, the apparent Km and Vmax for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM d,l-β-ABA, producing optically pure d-β-ABA (99% enantiomeric excess) with 53% conversion. PMID:15066855

Amonia gas: an improved reagent for chemical ionization mass spectrometry of bile acid methyl ester acetates

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeMark, B.R.; Klein, P.D.

1981-01-01

The ammonia chemical ionization mass spectra of 28 methyl ester acetate derivatives of bile acids and related compounds have been determined by gas-liquid chromatography-mass spectrometry. Advantages of ammonia ionization over the previously studied isobutane ionization include a 130 to 270% enhancement in the sensitivity of base peak monitoring, and direct determination of molecular weight from the base peak (M + NH/sub 4//sup +/) in the mass spectrum of any of the derivatives. Minor ions in the ammonia spectra also allow selective detection of 3-keto compounds and can indicate unsaturation or double bond conjugation in the molecule. The significance of thesemore » studies for the detection and quantitation of bile acids is discussed. 2 tables.« less

Synthesis, spectral studies, antimicrobial, antioxidant and insect antifeedant activities of some 9 H-fluorene-2-yl keto-oxiranes

NASA Astrophysics Data System (ADS)

Thirunarayanan, G.; Vanangamudi, G.

2011-10-01

Thirteen ee (α S, β R) 9 H-fluorene-2-yl keto-oxiranes (2-(9 H)-fluorene-4-yl[3-(substituted phenyl)oxiran-2-yl]methanones) have been synthesized by phase transfer catalysed epoxidation of 9 H-fluorene-2-yl chalcones. The yields of oxiranes are more than 95%. The synthesized oxiranes have been characterized by IR, 1H, 13C and GC-MS spectral data. The spectral data are correlated with Hammett substituent constants and Swain-Lupton parameters. From the regression analysis, the effect of substituents on the group frequencies has been predicted. The antimicrobial, antioxidant and insect antifeedant activities of all the synthesized oxiranes have been studied.

Estimation of free energy barriers in the cytoplasmic and mitochondrial aspartate aminotransferase reactions probed by hydrogen-exchange kinetics of C alpha-labeled amino acids with solvent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Julin, D.A.; Wiesinger, H.; Toney, M.D.

1989-05-02

The existence of the postulated quinonoid intermediate in the cytoplasmic aspartate amino-transferase catalyzed transamination of aspartate to oxaloacetate was probed by determining the extent of transfer of tritium from the C alpha position of tritiated L-aspartate to pyridoxamine 5'-phosphate in single turnover experiments in which washout from the back-reaction was obviated by product trapping. The maximum amount of transferred tritium observed was 0.7%, consistent either with a mechanism in which a fraction of the net transamination reaction proceeds through a quinonoid intermediate or with a mechanism in which this intermediate is formed off the main reaction pathway. It is shownmore » that transfer of labeled hydrogen from the amino acid to cofactor cannot be used to differentiate a stepwise from a concerted transamination mechanism. The amount of tritium transferred is a function of the rate constant for torsional equilibration about the epsilon-amino group of Lys-258, the presumptive abstractor of the C alpha proton; the relative rate constants for hydrogen exchange with solvent versus cofactor protonation; and the tritium isotope effect on this ratio. The free energy barriers facing the covalent intermediate between aldimine and keto acid product (i.e., ketimine and possibly quinonoid) were evaluated relatively by comparing the rates of C alpha-hydrogen exchange in starting amino acid with the rates of keto acid formation. The value of theta (= kexge/kprod) was found to be 2.6 for the reaction of cytoplasmic isozyme with aspartate and ca. 0.5 for that of the mitochondrial form with glutamate.« less

Accumulation of 2-Keto-L-Gulonate at 33°C by a Thermotolerant Gluconobacter Oxydans Mutant Obtained by Ion Beam Implantation

NASA Astrophysics Data System (ADS)

Yan, Bing; Xu, An; Zhang, Wan; Zhou, Wei; Wang, Jun; Yao, Jianming; Yu, Zengliang

2006-03-01

To obtain thermotolerant mutants of G. oxydans, which can enhance the transformation rate of L-sorbose to 2-Keto-L-gulonate (2-KLG) at 33oC in a two-step process of vitamin C manufacture, ion beam was used as a mutation source. Gluconobacter oxydans G0 and Bacillus megaterium B0 were used in this study. The original strain Gluconobacter oxydans G0 was mutated by the heavy ion implantation facility at the Institute of Plasma Physics, Chinese Academy of Sciences. Several mutants including Gluconobacter oxydans GI13 were isolated and cocultured with Bacillus megaterium B0 at 33oC in shaking flasks. The average transformation rate of the new mixed strain GI13-B0 in per gram-molecule reached 94.4% after seven passages in shaking flasks, which was increased by 7% when compared with the original mixed strain G0-B0 (Gluconobacter oxydans G0 and Bacillus megaterium B0). Moreover, the transformation rate of I13B0 was stable at 94% at temperatures ranging from 25oC to 33oC, which would be of much value in reducing energy consumption in the manufacture of L-ascorbic acid, especially in the season of summer. To clarify some mechanism of the mutation, the specific activities of L-sorbose dehydrogenase in both G0 and GI13 were estimated.

Production and characterization of murine models of classic and intermediate maple syrup urine disease

PubMed Central

Homanics, Gregg E; Skvorak, Kristen; Ferguson, Carolyn; Watkins, Simon; Paul, Harbhajan S

2006-01-01

Background Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model. Methods To create a murine model of classic MSUD, we used gene targeting and embryonic stem cell technologies to create a mouse line that lacked a functional E2 subunit gene of branched-chain keto acid dehydrogenase. To create a murine model of intermediate MSUD, we used transgenic technology to express a human E2 cDNA on the knockout background. Mice of both models were characterized at the molecular, biochemical, and whole animal levels. Results By disrupting the E2 subunit gene of branched-chain keto acid dehydrogenase, we created a gene knockout mouse model of classic MSUD. The homozygous knockout mice lacked branched-chain keto acid dehydrogenase activity, E2 immunoreactivity, and had a 3-fold increase in circulating branched-chain amino acids. These metabolic derangements resulted in neonatal lethality. Transgenic expression of a human E2 cDNA in the liver of the E2 knockout animals produced a model of intermediate MSUD. Branched-chain keto acid dehydrogenase activity was 5–6% of normal and was sufficient to allow survival, but was insufficient to normalize circulating branched-chain amino acids levels, which were intermediate between wildtype and the classic MSUD mouse model. Conclusion These mice represent important animal models that closely approximate the phenotype of humans with the classic and intermediate forms of MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease. PMID

Effectiveness and Mechanisms of Antagonism of Toxic Effects of Cyanide by Alpha-Keto Acids.

DTIC Science & Technology

1986-12-31

until the miss-w near death. Lethal blood levels of cyanide in alpha-KG treated animl. as levels of 5-7 mcg cyani0e, which so 5-7 times the expected...lethal levels . rwm these studies, alpha-KC is effettive in antagonising administered dos of CH of five time the lethal dose before the toxic effects are...parameters in the dog .................. 26 Table 6 The effects of cyanide on 2,3 diphosphoglyceric acid .......... 28 Table 7 Stability of solution of ci

Cloning of a Novel Aldo-Keto Reductase Gene from Klebsiella sp. Strain F51-1-2 and Its Functional Expression in Escherichia coli▿

PubMed Central

Jiang, Hong; Yang, Chao; Qu, Hong; Liu, Zheng; Fu, Q. S.; Qiao, Chuanling

2007-01-01

A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P=S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His6-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the Km and kcat values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 ± 47.0 μΜ and 25.7 ± 1.7 min−1, respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling. PMID:17575004

Enhancement of 5-keto-d-gluconate production by a recombinant Gluconobacter oxydans using a dissolved oxygen control strategy.

PubMed

Yuan, Jianfeng; Wu, Mianbin; Lin, Jianping; Yang, Lirong

2016-07-01

The rapid and incomplete oxidation of sugars, alcohols, and polyols by the gram-negative bacterium Gluconobacter oxydans facilitates a wide variety of biological applications. For the conversion of glucose to 5-keto-d-gluconate (5-KGA), a promising precursor of the industrial substance L-(+)-tartaric acid, G. oxydans DSM2343 was genetically engineered to strain ZJU2, in which the GOX1231 and GOX1081 genes were knocked out in a markerless fashion. Then, a secondary alcohol dehydrogenase (GCD) from Xanthomonas campestris DSM3586 was heterologously expressed in G. oxydans ZJU2. The 5-KGA production and cell yield were increased by 10% and 24.5%, respectively. The specific activity of GCD towards gluconate was 1.75±0.02 U/mg protein, which was 7-fold higher than that of the sldAB in G. oxydans. Based on the analysis of kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (qs) and specific 5-KGA production rate (qp), a dissolved oxygen (DO) control strategy was proposed. Finally, batch fermentation was carried out in a 15-L bioreactor using an initial agitation speed of 600 rpm to obtain a high μ for cell growth. Subsequently, DO was continuously maintained above 20% to achieve a high qp to ensure a high accumulation of 5-KGA. Under these conditions, the maximum concentration of 5-KGA reached 117.75 g/L with a productivity of 2.10 g/(L·h). Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Molecular basis of maple syrup urine disease: Novel mutations at the E1[alpha] locus that impair E1([alpha][sub 2][beta][sub 2]) assembly or decrease steady-state E1[alpha] mRNA levels of branched-chain [alpha]-keto acid dehydrogenase complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, J.L.; Fisher, C.R.; Chuang, D.T.

1994-08-01

The authors report the occurrence of three novel mutations in the E1[alpha] (BCKDHA) locus of the branched-chain [alpha]-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1[alpha] gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1[alpha] subunit. Both the 8-bp deletion and the single C insertionmore » generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1[alpha] mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1[alpha] subunit impairs its proper assembly with the normal E1[beta]. Unassembled as well as misassembled E1[alpha] and E1[beta] subunits are degraded in the cell. 32 refs., 8 figs.« less

Application of high-performance liquid chromatography to the determination of glyoxylate synthesis in chick embryo liver.

PubMed

Qureshi, A A; Elson, C E; Lebeck, L A

1982-11-19

The isolation and identification of three major alpha-keto end products (glyoxylate, pyruvate, alpha-ketoglutarate) of the isocitrate lyase reaction in 18-day chick embryo liver have been described. This was accomplished by the separation of these alpha-keto acids as their 2,4-dinitrophenylhydrazones (DNPHs) by high-performance liquid chromatography (HPLC). The DNPHs of alpha-keto acids were eluted with an isocratic solvent system of methanol-water-acetic acid (60:38.5:1.5) containing 5 mM tetrabutylammonium phosphate from a reversed-phase ultrasphere C18 (IP) and from a radial compression C18 column. The separation can be completed on the radial compression column within 15-20 min as compared to 30-40 min with a conventional reversed-phase column. Retention times and peak areas were integrated for both the assay samples and reference compounds. A relative measure of alpha-keto acid in the peak was calculated by comparison with the standard. The identification of each peak was done on the basis of retention time matching, co-chromatography with authentic compounds, and stopped flow UV-VIS scanning between 240 and 440 nm. Glyoxylate represented 5% of the total product of the isocitrate lyase reaction. Day 18 parallels the peak period of embryonic hepatic glycogenesis which occurs at a time when the original egg glucose reserve has been depleted.

Efficacy of the Essential Amino Acids and Keto-Analogues on the CKD progression rate in real practice in Russia - city nephrology registry data for outpatient clinic.

PubMed

Zemchenkov, Alexander; Konakova, Irina N

2016-07-07

Renal replacement therapy (RRT) is growing by 10 % per year in Russia, but pre-dialysis care which can retard CKD progression and delay the start of RRT remains limited. We evaluate the effect of Essential Amino Acids and Keto-analogues (EAA/KA) on CKD progression. The effect of low protein diet (LPD), supplemented by EAA/KA, on GFR slope changes between first and second treatment period (five sequential visits per period) in 96 patients withs CKD Stage 3B-5 was compared to GFR slope changes in the control group of 96 patients, randomly selected from matched (by gender, age, diagnosis and CKD Stage) cohort of 320 patients from the city Registry. The mean baseline eGFR was 23 ± 9 ml/min/1.73 m2; 29 % had CKD3B, 45 % - CKD4, 26 % - CKD5. The rate of eGFR decline changed from -2.71 ± 2.38 to -2.01 ± 2.26 ml/min/1.73 m2 per year in the treatment group and from -2.18 ± 2.01 to -2.04 ± 2.18 ml/min/1.73 m2 per year in the control group. Only in the treatment group the difference was significant (p = 0.04 and p = 0.6). Standardized effect size for intervention was significant in treatment group: -0.3 (of pooled SD), 95 % CI -0.58 ÷  -0.02 and non-significant in control group: -0.07 (-0.35 ÷ +0.22). The univariate and multivariate analysis of EAA/KA therapy effect demonstrated that it was probably more effective in patients of older age, with higher time-averaged proteinuria (PU), lower phosphate level, in patients with glomerular v. interstitial diseases, and in females. Only the latter factor was significant at pre-specified level (<0.05). LPD combined with EAA/KA supplementation lead to the decrease of the CKD progression both in well-designed clinical study and in real nephrology practice in wide variety diseases and settings. Registry data can be helpful to reveal patients with optimal chances for beneficial effect of LPD supplemented by EAA/KA. ISRCTN28190556 06/05/2016.

Two tautomeric forms of 2-amino-5,6-dimethylpyrimidin-4-one.

PubMed

Hall, Victoria M; Bertke, Jeffery A; Swift, Jennifer A

2016-06-01

Derivatives of 4-hydroxypyrimidine are an important class of biomolecules. These compounds can undergo keto-enol tautomerization in solution, though a search of the Cambridge Structural Database shows a strong bias toward the 3H-keto tautomer in the solid state. Recrystallization of 2-amino-5,6-dimethyl-4-hydroxypyrimidine, C6H9N3O, from aqueous solution yielded triclinic crystals of the 1H-keto tautomer, denoted form (I). Though not apparent in the X-ray data, the IR spectrum suggests that small amounts of the 4-hydroxy tautomer are also present in the crystal. Monoclinic crystals of form (II), comprised of a 1:1 ratio of both the 1H-keto and the 3H-keto tautomers, were obtained from aqueous solutions containing uric acid. Forms (I) and (II) exhibit one-dimensional and three-dimensional hydrogen-bonding motifs, respectively.

Pharmacogenetics of aldo-keto reductase 1C (AKR1C) enzymes.

PubMed

Alshogran, Osama Y

2017-10-01

Genetic variation in metabolizing enzymes contributes to variable drug response and disease risk. Aldo-keto reductase type 1C (AKR1C) comprises a sub-family of reductase enzymes that play critical roles in the biotransformation of various drug substrates and endogenous compounds such as steroids. Several single nucleotide polymorphisms have been reported among AKR1C encoding genes, which may affect the functional expression of the enzymes. Areas covered: This review highlights and comprehensively discusses previous pharmacogenetic reports that have examined genetic variations in AKR1C and their association with disease development, drug disposition, and therapeutic outcomes. The article also provides information about the effect of AKR1C genetic variants on enzyme function in vitro. Expert opinion: The current evidence that links the effect of AKR1C gene polymorphisms to disease progression and development is inconsistent and needs further validation, despite of the tremendous knowledge available. Information about association of AKR1C genetic variants and drug efficacy, safety, and pharmacokinetics is limited, thus, future studies that advance our understanding about these relationships and their clinical relevance are needed. It is imperative to achieve consistent findings before the potential translation and adoption of AKR1C genetic variants in clinical practice.

Metabolic characteristics of keto-adapted ultra-endurance runners.

PubMed

Volek, Jeff S; Freidenreich, Daniel J; Saenz, Catherine; Kunces, Laura J; Creighton, Brent C; Bartley, Jenna M; Davitt, Patrick M; Munoz, Colleen X; Anderson, Jeffrey M; Maresh, Carl M; Lee, Elaine C; Schuenke, Mark D; Aerni, Giselle; Kraemer, William J; Phinney, Stephen D

2016-03-01

Many successful ultra-endurance athletes have switched from a high-carbohydrate to a low-carbohydrate diet, but they have not previously been studied to determine the extent of metabolic adaptations. Twenty elite ultra-marathoners and ironman distance triathletes performed a maximal graded exercise test and a 180 min submaximal run at 64% VO2max on a treadmill to determine metabolic responses. One group habitually consumed a traditional high-carbohydrate (HC: n=10, %carbohydrate:protein:fat=59:14:25) diet, and the other a low-carbohydrate (LC; n=10, 10:19:70) diet for an average of 20 months (range 9 to 36 months). Peak fat oxidation was 2.3-fold higher in the LC group (1.54±0.18 vs 0.67±0.14 g/min; P=0.000) and it occurred at a higher percentage of VO2max (70.3±6.3 vs 54.9±7.8%; P=0.000). Mean fat oxidation during submaximal exercise was 59% higher in the LC group (1.21±0.02 vs 0.76±0.11 g/min; P=0.000) corresponding to a greater relative contribution of fat (88±2 vs 56±8%; P=0.000). Despite these marked differences in fuel use between LC and HC athletes, there were no significant differences in resting muscle glycogen and the level of depletion after 180 min of running (-64% from pre-exercise) and 120 min of recovery (-36% from pre-exercise). Compared to highly trained ultra-endurance athletes consuming an HC diet, long-term keto-adaptation results in extraordinarily high rates of fat oxidation, whereas muscle glycogen utilization and repletion patterns during and after a 3 hour run are similar. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Branched-chain amino acids in health and disease: metabolism, alterations in blood plasma, and as supplements.

PubMed

Holeček, Milan

2018-01-01

Branched-chain amino acids (BCAAs; valine, leucine, and isoleucine) are essential amino acids with protein anabolic properties, which have been studied in a number of muscle wasting disorders for more than 50 years. However, until today, there is no consensus regarding their therapeutic effectiveness. In the article is demonstrated that the crucial roles in BCAA metabolism play: (i) skeletal muscle as the initial site of BCAA catabolism accompanied with the release of alanine and glutamine to the blood; (ii) activity of branched-chain keto acid dehydrogenase (BCKD); and (iii) amination of branched-chain keto acids (BCKAs) to BCAAs. Enhanced consumption of BCAA for ammonia detoxification to glutamine in muscles is the cause of decreased BCAA levels in liver cirrhosis and urea cycle disorders. Increased BCKD activity is responsible for enhanced oxidation of BCAA in chronic renal failure, trauma, burn, sepsis, cancer, phenylbutyrate-treated subjects, and during exercise. Decreased BCKD activity is the main cause of increased BCAA levels and BCKAs in maple syrup urine disease, and plays a role in increased BCAA levels in diabetes type 2 and obesity. Increased BCAA concentrations during brief starvation and type 1 diabetes are explained by amination of BCKAs in visceral tissues and decreased uptake of BCAA by muscles. The studies indicate beneficial effects of BCAAs and BCKAs in therapy of chronic renal failure. New therapeutic strategies should be developed to enhance effectiveness and avoid adverse effects of BCAA on ammonia production in subjects with liver cirrhosis and urea cycle disorders. Further studies are needed to elucidate the effects of BCAA supplementation in burn, trauma, sepsis, cancer and exercise. Whether increased BCAA levels only markers are or also contribute to insulin resistance should be known before the decision is taken regarding their suitability in obese subjects and patients with type 2 diabetes. It is concluded that alterations in BCAA

Impact of Branched-Chain Amino Acid Catabolism on Fatty Acid and Alkene Biosynthesis in Micrococcus luteus.

PubMed

Surger, Maximilian J; Angelov, Angel; Stier, Philipp; Übelacker, Maria; Liebl, Wolfgang

2018-01-01

Micrococcus luteus naturally produces alkenes, unsaturated aliphatic hydrocarbons, and represents a promising host to produce hydrocarbons as constituents of biofuels and lubricants. In this work, we identify the genes for key enzymes of the branched-chain amino acid catabolism in M. luteus , whose first metabolic steps lead also to the formation of primer molecules for branched-chain fatty acid and olefin biosynthesis, and demonstrate how these genes can be used to manipulate the production of specific olefins in this organism. We constructed mutants of several gene candidates involved in the branched-chain amino acid metabolism or its regulation and investigated the resulting changes in the cellular fatty acid and olefin profiles by GC/MS. The gene cluster encoding the components of the branched-chain α-keto acid dehydrogenase (BCKD) complex was identified by deletion and promoter exchange mutagenesis. Overexpression of the BCKD gene cluster resulted in about threefold increased olefin production whereas deletion of the cluster led to a drastic reduction in branched-chain fatty acid content and a complete loss of olefin production. The specificities of the acyl-CoA dehydrogenases of the branched amino acid degradation pathways were deduced from the fatty acid and olefin profiles of the respective deletion mutant strains. In addition, growth experiments with branched amino acids as the only nitrogen source were carried out with the mutants in order to confirm our annotations. Both the deletion mutant of the BCKD complex, responsible for the further degradation of all three branched-chain amino acids, as well as the deletion mutant of the proposed isovaleryl-CoA dehydrogenase (specific for leucine degradation) were not able to grow on leucine in contrast to the parental strain. In conclusion, our experiments allow the unambigous assignment of specific functions to the genes for key enzymes of the branched-chain amino acid metabolism of M. luteus . We also show how

Olfactory sensitivity of Pacific Lampreys to lamprey bile acids

USGS Publications Warehouse

Robinson, T. Craig; Sorensen, Peter W.; Bayer, Jennifer M.; Seelye, James G.

2009-01-01

Pacific lampreys Lampetra tridentata are in decline throughout much of their historical range in the Columbia River basin. In support of restoration efforts, we tested whether larval and adult lamprey bile acids serve as migratory and spawning pheromones in adult Pacific lampreys, as they do in sea lampreys Petromyzon marinus. The olfactory sensitivity of adult Pacific lampreys to lamprey bile acids was measured by electro-olfactogram recording from the time of their capture in the spring until their spawning in June of the following year. As controls, we tested L-arginine and a non-lamprey bile acid, taurolithocholic acid 3-sulfate (TLS). Migrating adult Pacific lampreys were highly sensitive to petromyzonol sulfate (a component of the sea lamprey migratory pheromone) and 3-keto petromyzonol sulfate (a component of the sea lamprey sex pheromone) when first captured. This sensitivity persisted throughout their long migratory and overwinter holding period before declining to nearly unmeasurable levels by the time of spawning. The absolute magnitudes of adult Pacific lamprey responses to lamprey bile acids were smaller than those of the sea lamprey, and unlike the sea lamprey, the Pacific lamprey did not appear to detect TLS. No sexual dimorphism was noted in olfactory sensitivity. Thus, Pacific lampreys are broadly similar to sea lampreys in showing sensitivity to the major lamprey bile acids but apparently differ in having a longer period of sensitivity to those acids. The potential utility of bile acid-like pheromones in the restoration of Pacific lampreys warrants their further investigation in this species.

Synthesis and Characterization of 9-Hydroxyphenalenone Using 2D NMR Techniques

ERIC Educational Resources Information Center

Caes, Benjamin; Jensen, Dell, Jr.

2008-01-01

9-Hydroxyphenalenone is a planar multicyclic [beta]-keto-enol, which is synthesized via a Friedel-Crafts acylation followed by acid-catalyzed intramolecular Michael addition with the loss of a phenyl group in a one-pot reaction during a four-hour lab period. Tautomerization of the [beta]-keto-enol results in C[subscript 2v] symmetry on the NMR…

Oxidation of alginate and pectate biopolymers by cerium(IV) in perchloric and sulfuric acid solutions: A comparative kinetic and mechanistic study.

PubMed

Fawzy, Ahmed

2016-03-15

The kinetics of oxidation of alginate (Alg) and pectate (Pec) carbohydrate biopolymers was studied by spectrophotometry in aqueous perchloric and sulfuric acid solutions at fixed ionic strengths and temperature. In both acids, the reactions showed a first order dependence on [Ce(IV)], whereas the orders with respect to biopolymer concentrations are less than unity. In perchloric acid, the reactions exhibited less than unit orders with respect to [H(+)] whereas those proceeded in sulfuric acid showed negative fractional-first order dependences on [H(+)]. The effect of ionic strength and dielectric constant was studied. Probable mechanistic schemes for oxidation reactions were proposed. In both acids, the final oxidation products were characterized as mono-keto derivatives of both biopolymers. The activation parameters with respect to the slow step of the mechanisms were computed and discussed. The rate laws were derived and the reaction constants involved in the different steps of the mechanisms were calculated. Copyright © 2015 Elsevier Ltd. All rights reserved.

A combined experimental and computational study of the mechanism of fructose dehydration to 5-hydroxymethylfurfural in dimethylsulfoxide using Amberlyst 70, PO 4 3-/niobic acid, or sulfuric acid catalysts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jing; Das, Anirban; Assary, Rajeev S.

We report on a combined experimental and theoretical study of the acid catalyzed dehydration of d-fructose in dimethylsulfoxide (DMSO) using; Amberlyst 70, PO 4 3-/niobic acid, and sulfuric acid as catalysts. The reaction has been studied and intermediates characterized using; 13C, 1H, and 17O NMR, and high resolution electrospray ionization mass spectrometry (HR ESI–MS). High level G4MP2 theory calculations are used to understand the thermodynamic landscape for the reaction mechanism in DMSO. We have experimentally identified two key intermediates in the dehydration of fructose to form HMF that were also identified, using theory, as local minima on the potential surfacemore » for reaction. A third intermediate, a species capable of undergoing keto–enol tautomerism, was also experimentally detected. However, it was not possible to experimentally distinguish between the keto and the enol forms. These data with different catalysts are consistent with common intermediates along the reaction pathway from fructose to HMF in DMSO. The role of oxygen in producing acidic species in reactions carried out in DMSO in presence of air is also discussed.« less

Catabolism of 6-ketoprostaglandin F1alpha by the rat kidney cortex.

PubMed

Pace-Asciak, C R; Domazet, Z; Carrara, M

1977-05-25

Homogenates of the rat kidney cortex converted 5,8,9,11,12,14,15-hepta-tritiated 6-ketoprostaglandin F 1alpha into one major product identified by gas chromatography-mass spectrometry of the methoxime-methyl ester trimethylsilyl ether derivative as 6,15-diketo-9,11-dihydroxyprost-13-enoic acid. The sequence of derivatisation i.e. methoximation prior to methylation, was crucial as methylation of 15-keto catabolites of the E, F and 6-keto-F series affords degradation products. The corresponding 15-keto-13,14-dihydro catabolite was formed in much smaller quantities. Time course studies indicated that 6-keto-prostaglandin F1alpha was catabolised at a slower rate (about 2-5 fold) than prostaglandin F1alpha. The catabolic activity was blocked by NADH.

Substituent effect on supramolecular motifs in series of succinimide polycyclic keto derivatives - Spectroscopic, theoretical and crystallographic studies

NASA Astrophysics Data System (ADS)

Miroslaw, Barbara; Koziol, Anna E.; Bielenica, Anna; Dziuba, Kamil; Struga, Marta

2014-09-01

The substituent effect on the supramolecular arrangement in a series of polycyclic monoimide keto derivatives crystals was studied. Single crystal X-ray diffraction and IR spectroscopic experiments were performed for seven related compounds, as well as the Hirshfeld surface analysis and quantum chemical calculations at HF and DFT levels in vacuo, in solution and for small clusters. The presence of Cdbnd O group at the bridge of the main hydrocarbon skeleton implied the catemer motif of the Nimidesbnd H⋯Oimide hydrogen bond in case of smaller substituents (Hsbnd , MeOsbnd , EtOsbnd ). For more voluminous groups (iBuOsbnd ) or additional hydrogen bond acceptors (AcOsbnd , Odbnd ) the steric hindrance increased and the imide⋯imide interactions were no longer present in the solid state. The Nimidesbnd H⋯Oketo or Nimidesbnd H⋯Oester hydrogen bonds were formed instead. The binding energy per one Nsbnd H⋯O interaction calculated for supramolecular clusters at HF/6-31G(d,p) level was ca. 20 kJ mol-1, indicating moderate strength of this hydrogen bond. The solvation free energies and induced dipole moments were computed at B3LYP/6-311+G(d,p) level using the integral equation formalism model (IEF PCM) considering three solvents of various polarity: non-polar chloroform, polar aprotic dimethyl sulfoxide (DMSO) and polar protic water. The relations between the vibrational spectra and the crystal structure have been discussed. The following sequence of carbonyl stretching modes in IR spectra has been derived from quantum chemical calculations: (1) at the highest frequencies - the symmetric vibration of two imide Cdbnd O bonds, (2) the vibrations of keto Cdbnd O bonds attached directly to the polycyclic hydrocarbon skeleton, (3) the asymmetric vibration of two imide Cdbnd O bonds, and (4) at the lowest frequencies - the vibration of ester Cdbnd O group. The characteristic peaks observed in imide experimental IR spectra at about 3080 cm-1 have been

Inhibitory effects of fenretinide metabolites N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR) on fenretinide molecular targets β-carotene oxygenase 1, stearoyl-CoA desaturase 1 and dihydroceramide Δ4-desaturase 1

PubMed Central

Poliakov, Eugenia; Samuel, William; Duncan, Todd; Gutierrez, Danielle B.; Mata, Nathan L.; Redmond, T. Michael

2017-01-01

The therapeutic capacity of fenretinide (N-[4-hydroxyphenyl] retinamide; 4-HPR) has been demonstrated for several conditions, including cancer, obesity, diabetes, and ocular disease. Yet, the mechanisms of action for its pleiotropic effects are still undefined. We hypothesized that investigation of two of the major physiological metabolites of fenretinide, N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR), might begin to resolve the multifaceted effects of this synthetic retinoid. We analyzed the effects of fenretinide, MPR, 3-keto-HPR, and the non-retinoid RBP4 ligand A1120, on the activity of known targets of fenretinide, stearoyl-CoA desaturase 1 (SCD1) and dihydroceramide Δ4-desaturase 1 (DES1) in ARPE-19 cells, and purified recombinant mouse beta-carotene oxygenase 1 (BCO1) in vitro. Lipids and retinoids were extracted and quantified by liquid chromatography-mass spectrometry and reversed phase HPLC, respectively. The data demonstrate that while fenretinide is an inhibitor of the activities of these three enzymes, that 3-keto-HPR is a more potent inhibitor of all three enzymes, potentially mediating most of the in vivo beneficial effects of fenretinide. However, while MPR does not affect SCD1 and DES1 activity, it is a potent specific inhibitor of BCO1. We conclude that a deeper understanding of the mechanisms of action of fenretinide and its metabolites provides new avenues for therapeutic specificity. For example, administration of 3-keto-HPR instead of fenretinide may be preferential if inhibition of SCD1 or DES1 activity is the goal (cancer), while MPR may be better for BCO1 modulation (carotenoid metabolism). Continued investigation of fenretinide metabolites in the context of fenretinide’s various therapeutic uses will begin to resolve the pleotropic nature of this compound. PMID:28448568

A Single Acyl-CoA Dehydrogenase Is Required For Catabolism Of Isoleucine, Valine And Short-Chain Fatty Acids In Aspergillus nidulans

PubMed Central

Maggio-Hall, Lori A.; Lyne, Paul; Wolff, Jon A.; Keller, Nancy P.

2010-01-01

An acyl-CoA dehydrogenase has been identified as part of the mitochondrial β-oxidation pathway in the ascomycete fungus Aspergillus nidulans. Disruption of the scdA gene prevented use of butyric acid (C4) and hexanoic acid (C6) as carbon sources and reduced cellular butyryl-CoA dehydrogenase activity by 7.5-fold. While the mutant strain exhibited wild-type levels of growth on erucic acid (C22:1) and oleic acid (C18:1), some reduction in growth was observed with myristic acid (C14). The ΔscdA mutation was found to be epistatic to a mutation downstream in the β-oxidation pathway (disruption of enoyl-CoA hydratase). The ΔscdA mutant was also unable to use isoleucine or valine as a carbon source. Transcription of scdA was observed in the presence of either fatty acids or amino acids. When the mutant was grown in medium containing either isoleucine or valine, organic acid analysis of culture supernatants showed accumulation of 2-oxo acid intermediates of branched chain amino acid catabolism, suggesting feedback inhibition of the upstream branched-chain α-keto acid dehydrogenase. PMID:17656140

[Suplemented restricted diet in old patients with chronic renal disease].

PubMed

Teplan, Vladimír

2016-01-01

In last decades was confirmed remarkable increase in number of old patients with chronic kidney disease. Despide of developments in dialysis technology and kidney transplantation there is a growing number of old patients who are not suitable for these methods. Recently were published data showing long-term effect of protein restricted diet supplemented with keto amino acids in elderly. Based on our results obtained in re-analysis of 3 000 patients we can confirm also good compliance and low risk of malnutrition.Key words: chronic kidney disease - keto amino acids - old age - restricted diet.

Sequential derivatization of polar organic compounds in cloud water using O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride, N,O-bis(trimethylsilyl)trifluoroacetamide, and gas-chromatography/mass spectrometry analysis.

PubMed

Sagona, Jessica A; Dukett, James E; Hawley, Harmonie A; Mazurek, Monica A

2014-10-03

Cloud water samples from Whiteface Mountain, NY were used to develop a combined sampling and gas chromatography-mass spectrometric (GCMS) protocol for evaluating the complex mixture of highly polar organic compounds (HPOC) present in this atmospheric medium. Specific HPOC of interest were mono- and di keto-acids which are thought to originate from photochemical reactions of volatile unsaturated hydrocarbons from biogenic and manmade emissions and be a major fraction of atmospheric carbon. To measure HPOC mixtures and the individual keto-acids in cloud water, samples first must be derivatized for clean elution and measurement, and second, have low overall background of the target species as validated by GCMS analysis of field and laboratory blanks. Here, we discuss a dual derivatization method with PFBHA and BSTFA which targets only organic compounds that contain functional groups reacting with both reagents. The method also reduced potential contamination by minimizing the amount of sample processing from the field through the GCMS analysis steps. Once derivatized only gas chromatographic separation and selected ion monitoring (SIM) are needed to identify and quantify the polar organic compounds of interest. Concentrations of the detected total keto-acids in individual cloud water samples ranged from 27.8 to 329.3ngmL(-1) (ppb). Method detection limits for the individual HPOC ranged from 0.17 to 4.99ngmL(-1) and the quantification limits for the compounds ranged from 0.57 to 16.64ngmL(-1). The keto-acids were compared to the total organic carbon (TOC) results for the cloud water samples with concentrations of 0.607-3.350mgL(-1) (ppm). GCMS analysis of all samples and blanks indicated good control of the entire collection and analysis steps. Selected ion monitoring by GCMS of target keto-acids was essential for screening the complex organic carbon mixtures present at low ppb levels in cloud water. It was critical for ensuring high levels of quality assurance and

Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase.

PubMed

Sutter, Jan-Moritz; Tästensen, Julia-Beate; Johnsen, Ulrike; Soppa, Jörg; Schönheit, Peter

2016-08-15

The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcanii The work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii The functional involvement of the three enzymes was proven by analyses of the

Duodenal prostaglandin synthesis and acid load in health and in duodenal ulcer disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahlquist, D.A.; Dozois, R.R.; Zinsmeister, A.R.

1983-09-01

We sought to test the hypothesis that duodenal ulcer disease results from an imbalance between duodenal acid load, an injurious force, and mucosal prostaglandin generation, a protective factor. Ten patients with duodenal ulcer and 8 healthy controls were studied. The duodenal acid load after an amino acid soup was quantified by a double-marker technique. Mucosal biopsy specimens were taken endoscopically from the duodenal bulb before and after the test meal. Prostaglandin synthesis activity was measured by incubating biopsy homogenates in excess (/sup 14/C)arachidonic acid. Although mean duodenal acid load was higher in duodenal ulcer, ranges overlapped. Neither the qualitative normore » quantitative profile of mucosal prostaglandin synthesis activities differed significantly between test groups. Prostaglandin synthesis activities, however, tended to increase post cibum in controls, but change little or decrease in duodenal ulcer. Only by comparing the responses with a meal of both parameters together (duodenal acid load and the change in prostaglandin synthesis activities) was there complete or nearly complete separation of duodenal ulcer from controls. Greatest discrimination was observed with prostacyclin (6-keto-PGF1 alpha). We conclude that in health, mucosal prostaglandin generation in the duodenum is induced post cibum in relation to duodenal acid load; this may be a physiologic example of adaptive cytoprotection. In duodenal ulcer there may be a defect in such a mechanism.« less

Medium-chain dehydrogenase/reductase and aldo-keto reductase scavenge reactive carbonyls in Synechocystis sp. PCC 6803.

PubMed

Shimakawa, Ginga; Kohara, Ayaka; Miyake, Chikahiro

2018-03-01

Reactive carbonyls (RCs), which are inevitably produced during respiratory and photosynthetic metabolism, have the potential to cause oxidative damage to photosynthetic organisms. Previously, we proposed a scavenging model for RCs in the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803). In the current study, we constructed mutants deficient in the enzymes medium-chain dehydrogenase/reductase (ΔMDR) and aldo-keto reductase (ΔAKR) to investigate their contributions to RC scavenging in vivo. We found that treatment with the lipid-derived RC acrolein causes growth inhibition and promotes greater protein carbonylation in ΔMDR, compared with the wild-type and ΔAKR. In both ΔMDR and ΔAKR, photosynthesis is severely inhibited in the presence of acrolein. These results suggest that these enzymes function as part of the scavenging systems for RCs in S. 6803 in vivo. © 2018 Federation of European Biochemical Societies.

[Acetyl-11-keto-beta-boswellic acid and arsenic trioxide regulate the productions and activities of matrix metalloproteinases in human skin fibroblasts and human leukemia cell line THP-1].

PubMed

Liang, Ya-hui; Li, Ping; Zhao, Jing-xia; Liu, Xin; Huang, Qi-fu

2010-11-01

In order to reveal the treatment mechanism of Chinese medicine with the effect of activating blood and resolving putridity, we selected acetyl-11-keto-beta-boswellic acid (AKBA) and arsenic trioxide (ATO), the main monomeric components of frankincense and arsenolite which are two most commonly used Chinese medicine with effect of activating blood and resolving putridity. We combined AKBA and ATO as a compound, and explored its regulatory role in productions and activities of matrix metalloproteinase (MMP)-1, MMP-2 and MMP-9 in human skin fibroblasts (HSFbs) and human acute monocytic leukemia cell line THP-1 in inflammatory state. In order to simulate the inflammatory micro-environment of chronic wounds, we established 3 cell models: HSFb model activated by tumor necrosis factor-alpha (TNF-α), THP-1 cell model activated by phorbol-12-myristate-13-acetate (PMA) and HSFb-THP-1 cell coculture system. AKBA and ATO were cocultured with these cell models. Enzyme-linked immunosorbent assay (ELISA), gelatin zymography assay and reverse transcription-polymerase chain reaction (RT-PCR) were used to test the secretions, activities and mRNA expressions of MMP-1, MMP-2 and MMP-9. In the study of the regulatory mechanism of AKBA and ATO on MMPs, AKBA and ATO were cocultured with the cell models. ELISA was used to test the secretions of TNF-α and interleukin-1beta (IL-β) and Western blot was used to test the phosphorylation levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated proteinkinase (p38MAPK). Compound of AKBA and ATO inhibited MMP-1, MMP-2 and MMP-9 mRNA expressions, secretions and activities respectively in HSFbs and THP-1 cells in inflammatory state (P<0.05, P<0.01). Also compound of AKBA and ATO inhibited secretions of TNF-α and IL-1β in THP-1 cells and cell coculture system (P<0.01). It also decreased the phosphorylation of ERK1/2 and p38 MAPK in HSFbs and THP-1 cells (P<0.05, P<0.01). The combined use of AKBA and ATO which

Synthesis of new C-25 and C-26 steroidal acids as potential ligands of the nuclear receptors DAF-12, LXR and GR.

PubMed

Dansey, María V; Del Fueyo, María C; Veleiro, Adriana S; Di Chenna, Pablo H

2017-05-01

A new methodology to obtain C-25 and C-26 steroidal acids starting from pregnenolone is described. Construction of the side chain was achieved by applying the Mukaiyama aldol reaction with a non-hydrolytic work-up to isolate the trapped silyl enol ether with higher yields. Using this methodology we synthesized three new steroidal acids as potential ligands of DAF-12, Liver X and Glucocorticoid nuclear receptors and studied their activity in reporter gene assays. Our results show that replacement of the 21-CH 3 by a 20-keto group in the side chains of the cholestane scaffold of DAF-12 or Liver X receptors ligands causes the loss of the activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Production of C4 and C5 branched-chain alcohols by engineered Escherichia. coli.

PubMed

Chen, Xiaoyan; Xu, Jingliang; Yang, Liu; Yuan, Zhenhong; Xiao, Shiyuan; Zhang, Yu; Liang, Cuiyi; He, Minchao; Guo, Ying

2015-11-01

Higher alcohols, longer chain alcohols, contain more than 3 carbon atoms, showed close energy advantages as gasoline, and were considered as the next generation substitution for chemical fuels. Higher alcohol biosynthesis by native microorganisms mainly needs gene expression of heterologous keto acid decarboxylase and alcohol dehydrogenases. In the present study, branched-chain α-keto acid decarboxylase gene from Lactococcus lactis subsp. lactis CICC 6246 (Kivd) and alcohol dehydrogenases gene from Zymomonas mobilis CICC 41465 (AdhB) were transformed into Escherichia coli for higher alcohol production. SDS-PAGE results showed these two proteins were expressed in the recombinant strains. The resulting strain was incubated in LB medium at 37 °C in Erlenmeyer flasks and much more 3-methyl-1-butanol (104 mg/L) than isobutanol (24 mg/L) was produced. However, in 5 g/L glucose-containing medium, the production of two alcohols was similar, 156 and 161 mg/L for C4 (isobutanol) and C5 (3-methyl-1-butanol) alcohol, respectively. Effects of fermentation factors including temperature, glucose content, and α-keto acid on alcohol production were also investigated. The increase of glucose content and the adding of α-keto acids facilitated the production of C4 and C5 alcohols. The enzyme activities of pure Kivd on α-ketoisovalerate and α-ketoisocaproate were 26.77 and 21.24 μmol min(-1) mg(-1), respectively. Due to its ability on decarboxylation of α-ketoisovalerate and α-ketoisocaproate, the recombinant E. coli strain showed potential application on isoamyl alcohol and isobutanol production.

Control of light-dependent keto carotenoid biosynthesis in Nostoc 7120 by the transcription factor NtcA.

PubMed

Sandmann, Gerhard; Mautz, Jürgen; Breitenbach, Jürgen

2016-09-01

In Nostoc PCC 7120, two different ketolases, CrtW and CrtO are involved in the formation of keto carotenoids from β-carotene. In contrast to other cyanobacteria, CrtW catalyzes the formation of monoketo echinenone whereas CrtO is the only enzyme for the synthesis of diketo canthaxanthin. This is the major photo protective carotenoid in this cyanobacterium. Under high-light conditions, basic canthaxanthin formation was transcriptionally up-regulated. Upon transfer to high light, the transcript levels of all investigated carotenogenic genes including those coding for phytoene synthase, phytoene desaturase and both ketolases were increased. These transcription changes proceeded via binding of the transcription factor NtcA to the promoter regions of the carotenogenic genes. The binding was absolutely dependent on the presence of reductants and oxo-glutarate. Light-stimulated transcript formation was inhibited by DCMU. Therefore, photosynthetic electron transport is proposed as the sensor for high-light and a changing redox state as a signal for NtcA binding.

Enzyme-Inspired Chiral Secondary-Phosphine-Oxide Ligand with Dual Noncovalent Interactions for Asymmetric Hydrogenation.

PubMed

Chen, Caiyou; Zhang, Zhefan; Jin, Shicheng; Fan, Xiangru; Geng, Mingyu; Zhou, Yan; Wen, Songwei; Wang, Xinrui; Chung, Lung Wa; Dong, Xiu-Qin; Zhang, Xumu

2017-06-06

Inspired by the unique character of enzymes, we developed novel chiral SPO (secondary-phosphine-oxide) ligand (SPO-Wudaphos) which can enter into both ion pair and H-bond noncovalent interactions. The novel chiral SPO-Wudaphos exhibited excellent results in the asymmetric hydrogenation of α-methylene-γ-keto carboxylic acids, affording the chiral γ-keto acids with up to over 99 % ee. A series of control experiments and DFT calculations were conducted to illustrate the critical roles of both the ion pair and H-bond noncovalent interactions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Aldo-Keto Reductases as Early Biomarkers of Hepatocellular Carcinoma: A Comparison Between Animal Models and Human HCC.

PubMed

Torres-Mena, Julia Esperanza; Salazar-Villegas, Karla Noemí; Sánchez-Rodríguez, Ricardo; López-Gabiño, Belém; Del Pozo-Yauner, Luis; Arellanes-Robledo, Jaime; Villa-Treviño, Saúl; Gutiérrez-Nava, María Angélica; Pérez-Carreón, Julio Isael

2018-04-01

The intrinsic heterogeneity of hepatocellular carcinoma (HCC) represents a great challenge for its molecular classification and for detecting predictive biomarkers. Aldo-keto reductase (Akr) family members have shown differential expression in human HCC, while AKR1B10 overexpression is considered a biomarker; AKR7A3 expression is frequently reduced in HCC. To investigate the time-course expression of Akr members in the experimental hepatocarcinogenesis. Using DNA-microarray data, we analyzed the time-course gene expression profile from nodules to tumors (4-17 months) of 17 Akr members induced by the resistant hepatocyte carcinogenesis model in the rat. The expression of six members (Akr1c19, Akr1b10, Akr7a3, Akr1b1, Akr1cl1, and Akr1b8) was increased, comparable to that of Ggt and Gstp1, two well-known liver cancer markers. In particular, Akr7a3 and Akr1b10 expression also showed a time-dependent increment at mRNA and protein levels in a second hepatocarcinogenesis model induced with diethylnitrosamine. We confirmed that aldo-keto reductases 7A3 and 1B10 were co-expressed in nine biopsies of human HCC, independently from the presence of glypican-3 and cytokeratin-19, two well-known HCC biomarkers. Because it has been suggested that expression of Akr members is regulated through NRF2 activity at the antioxidant response element (ARE) sequences, we searched and identified at least two ARE sites in Akr1b1, Akr1b10, and Akr7a3 from rat and human gene sequences. Moreover, we observed higher NRF2 nuclear translocation in tumors as compared with non-tumor tissues. Our results demonstrate that Akr7a3 mRNA and protein levels are consistently co-expressed along with Akr1b10, in both experimental liver carcinogenesis and some human HCC samples. These results highlight the presence of AKR7A3 and AKR1B10 from early stages of the experimental HCC and introduce them as a potential application for early diagnosis, staging, and prognosis in human cancer.

Regulation of branched-chain amino acid catabolism in rat models for spontaneous type 2 diabetes mellitus.

PubMed

Kuzuya, Teiji; Katano, Yoshiaki; Nakano, Isao; Hirooka, Yoshiki; Itoh, Akihiro; Ishigami, Masatoshi; Hayashi, Kazuhiko; Honda, Takashi; Goto, Hidemi; Fujita, Yuko; Shikano, Rie; Muramatsu, Yuji; Bajotto, Gustavo; Tamura, Tomohiro; Tamura, Noriko; Shimomura, Yoshiharu

2008-08-15

The branched-chain alpha-keto acid dehydrogenase (BCKDH) complex is the most important regulatory enzyme in branched-chain amino acid (BCAA) catabolism. We examined the regulation of hepatic BCKDH complex activity in spontaneous type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Zucker diabetic fatty rats. Hepatic BCKDH complex activity in these rats was significantly lower than in corresponding control rats. The amount of BCKDH complex in OLETF rats corresponded to the total activity of the complex. Activity and abundance of the bound form of BCKDH kinase, which is responsible for inactivation of the complex, showed an inverse correlation to BCKDH complex activity in OLETF rats. Dietary supplementation of 5% BCAAs for 10 weeks markedly increased BCKDH complex activity, and decreased the activity and bound form of BCKDH kinase in the rats. These results suggest that BCAA catabolism in type 2 diabetes is downregulated and enhanced by BCAA supplementation.

The intra-annular acylamide chelate-coordinated compound: The keto-tautomer of metal (II) milrinone complex

NASA Astrophysics Data System (ADS)

Gong, Yun; Liu, Jinzhi; Tang, Wang; Hu, Changwen

2008-03-01

In the presence of N, N'-dimethyllformamide (DMF), two isostructural metal (II)-milrinone complexes formulated as M(C 12H 8N 3O) 2 (M = Co 1 and Ni 2) have been synthesized and characterized by elemental analysis, IR, TG and single crystal X-ray diffraction. The two compounds crystallize in the tetragonal system, chiral space group P4 32 12. They exhibit similar two dimensional (2D) square grid-like framework, in which milrinone acts as a ditopic ligand with its terminal pyridine and intra-annular acylamide groups covalently bridging different metal centers. The intra-annular acylamide ligand shows a chelate-coordinated mode. Compounds 1 and 2 are stable under 200 °C. Compound 3 formulated as (C 12H 9N 3O) 4·H 2O was obtained in the presence of water, the water molecule in the structure leads to the racemization of compound 3 and it crystallizes in the monoclinic system, non-chiral space group P2 1/ c. Milrinone exhibits a keto-form in the three compounds and compounds 1- 3 exhibit different photoluminescence properties.

An allene oxide and 12-oxophytodienoic acid are key intermediates in jasmonic acid biosynthesis by Fusarium oxysporum.

PubMed

Oliw, Ernst H; Hamberg, Mats

2017-08-01

Fungi can produce jasmonic acid (JA) and its isoleucine conjugate in large quantities, but little is known about the biosynthesis. Plants form JA from 18:3 n -3 by 13 S -lipoxygenase (LOX), allene oxide synthase, and allene oxide cyclase. Shaking cultures of Fusarium oxysporum f. sp. tulipae released over 200 mg of jasmonates per liter. Nitrogen powder of the mycelia expressed 10 R -dioxygenase-epoxy alcohol synthase activities, which was confirmed by comparison with the recombinant enzyme. The 13 S -LOX of F. oxysporum could not be detected in the cell-free preparations. Incubation of mycelia in phosphate buffer with [17,17,18,18,18- 2 H 5 ]18:3 n -3 led to biosynthesis of a [ 2 H 5 ]12-oxo-13-hydroxy-9 Z ,15 Z -octadecadienoic acid (α-ketol), [ 2 H 5 ]12-oxo-10,15 Z -phytodienoic acid (12-OPDA), and [ 2 H 5 ]13-keto- and [ 2 H 5 ]13 S -hydroxyoctadecatrienoic acids. The α-ketol consisted of 90% of the 13 R stereoisomer, suggesting its formation by nonenzymatic hydrolysis of an allene oxide with 13 S configuration. Labeled and unlabeled 12-OPDA were observed following incubation with 0.1 mM [ 2 H 5 ]18:3 n -3 in a ratio from 0.4:1 up to 47:1 by mycelia of liquid cultures of different ages, whereas 10 times higher concentration of [ 2 H 5 ]13 S -hydroperoxyoctadecatrienoic acid was required to detect biosynthesis of [ 2 H 5 ]12-OPDA. The allene oxide is likely formed by a cytochrome P450 or catalase-related hydroperoxidase. We conclude that F. oxysporum , like plants, forms jasmonates with an allene oxide and 12-OPDA as intermediates. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Clofibric acid stimulates branched-chain amino acid catabolism by three mechanisms.

PubMed

Kobayashi, Rumi; Murakami, Taro; Obayashi, Mariko; Nakai, Naoya; Jaskiewicz, Jerzy; Fujiwara, Yoko; Shimomura, Yoshiharu; Harris, Robert A

2002-11-15

Clofibrate promotes catabolism of branched-chain amino acids by increasing the activity of the branched-chain alpha-keto acid dehydrogenase [BCKDH] complex. Depending upon the sex of the rats, nutritional state, and tissue being studied, clofibrate can affect BCKDH complex activity by three different mechanisms. First, by directly inhibiting BCKDH kinase activity, clofibrate can increase the proportion of the BCKDH complex in the active, dephosphorylated state. This occurs in situations in which the BCKDH complex is largely inactive due to phosphorylation, e.g., in the skeletal muscle of chow-fed rats or in the liver of female rats late in the light cycle. Second, by increasing the levels at which the enzyme components of the BCKDH complex are expressed, clofibrate can increase the total enzymatic activity of the BCKDH complex. This is readily demonstrated in livers of rats fed a low-protein diet, a nutritional condition that induces a decrease in the level of expression of the BCKDH complex. Third, by decreasing the amount of BCKDH kinase expressed and therefore its activity, clofibrate induces an increase in the percentage of the BCKDH complex in the active, dephosphorylated state. This occurs in the livers of rats fed a low-protein diet, a nutritional condition that causes inactivation of the BCKDH complex due to upregulation of the amount of BCKDH kinase. WY-14,643, which, like clofibric acid, is a ligand for the peroxisome-proliferator-activated receptor alpha [PPARalpha], does not directly inhibit BCKDH kinase but produces the same long-term effects as clofibrate on expression of the BCKDH complex and its kinase. Thus, clofibrate is unique in its capacity to stimulate BCAA oxidation through inhibition of BCKDH kinase activity, whereas PPARalpha activators in general promote BCAA oxidation by increasing expression of components of the BCKDH complex and decreasing expression of the BCKDH kinase.

Conditional knock-out of lipoic acid protein ligase 1 reveals redundancy pathway for lipoic acid metabolism in Plasmodium berghei malaria parasite.

PubMed

Wang, Min; Wang, Qiong; Gao, Xiang; Su, Zhong

2017-06-27

Lipoic acid is a cofactor for α-keto acid dehydrogenase system that is involved in the central energy metabolism. In the apicomplexan parasite, Plasmodium, lipoic acid protein ligase 1 (LplA1) and LplA2 catalyse the ligation of acquired lipoic acid to the dehydrogenase complexes in the mitochondrion. The enzymes LipB and LipA mediate lipoic acid synthesis and ligation to the enzymes in the apicoplast. These enzymes in the lipoic acid metabolism machinery have been shown to play important roles in the biology of Plasmodium parasites, but the relationship between the enzymes is not fully elucidated. We used an anhydrotetracycline (ATc)-inducible transcription system to generate transgenic P. berghei parasites in which the lplA1 gene was conditionally knocked out (LplA1-cKO). Phenotypic changes and the lplA1 and lplA2 gene expression profiles of cloned LplA1-cKO parasites were analysed. LplA1-cKO parasites showed severely impaired growth in vivo in the first 8 days of infection, and retarded blood-stage development in vitro, in the absence of ATc. However, these parasites resumed viability in the late stage of infection and mounted high levels of parasitemia leading to the death of the hosts. Although lplA1 mRNA expression was regulated tightly by ATc during the whole course of infection, lplA2 mRNA expression was significantly increased in the late stage of infection only in the LplA1-cKO parasites that were not exposed to ATc. The lplA2 gene can be activated as an alternative pathway to compensate for the loss of LplA1 activity and to maintain lipoic acid metabolism.

Decarboxylation of Δ 9-tetrahydrocannabinol: Kinetics and molecular modeling

NASA Astrophysics Data System (ADS)

Perrotin-Brunel, Helene; Buijs, Wim; van Spronsen, Jaap; van Roosmalen, Maaike J. E.; Peters, Cor J.; Verpoorte, Rob; Witkamp, Geert-Jan

2011-02-01

Efficient tetrahydrocannabinol (Δ 9-THC) production from cannabis is important for its medical application and as basis for the development of production routes of other drugs from plants. This work presents one of the steps of Δ 9-THC production from cannabis plant material, the decarboxylation reaction, transforming the Δ 9-THC-acid naturally present in the plant into the psychoactive Δ 9-THC. Results of experiments showed pseudo-first order reaction kinetics, with an activation barrier of 85 kJ mol -1 and a pre-exponential factor of 3.7 × 10 8 s -1. Using molecular modeling, two options were identified for an acid catalyzed β-keto acid type mechanism for the decarboxylation of Δ 9-THC-acid. Each of these mechanisms might play a role, depending on the actual process conditions. Formic acid proved to be a good model for a catalyst of such a reaction. Also, the computational idea of catalysis by water to catalysis by an acid, put forward by Li and Brill, and Churchev and Belbruno was extended, and a new direct keto-enol route was found. A direct keto-enol mechanism catalyzed by formic acid seems to be the best explanation for the observed activation barrier and the pre-exponential factor of the decarboxylation of Δ 9-THC-acid. Evidence for this was found by performing an extraction experiment with Cannabis Flos. It revealed the presence of short chain carboxylic acids supporting this hypothesis. The presented approach is important for the development of a sustainable production of Δ 9-THC from the plant.

An Iron 13S-Lipoxygenase with an α-Linolenic Acid Specific Hydroperoxidase Activity from Fusarium oxysporum

PubMed Central

Brodhun, Florian; Cristobal-Sarramian, Alvaro; Zabel, Sebastian; Newie, Julia; Hamberg, Mats; Feussner, Ivo

2013-01-01

Jasmonates constitute a family of lipid-derived signaling molecules that are abundant in higher plants. The biosynthetic pathway leading to plant jasmonates is initiated by 13-lipoxygenase-catalyzed oxygenation of α-linolenic acid into its 13-hydroperoxide derivative. A number of plant pathogenic fungi (e.g. Fusarium oxysporum) are also capable of producing jasmonates, however, by a yet unknown biosynthetic pathway. In a search for lipoxygenase in F. oxysporum, a reverse genetic approach was used and one of two from the genome predicted lipoxygenases (FoxLOX) was cloned. The enzyme was heterologously expressed in E. coli, purified via affinity chromatography, and its reaction mechanism characterized. FoxLOX was found to be a non-heme iron lipoxygenase, which oxidizes C18-polyunsaturated fatty acids to 13S-hydroperoxy derivatives by an antarafacial reaction mechanism where the bis-allylic hydrogen abstraction is the rate-limiting step. With α-linolenic acid as substrate FoxLOX was found to exhibit a multifunctional activity, because the hydroperoxy derivatives formed are further converted to dihydroxy-, keto-, and epoxy alcohol derivatives. PMID:23741422

11β-Hydroxysteroid dehydrogenase type 1 contributes to the balance between 7-keto- and 7-hydroxy-oxysterols in vivo.

PubMed

Mitić, Tijana; Shave, Steven; Semjonous, Nina; McNae, Iain; Cobice, Diego F; Lavery, Gareth G; Webster, Scott P; Hadoke, Patrick W F; Walker, Brian R; Andrew, Ruth

2013-07-01

11β-Hydroxysteroid dehydrogenase 1 (11βHSD1; EC 1.1.1.146) generates active glucocorticoids from inert 11-keto metabolites. However, it can also metabolize alternative substrates, including 7β-hydroxy- and 7-keto-cholesterol (7βOHC, 7KC). This has been demonstrated in vitro but its consequences in vivo are uncertain. We used genetically modified mice to investigate the contribution of 11βHSD1 to the balance of circulating levels of 7KC and 7βOHC in vivo, and dissected in vitro the kinetics of the interactions between oxysterols and glucocorticoids for metabolism by the mouse enzyme. Circulating levels of 7KC and 7βOHC in mice were 91.3±22.3 and 22.6±5.7 nM respectively, increasing to 1240±220 and 406±39 nM in ApoE(-/-) mice receiving atherogenic western diet. Disruption of 11βHSD1 in mice increased (p<0.05) the 7KC/7βOHC ratio in plasma (by 20%) and also in isolated microsomes (2 fold). The 7KC/7βOHC ratio was similarly increased when NADPH generation was restricted by disruption of hexose-6-phosphate dehydrogenase. Reduction and oxidation of 7-oxysterols by murine 11βHSD1 proceeded more slowly and substrate affinity was lower than for glucocorticoids. in vitro 7βOHC was a competitive inhibitor of oxidation of corticosterone (Ki=0.9 μM), whereas 7KC only weakly inhibited reduction of 11-dehydrocorticosterone. However, supplementation of 7-oxysterols in cultured cells, secondary to cholesterol loading, preferentially slowed reduction of glucocorticoids, rather than oxidation. Thus, in mouse, 11βHSD1 influenced the abundance and balance of circulating and tissue levels of 7βOHC and 7KC, promoting reduction of 7KC. In health, 7-oxysterols are unlikely to regulate glucocorticoid metabolism. However, in hyperlipidaemia, 7-oxysterols may inhibit glucocorticoid metabolism and modulate signaling through corticosteroid receptors. Copyright © 2013 Elsevier Inc. All rights reserved.

6-Iso-chlortetracycline or keto form of chlortetracycline? Need for clarification for relevant monitoring of chlortetracycline residues in food.

PubMed

Gaugain, Murielle; Gautier, Sophie; Bourcier, Sophie; Jacques, Anne-Marie; Laurentie, Michel; Abjean, Jean-Pierre; Hurtaud-Pessel, Dominique; Verdon, Eric

2015-01-01

Chlortetracycline (CTC) is a broad-spectrum antibiotic used in veterinary medicine for pulmonary or digestive infections and having a regulatory maximum residue limit (MRL) necessitating an official analytical control method. The purpose of this study was to clarify the identification of different forms of CTC observed in standard solution, in spiked muscle samples and in naturally incurred muscle samples of pigs analysed by LC-MS/MS and to demonstrate the in vivo formation of 6-iso-chlortetracycline and 4-epi-6-iso-CTC as a metabolite of CTC and 4-epi-CTC in muscle. The six following forms were identified, all being isobaric with a protonated molecule at m/z 479 (precursor ion): the keto-enol forms of CTC and the keto-enol forms of 4-epi-chlortetracycline (4-epi-CTC), 6-iso-chlortetracycline (6-iso-CTC) and 4-epi-6-iso-chlortetracycline (4-epi-6-iso-CTC). The 6-iso-CTC and 4-epi-6-iso-CTC were observed only in incurred pig samples so were identified for the first time as metabolites of CTC and 4-epi-CTC. Identification of the different forms was obtained by comparing incurred muscle samples with standard solutions and with spiked samples. Then the differences between the features of the chromatograms obtained by LC-TQ-MS and the fragmentation study of the different forms of CTC obtained by LC-Q-TOF-MS helped us to support this identification. The extraction steps and the LC-MS/MS conditions developed to analyse muscle tissue samples are described. This clarification concerning the rigorous identification of chromatographic peaks allowed us to evaluate the relevance of our monitoring method with regard to the regulations in place in the European Union and could be of help to laboratories involved in official control of antibiotic residues in food of animal origin. Additional results are also presented highlighting the transformation of the CTC when prepared in a mixture with other antibiotics.

UDP-4-Keto-6-Deoxyglucose, a Transient Antifungal Metabolite, Weakens the Fungal Cell Wall Partly by Inhibition of UDP-Galactopyranose Mutase

PubMed Central

Ma, Liang; Salas, Omar; Bowler, Kyle

2017-01-01

ABSTRACT Can accumulation of a normally transient metabolite affect fungal biology? UDP-4-keto-6-deoxyglucose (UDP-KDG) represents an intermediate stage in conversion of UDP-glucose to UDP-rhamnose. Normally, UDP-KDG is not detected in living cells, because it is quickly converted to UDP-rhamnose by the enzyme UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase (ER). We previously found that deletion of the er gene in Botrytis cinerea resulted in accumulation of UDP-KDG to levels that were toxic to the fungus due to destabilization of the cell wall. Here we show that these negative effects are at least partly due to inhibition by UDP-KDG of the enzyme UDP-galactopyranose mutase (UGM), which reversibly converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). An enzymatic activity assay showed that UDP-KDG inhibits the B. cinerea UGM enzyme with a Ki of 221.9 µM. Deletion of the ugm gene resulted in strains with weakened cell walls and phenotypes that were similar to those of the er deletion strain, which accumulates UDP-KDG. Galf residue levels were completely abolished in the Δugm strain and reduced in the Δer strain, while overexpression of the ugm gene in the background of a Δer strain restored Galf levels and alleviated the phenotypes. Collectively, our results show that the antifungal activity of UDP-KDG is due to inhibition of UGM and possibly other nucleotide sugar-modifying enzymes and that the rhamnose metabolic pathway serves as a shunt that prevents accumulation of UDP-KDG to toxic levels. These findings, together with the fact that there is no Galf in mammals, support the possibility of developing UDP-KDG or its derivatives as antifungal drugs. PMID:29162710

Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis: purification and characterization of CDP-4-keto-6-deoxy-D-glucose-3-dehydrase.

PubMed

Weigel, T M; Liu, L D; Liu, H W

1992-02-25

CDP-4-keto-6-deoxy-D-glucose-3-dehydrase (E1) is a PMP-dependent enzyme which plays an essential role in C-O bond cleavage leading to the formation of 3,6-dideoxyhexoses. Although E1 catalysis has long been recognized as a unique biological deoxygenation reaction, the catalytic mechanism of this unusual enzyme has never been fully elucidated. The lack of methods that would allow this enzyme's activity to be monitored directly has been an impediment to E1 purification and has consequently hampered the mechanistic studies. In order to circumvent this problem, we have developed a few convenient and sensitive methods to facilitate the E1 assay. The first method relies on the fact that E1-catalyzed dehydration is initiated by a proton abstraction from C-4' of the PMP-substrate adduct. By using a tritium-labeled cofactor in the incubation that was later quenched with charcoal, the amount of E1 present could be determined from the amount of released tritium in the supernatant. The second method was designed on the basis of the expectation that E1 will bind and rupture the C-F bond of a substrate analogue, CDP-4-keto-3,6-dideoxy-3-fluoro-D-glucose, which was derived from CDP-3-deoxy-3-fluoro-D-glucose. Since the bond length and electronegativity of the C-F group are similar to those of a C-OH group, we anticipated that the proposed compound would be processed by E1, an assumption which was later substantiated. Another assay useful for measuring E1 activity couples the E1 transformation with the subsequent reduction step catalyzed by CDP-6-deoxy-delta 3,4-D-glucoseen reductase (E3) to a thiobarbituric acid (TBA) reaction. Since the condensation product of TBA and malonaldehyde derived from oxidative degradation of the E1/E3 product gave a pink chromophore at 532 nm with a known absorption coefficient, the yield of deoxysugar formation could be directly deduced on the basis of the observed absorbance. The most conclusive evidence confirming the role of E1 was attained by a

Diabetes and branched-chain amino acids: What is the link?

PubMed

Bloomgarden, Zachary

2018-05-01

Branched-chain amino acids (BCAA) have increasingly been studied as playing a role in diabetes, with the PubMed search string "diabetes" AND "branched chain amino acids" showing particular growth in studies of the topic over the past decade (Fig. ). In the Young Finn's Study, BCAA and, to a lesser extent, the aromatic amino acids phenylalanine and tyrosine were associated with insulin resistance (IR) in men but not in women, whereas the gluconeogenic amino acids alanine, glutamine, or glycine, and several other amino acids (i.e. histidine, arginine, and tryptophan) did not show an association with IR. Obesity may track more strongly than metabolic syndrome and diabetes with elevated BCAA. In a study of 1302 people aged 40-79; higher levels of BCAA tracked with older age, male sex, and metabolic syndrome, as well as with obesity, cardiovascular risk, dyslipidemia, hypertension, and uric acid. Medium- and long-chain acylcarnitines, by-products of mitochondrial catabolism of BCAAs, as well as branched-chain keto acids and the BCAA themselves distinguished obese people having versus not having features of IR, and in a study of 898 patients with essential hypertension, the BCAA and tyrosine and phenylalanine were associated with metabolic syndrome and impaired fasting glucose. In a meta-analysis of three genome-wide association studies, elevations in BCAA and, to a lesser extent, in alanine tracked with IR, whereas higher levels of glutamine and glycine were associated with lesser likelihood of IR. Given these associations with IR, it is not surprising that a number of studies have shown higher BCAA levels in people with and prior to development of type 2 diabetes (T2D), although this has particularly been shown in Caucasian and Asian ethnic groups while not appearing to occur in African Americans. Similarly, higher BCAA levels track with cardiovascular disease. [Figure: see text] The metabolism of BCAA involves two processes: (i) a reversible process catalysed by a

A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis.

PubMed

Braga, Daniel; Hoffmeister, Dirk; Nett, Markus

2016-01-01

Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

Prevention of DNA damage by L-carnitine induced by metabolites accumulated in maple syrup urine disease in human peripheral leukocytes in vitro.

PubMed

Mescka, Caroline Paula; Wayhs, Carlos Alberto Yasin; Guerreiro, Gilian; Manfredini, Vanusa; Dutra-Filho, Carlos Severo; Vargas, Carmen Regla

2014-09-15

Maple syrup urine disease (MSUD) is an inherited aminoacidopathy caused by a deficiency in branched-chain α-keto acid dehydrogenase complex activity that leads to the accumulation of the branched-chain amino acids (BCAAs) leucine (Leu), isoleucine, and valine and their respective α-keto-acids, α-ketoisocaproic acid (KIC), α keto-β-methylvaleric acid, and α-ketoisovaleric acid. The major clinical features presented by MSUD patients include ketoacidosis, failure to thrive, poor feeding, apnea, ataxia, seizures, coma, psychomotor delay, and mental retardation; however, the pathophysiology of this disease is poorly understood. MSUD treatment consists of a low protein diet supplemented with a mixture containing micronutrients and essential amino acids but excluding BCAAs. Studies have shown that oxidative stress may be involved in the neuropathology of MSUD, with the existence of lipid and protein oxidative damage in affected patients. In recent years, studies have demonstrated the antioxidant role of L-carnitine (L-Car), which plays a central function in cellular energy metabolism and for which MSUD patients have a deficiency. In this work, we investigated the in vitro effect of Leu and KIC in the presence or absence of L-Car on DNA damage in peripheral whole blood leukocytes using the alkaline comet assay with silver staining and visual scoring. Leu and KIC resulted in a DNA damage index that was significantly higher than that of the control group, and L-Car was able to significantly prevent this damage, mainly that due to KIC. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

González, Laura; García-Huertas, Paola; Triana-Chávez, Omar; García, Gabriela Andrea; Murta, Silvane Maria Fonseca; Mejía-Jaramillo, Ana M

2017-12-01

The improvement of Chagas disease treatment is focused not only on the development of new drugs but also in understanding mechanisms of action and resistance to drugs conventionally used. Thus, some strategies aim to detect specific changes in proteins between sensitive and resistant parasites and to evaluate the role played in these processes by functional genomics. In this work, we used a natural Trypanosoma cruzi population resistant to benznidazole, which has clones with different susceptibilities to this drug without alterations in the NTR I gene. Using 2DE-gel electrophoresis, the aldo-keto reductase and the alcohol dehydrogenase proteins were found up regulated in the natural resistant clone and therefore their possible role in the resistance to benznidazole and glyoxal was investigated. Both genes were overexpressed in a drug sensitive T. cruzi clone and the biological changes in response to these compounds were evaluated. The results showed that the overexpression of these proteins enhances resistance to benznidazole and glyoxal in T. cruzi. Moreover, a decrease in mitochondrial and cell membrane damage was observed, accompanied by a drop in the intracellular concentration of reactive oxygen species after treatment. Our results suggest that these proteins are involved in the mechanism of action of benznidazole. © 2017 John Wiley & Sons Ltd.

Plasma concentrations of 15-keto-13,14-dihydro-PGF-2 alpha, oestrone sulphate, oestradiol-17 beta and progesterone in pregnant guinea-pigs treated with polychlorinated biphenyls.

PubMed

Lundkvist, U; Kindahl, H

1989-09-01

Guinea-pigs treated by gavage with a total dose of 100 mg polychlorinated biphenyls (PCB: Clophen A50) during Days 17-61 of gestation had higher plasma concentrations of 15-keto-13,14-dihydroprostaglandin F-2 alpha, oestrone sulphate and oestradiol-17 beta during the later stages of gestation than did vehicle-treated guinea-pigs. No changes were observed in plasma progesterone concentrations. Our results provide no support for the hypothesis that an enzyme-induced decrease in progesterone concentrations is the main cause of the fetal death observed in PCB-treated guinea-pigs.

Metabolites of arachidonic acid formed by human gastrointestinal tissues and their actions on the muscle layers.

PubMed Central

Bennett, A.; Hensby, C. N.; Sanger, G. J.; Stamford, I. F.

1981-01-01

1. Gas chromatography-mass spectrometry demonstrated the presence of arachidonic acid (AA), 6-keto-prostaglandin F1 alpha and thromboxane B2 (TxB2) in all extracts of homogenized muscle or mucosa from human stomach, terminal ileum or sigmoid colon. Prostaglandin D2 (PGD2), PGE2 or PGF2 alpha were usually found more often in the mucosal extracts. The 12-hydroxy-derivative of AA (12-HETE) was detected in all extracts of the colon but in only some of the other tissues. 2. Most prostanoids tested contracted the longitudinal muscle, the order of potency being U-46619 (an epoxymethano analogue of PGH2) greater than PGE2 greater than PGF2 alpha greater than PGD2; PGI2 usually caused relaxation, whereas its breakdown products or TxB2 had weak and variable effects. 3. U-46619 or, less potently, PGF2 alpha contracted the circular muscle, whereas PGI2 and usually PGE2 caused relaxation. PGD2, 6-keto-PGF1 alpha, 6,15-diketo-PGF1 alpha or TxB2 usually had little or no effect. 4. PGI2 antagonized contractions to some excitatory prostanoids, without greatly affecting contractions to acetylcholine. 5. For both muscle layers there was a gradient in sensitivity to prostanoids along the gastrointestinal tract. The sensitivities were stomach greater than distal ileum greater than sigmoid colon. 6. The results are discussed in relation to gastrointestinal physiology and pathophysiology. PMID:7317691

Acetolactate metabolism and the presence of a dehydroxy acid dehydratase in micro-organisms

PubMed Central

Wixom, R. L.

1965-01-01

1. The growth characteristics of nine micro-organisms on complex broth and defined media, usually with a single nitrogen source (other than vitamins), were examined as a necessary step before growth of cells for enzyme assays. Six of these bacteria gave a positive colour test with a creatine–potassium hydroxide reagent, indicating the presence of acetoin, which other investigators have shown is formed via the intermediate, α-acetolactate. 2. Cell-free extracts of exponential-phase cells of Bacillus subtilis, Staphylococcus aureus, Proteus morganii, Acetobacter rancens (two strains), A. kuetzingianus, A. acetosus, Acetomonas (Acetobacter) melanogenus and Acetomonas (Acetobacter) suboxydans (A.T.C.C. no. 621) were found to contain the enzyme, dihydroxy acid dehydratase (2,3-dihydroxy acid hydro-lyase). 3. The specific activity of the dehydratase from organisms grown on valine- and isoleucine-deficient media was greater than those grown on a complex broth or media containing complete amino acid mixtures. The omission of valine plus isoleucine from a medium containing 19 amino acids caused an increase in the dehydratase specific activity of Staphylococcus aureus and Proteus morganii. 4. The rate of keto acid formation from αβ-dihydroxyisovalerate by extracts of six of the above-named organisms was faster than, but somewhat proportional to, the similar rate from αβ-dihydroxy-β-methyl-n-valerate as substrate. 5. These findings may be related to acetolactate synthesis, acetoin formation and valine–isoleucine biosynthesis in the above-mentioned micro-organisms. PMID:14348203

Efficient Enzymatic Preparation of (13) N-Labelled Amino Acids: Towards Multipurpose Synthetic Systems.

PubMed

da Silva, Eunice S; Gómez-Vallejo, Vanessa; Baz, Zuriñe; Llop, Jordi; López-Gallego, Fernando

2016-09-12

Nitrogen-13 can be efficiently produced in biomedical cyclotrons in different chemical forms, and its stable isotopes are present in the majority of biologically active molecules. Hence, it may constitute a convenient alternative to Fluorine-18 and Carbon-11 for the preparation of positron-emitter-labelled radiotracers; however, its short half-life demands for the development of simple, fast, and efficient synthetic processes. Herein, we report the one-pot, enzymatic and non-carrier-added synthesis of the (13) N-labelled amino acids l-[(13) N]alanine, [(13) N]glycine, and l-[(13) N]serine by using l-alanine dehydrogenase from Bacillus subtilis, an enzyme that catalyses the reductive amination of α-keto acids by using nicotinamide adenine dinucleotide (NADH) as the redox cofactor and ammonia as the amine source. The integration of both l-alanine dehydrogenase and formate dehydrogenase from Candida boidinii in the same reaction vessel to facilitate the in situ regeneration of NADH during the radiochemical synthesis of the amino acids allowed a 50-fold decrease in the concentration of the cofactor without compromising reaction yields. After optimization of the experimental conditions, radiochemical yields were sufficient to carry out in vivo imaging studies in small rodents. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An aldo-keto reductase, Bbakr1, is involved in stress response and detoxification of heavy metal chromium but not required for virulence in the insect fungal pathogen, Beauveria bassiana.

PubMed

Wang, Huifang; He, Zhangjiang; Luo, Linli; Zhao, Xin; Lu, Zhuoyue; Luo, Tingying; Li, Min; Zhang, Yongjun

2018-02-01

The aldo-keto reductases (AKRs) belong to the NADP-dependent oxidoreductase superfamily, which play important roles in various physiological functions in prokaryotic and eukaryotic organisms. However, many AKR superfamily members remain uncharacterized. Here, a downstream target gene of the HOG1 MAPK pathways coding for an aldo-keto reductase, named Bbakr1, was characterized in the insect fungal pathogen, Beauveria bassiana. Bbakr1 expression increased in response to osmotic and salt stressors, and oxidative and heavy metal (chromium) stress. Deletion of Bbakr1 caused a reduction in conidiation, as well as delayed conidial germination. ΔBbakr1 displayed increased sensitivity to osmotic/high-salt stress with decreased compatible solute accumulation. In addition, the mutant was more sensitive to high concentrations of the heavy metal, chromium, and to oxidative stress than the wild type cells, with impaired ability to detoxify active aldehyde that might accumulate due to lipid peroxidation. However, over-expressing Bbakr1 in either the wild type strain or a ΔBbhog1 background did not cause any obvious changes in phenotypes as compared to their controls. Little effect on virulence was seen for either the ΔBbakr1 or overexpression strains in insect bioassays via cuticle infection or intrahemocoel injection assays, suggesting that Bbakr1 is not required for virulence. Copyright © 2018 Elsevier Inc. All rights reserved.

Biotechnological applications of acetic acid bacteria.

PubMed

Raspor, Peter; Goranovic, Dusan

2008-01-01

products of biotransformations by AAB or their enzymes include 2-keto-L-gulonic acid, which is used for the production of vitamin C; D-tagatose, which is used as a bulking agent in food and a noncalorific sweetener; and shikimate, which is a key intermediate for a large number of antibiotics. Recently, for the first time, a pathogenic acetic acid bacterium was described, representing the newest and tenth genus of AAB.

Activation of RAS/ERK alone is insufficient to inhibit RXRα function and deplete retinoic acid in hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ai-Guo, E-mail: wangaiguotl@hotmail.com; Song, Ya-Nan; Chen, Jun

2014-09-26

Highlights: • The activation of RAS/ERK is insufficient to inhibit RXRα function and deplete RA. • The retinoid metabolism-related genes are down-regulated by ras oncogene. • The atRA has no effect on preventing hepatic tumorigenesis or curing the developed hepatic nodules. - Abstract: Activation of RAS/ERK signaling pathway, depletion of retinoid, and phosphorylation of retinoid X receptor alpha (RXRα) are frequent events found in liver tumors and thought to play important roles in hepatic tumorigenesis. However, the relationships among them still remained to be elucidated. By exploring the transgenic mouse model of hepatic tumorigenesis induced by liver-specific expression of H-ras12Vmore » oncogene, the activation of RAS/ERK, the mRNA expression levels of retinoid metabolism-related genes, the contents of retinoid metabolites, and phosphorylation of RXRα were determined. RAS/ERK signaling pathway was gradually and significantly activated in hepatic tumor adjacent normal liver tissues (P) and hepatic tumor tissues (T) of H-ras12V transgenic mice compared with normal liver tissues (Wt) of wild type mice. On the contrary, the mRNA expression levels of retinoid metabolism-related genes were significantly reduced in P and T compared with Wt. Interestingly, the retinoid metabolites 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (atRA), the well known ligands for nuclear transcription factor RXR and retinoic acid receptor (RAR), were significantly decreased only in T compared with Wt and P, although the oxidized polar metabolite of atRA, 4-keto-all-trans-retinoic-acid (4-keto-RA) was significantly decreased in both P and T compared with Wt. To our surprise, the functions of RXRα were significantly blocked only in T compared with Wt and P. Namely, the total protein levels of RXRα were significantly reduced and the phosphorylation levels of RXRα were significantly increased only in T compared with Wt and P. Treatment of H-ras12V transgenic mice at 5

Elucidating the substrate specificities of acyl-lipid thioesterases from diverse plant taxa.

PubMed

Kalinger, Rebecca S; Pulsifer, Ian P; Rowland, Owen

2018-06-01

Acyl-ACP thioesterase enzymes, which cleave fatty acyl thioester bonds to release free fatty acids, contribute to much of the fatty acid diversity in plants. In Arabidopsis thaliana, a family of four single hot-dog fold domain, plastid-localized acyl-lipid thioesterases (AtALT1-4) generate medium-chain (C6-C14) fatty and β-keto fatty acids as secondary metabolites. These volatile products may serve to attract insect pollinators or deter predatory insects. Homologs of AtALT1-4 are present in all plant taxa, but are nearly all uncharacterized. Despite high sequence identity, AtALT1-4 generate different lipid products, suggesting that ALT homologs in other plants also have highly varied activities. We investigated the catalytic diversity of ALT-like thioesterases by screening the substrate specificities of 15 ALT homologs from monocots, eudicots, a lycophyte, a green microalga, and the ancient gymnosperm Gingko biloba, via expression in Escherichia coli. Overall, these enzymes had highly varied substrate preferences compared to one another and to AtALT1-4, and could be classified into four catalytic groups comprising members from diverse taxa. Group 1 ALTs primarily generated 14:1 β-keto fatty acids, Group 2 ALTs produced 6-10 carbon fatty/β-keto fatty acids, Group 3 ALTs predominantly produced 12-14 carbon fatty acids, and Group 4 ALTs mainly generated 16 carbon fatty acids. Enzymes in each group differed significantly in the quantities of lipids and types of minor products they generated in E. coli. Medium-chain fatty acids are used to manufacture insecticides, pharmaceuticals, and biofuels, and ALT-like proteins are ideal candidates for metabolic engineering to produce specific fatty acids in significant quantities. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

PubMed Central

Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

2012-01-01

Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. PMID:22619179

Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases.

PubMed

Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

2012-08-01

Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. Copyright © 2012 The Protein Society.

Characterization of a dual specificity aryl acid adenylation enzyme with dual function in nikkomycin biosynthesis.

PubMed

Moon, Mary; Van Lanen, Steven G

2010-09-01

Nikkomycin Z is a dipeptide antifungal antibiotic characterized by two nonproteinogenic amino acids, nikkomycin C(Z) and 4-(4'-hydroxy-2'-pyridinyl)-homothreonine (HPHT). The HPHT scaffold is assembled by an aldol reaction between 2-oxobutyrate and picolinaldehyde, the latter of which is derived from picolinic acid that is activated and loaded to coenzyme A by the aryl-activating adenylation enzyme, NikE. We now provide evidence that NikE is also involved in the activation and loading of the alpha-keto acid precursor, 4-(2'-pyridinyl)-2-oxo-4-hydroxyisovalerate (POHIV), to a phosphopantetheinyl group of an acyl carrier protein domain of NikT. POHIV was synthesized using Escherichia coli 2-dehydro-3-deoxy-phosphogluconate aldolase, and phenylalanine dehydrogenase from Bacillus sp. NRRL B-14911 was used to prepare the alpha-amino acid, 4-(2'-pyridinyl)-homothreonine (PHT). Using the carboxylic acid-dependent, ATP-[(32)P]PP(i) exchange assay, NikE is shown to activate both picolinic acid and POHIV but not PHT. Furthermore, NikE loads POHIV to holo-NikT to generate a new thioester-linked intermediate, which was not observed using a NikT(S33A) mutant. Thus, NikE activates two distinct carboxylic acids to form two new thioester intermediates, one of which is subsequently reduced to the aldehyde and the other that likely serves as a substrate for the aminotransferase domain of NikT prior to condensation with nikkomycin C(Z) to yield the dipeptide. Copyright 2010 Wiley Periodicals, Inc.

Preliminary study on health-related lipid components of bakery products.

PubMed

Cercaci, Luisito; Conchillo, Ana; Rodriguez-Estrada, Maria Teresa; Ansorena, Diana; Astiasarán, Iciar; Lercker, Giovanni

2006-06-01

The purpose of this study was to evaluate the presence of health-related lipid components, in particular trans fatty acids and sterol oxidation products, in four bakery products. Both types of components are known for their adverse biological effects, especially the increase of atherogenic risk, and therefore it is advisable to monitor their presence in food products. Trans fatty acids were determined by silver-ion thin-layer chromatography-gas chromatography, whereas sterol oxidation was assessed by gas chromatography and gas chromatography-mass spectrometry determination of 7-keto derivatives (tracers of sterol oxidation). The amount of trans fatty acids (0.02 to 3.13 g/100 g of product), sterols (34.9 to 128.3 mg/100 g of product), and 7-keto derivatives of sterols (1.88 to 3.14 mg/kg of product) varied considerably among samples. The supply of phytosterols (22.5 to 64.0 mg/100 g of product) was not significant, and the extent of oxidation of most phytosterols to its corresponding 7-keto derivative was low (0.29 to 0.84%), except for that of brassicasterol (2.01 to 3.11%). The quality of ingredients and raw materials seems to have greatly influenced the fatty acid profile, stability, safety, and quality of the final product; these ingredients should be chosen with extreme care to decrease their potential negative health effects and to increase safety of these products.

Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches.

PubMed

Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng

2015-10-01

Phenylpyruvic acid (PPA) is an important organic acid that has a wide range of applications. In this study, the membrane-bound L-amino acid deaminase (L-AAD) gene from Proteus mirabilis KCTC 2566 was expressed in Escherichia coli BL21(DE3) and then the L-AAD was purified. After that, we used the purified enzyme and the recombinant E. coli whole-cell biocatalyst to produce PPA via a one-step biotransformation from L-phenylalanine. L-AAD was solubilized from the membrane and purified 52-fold with an overall yield of 13 %, which corresponded to a specific activity of 0.94 ± 0.01 μmol PPA min(-1)·mg(-1). Then, the biotransformation conditions for the pure enzyme and the whole-cell biocatalyst were optimized. The maximal production was 2.6 ± 0.1 g·L(-1) (specific activity of 1.02 ± 0.02 μmol PPA min(-1)·mg(-1) protein, 86.7 ± 5 % mass conversion rate, and 1.04 g·L(-1)·h(-1) productivity) and 3.3 ± 0.2 g L(-1) (specific activity of 0.013 ± 0.003 μmol PPA min(-1)·mg(-1) protein, 82.5 ± 4 % mass conversion rate, and 0.55 g·L(-1)·h(-1) productivity) for the pure enzyme and whole-cell biocatalyst, respectively. Comparative studies of the enzymatic and whole-cell biotransformation were performed in terms of specific activity, production, conversion, productivity, stability, need of external cofactors, and recycling. We have developed two eco-friendly and efficient approaches for PPA production. The strategy described herein may aid the biotransformational synthesis of other α-keto acids from their corresponding amino acids.

Analysis of variability of concentrations of valproic acid (VPA) and its selected metabolites in the blood serum of patients treated with VPA and patients hospitalized because of VPA poisoning.

PubMed

Wilimowska, J; Kłys, M; Jawień, W

2014-01-01

To compare the metabolic profile of valproic acid (VPA) in the studied groups of cases through an analysis of variability of concentrations of VPA with its selected metabolites (2-ene-VPA, 4-ene-VPA, 3-keto-VPA). Blood serum samples collected from 27 patients treated with VPA drugs in the Psychiatry Unit and in the Neurology and Cerebral Strokes Unit at the Ludwik Rydygier Provincial Specialist Hospital in Krakow, and blood serum samples collected from 26 patients hospitalized because of suspected acute VPA poisoning at the Toxicology Department, Chair of Toxicology and Environmental Diseases, Jagiellonian University Medical College in Krakow. The analysis of concentrations of VPA and its selected metabolites has shown that the metabolic profile of VPA determined in cases of acute poisoning is different from cases of VPA therapy. One of VPA's metabolic pathways - the process of desaturation - is unchanged in acute poisoning and prevails over the process of β-oxidation. The ingestion of toxic VPA doses results in an increased formation of 4-ene-VPA, proportional to an increase in VPA concentration. Acute VPA poisoning involves the saturation of VPA's metabolic transformations at the stage of β-oxidation. The process of oxidation of 2-ene-VPA to 3-keto-VPA is slowed down after the ingestion of toxic doses.

Small-Molecule Inhibitor Leads of Ribosome-Inactivating Proteins Developed Using the Doorstop Approach

DTIC Science & Technology

2011-03-01

stereochemistry of R22 used in our virtual screen. R20 or R20b was prepared by coupling 4-formylbenzoic acid with a substituted pyrrole in the keto form for R20 or...a mixture of keto and enol forms for R20b according to a reported process [42] (Figure 4). The substituted pyrrole was obtained via cyclization of 2...1H- pyrrole -3-carboxylate—a close ana- log of R20b—has been reported to have the Z stereochemistry [45]. Therefore, identification of R20 as an active

Effect of alpha-ketoglutarate and oxaloacetate on brain mitochondrial DNA damage and seizures induced by kainic acid in mice.

PubMed

Yamamoto, Hiro-aki; Mohanan, Parayanthala V

2003-07-20

The effects of alpha-ketoglutarate and oxaloacetate on brain mitochondrial DNA (mtDNA) damage and seizures induced by kainic acid were examined both in vivo and in vitro. An intraperitoneal (ip) injection of kainic acid (45 mg/kg) produced broad-spectrum limbic and severe sustained seizures in all of the treated mice. The seizures were abolished when alpha-ketoglutarate (2 g/kg) or oxaloacetate (1 g/kg) was injected intraperitoneally in the animals 1 min before kainic acid administration. In addition, the administration of kainic acid caused damage to mtDNA in brain frontal and middle cortex of mice. These effects were completely abolished by the ip preinjection of alpha-ketoglutarate (2 g/kg) or oxaloacetate (1 g/kg). In vitro exposure of kainic acid (0.25, 0.5 or 1.0 mM) to brain homogenate inflicted damage to mtDNA in a concentration-dependent manner. The damage of mtDNA induced by 1.0 mM kainic acid was attenuated by the co-treatment with alpha-ketoglutarate (2.5 or 5.0 mM) or oxaloacetate (0.75 or 1.0 mM). Furthermore, in vivo and in vitro exposure of kainic acid elicited an increase in lipid peroxidation. However, the increased lipid peroxidation was completely inhibited by cotreatment of alpha-ketoglutarate or oxaloacetate. These results suggest that alpha-keto acids such as alpha-ketoglutarate and oxaloacetate play a role in the inhibition of seizures and subsequent mtDNA damage induced by the excitotoxic/neurotoxic agent, kainic acid.

Amino acid fermentation at the origin of the genetic code.

PubMed

de Vladar, Harold P

2012-02-10

There is evidence that the genetic code was established prior to the existence of proteins, when metabolism was powered by ribozymes. Also, early proto-organisms had to rely on simple anaerobic bioenergetic processes. In this work I propose that amino acid fermentation powered metabolism in the RNA world, and that this was facilitated by proto-adapters, the precursors of the tRNAs. Amino acids were used as carbon sources rather than as catalytic or structural elements. In modern bacteria, amino acid fermentation is known as the Stickland reaction. This pathway involves two amino acids: the first undergoes oxidative deamination, and the second acts as an electron acceptor through reductive deamination. This redox reaction results in two keto acids that are employed to synthesise ATP via substrate-level phosphorylation. The Stickland reaction is the basic bioenergetic pathway of some bacteria of the genus Clostridium. Two other facts support Stickland fermentation in the RNA world. First, several Stickland amino acid pairs are synthesised in abiotic amino acid synthesis. This suggests that amino acids that could be used as an energy substrate were freely available. Second, anticodons that have complementary sequences often correspond to amino acids that form Stickland pairs. The main hypothesis of this paper is that pairs of complementary proto-adapters were assigned to Stickland amino acids pairs. There are signatures of this hypothesis in the genetic code. Furthermore, it is argued that the proto-adapters formed double strands that brought amino acid pairs into proximity to facilitate their mutual redox reaction, structurally constraining the anticodon pairs that are assigned to these amino acid pairs. Significance tests which randomise the code are performed to study the extent of the variability of the energetic (ATP) yield. Random assignments can lead to a substantial yield of ATP and maintain enough variability, thus selection can act and refine the assignments

Enzymatic characterization of a novel bovine liver dihydrodiol dehydrogenase--reaction mechanism and bile acid dehydrogenase activity.

PubMed

Nanjo, H; Adachi, H; Morihana, S; Mizoguchi, T; Nishihara, T; Terada, T

1995-05-11

Bovine liver cytosolic dihydrodiol dehydrogenase (DD3) has been characterized by its unique dihydrodiol dehydrogenase activity for trans-benzenedihydrodiol (trans-1,2-dihydrobenzene-1,2-diol) with the highest affinity and the greatest velocity among three multiple forms of dihydrodiol dehydrogenases (DD1-DD3). It is the first time that DD3 has shown a significant dehydrogenase activity for (S)-(+)-1-indanol with low Km value (0.33 +/- 0.022 mM) and high K(cat) value (25 +/- 0.79 min-1). The investigation of the product inhibition of (S)-(+)-1-indanol with NADP+ versus 1-indanone and NADPH clearly showed that the enzymatic reaction of DD3 may follow a typical ordered Bi Bi mechanism similar to many aldo/keto reductases. Additionally, DD3 was shown to catalyze the dehydrogenation of bile acids (lithocholic acid, taurolithocholic acid and taurochenodeoxycholic acid) having no 12-hydroxy groups with low Km values (17 +/- 0.65, 33 +/- 1.9 and 890 +/- 73 microM, respectively). In contrast, DD1, 3 alpha-hydroxysteroid dehydrogenase, shows a broad substrate specificity for many bile acids with higher affinity than those of DD3. Competitive inhibition of DD3 with androsterone against dehydrogenase activity for (S)-(+)-1-indanol, trans-benzenedihydrodiol or lithocholic acid suggests that these three substrates bind to the same substrate binding site of DD3, different from the case of human liver bile acid binder/dihydrodiol dehydrogenase (Takikawa, H., Stolz, A., Sugiyama, Y., Yoshida, H., Yamamoto, M. and Kaplowitz, N. (1990) J. Biol. Chem. 265, 2132-2136). Considering the reaction mechanism, DD3 may also play an important role in bile acids metabolism as well as the detoxication of aromatic hydrocarbons.

Compositional, Atomic and Molecular Analysis in Support of Materials Needs of the U.S. Air Force.

DTIC Science & Technology

1982-09-01

internally hydrogen-bonded monomer in .which the keto group is involved in the hydrogen bond but the acid carbonyl is not. 3 ) 3 - Bromopyruvic Acid...The spectra and structure of 3 - bromopyruvic acid were investigated and compared to those of pyruvic acid. It has been found that the spectra of 3 ...phase, cyclic monomer in dilute solution). The solid state spectra are quite different, however. The solid states spectra of 3 - bromopyruvic acid show a

Multiple turnovers of the nicotino-enzyme PdxB require α-keto acids as co-substrates

PubMed Central

Rudolph, Johannes; Kim, Juhan; Copley, Shelley D.

2012-01-01

PdxB catalyzes the second step in the biosynthesis of pyridoxal phosphate by oxidizing 4-phospho-D-erythronate (4PE) to 2-oxo-3-hydroxy-4-phospho-butanoate (OHPB) with concomitant reduction of NAD+ to NADH. PdxB is a nicotino-enzyme wherein the NAD(H) cofactor remains tightly bound to PdxB. It has been a mystery how PdxB performs multiple turnovers since addition of free NAD+ does not re-oxidize the enzyme-bound NADH following conversion of 4PE to OHPB. We have solved this mystery by demonstrating that a variety of physiologically available α-ketoacids serve as oxidants of PdxB to sustain multiple turnovers. In a coupled assay using the next two enzymes of the biosynthetic pathway for pyridoxal phosphate (SerC and PdxA), we have found that α-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (kcat/Km values ~ 1 × 104 M-1s-1). The kinetic parameters for the substrate 4PE include a kcat of 1.4 s-1, a Km of 2.9 μM, and a kcat/Km of 6.7 × 106 M-1s-1. Additionally, we have characterized the stereochemistry of α-ketoglutarate reduction by showing that D-2-HGA, but not L-2-HGA, is a competitive inhibitor vs. 4PE and a noncompetitive inhibitor vs. α-ketoglutarate. PMID:20831184

Asymmetric allylation of α-ketoester-derived N-benzoylhydrazones promoted by chiral sulfoxides/N-oxides Lewis bases: highly enantioselective synthesis of quaternary α-substituted α-allyl-α-amino acids.

PubMed

Reyes-Rangel, Gloria; Bandala, Yamir; García-Flores, Fred; Juaristi, Eusebio

2013-09-01

Chiral sulfoxides/N-oxides (R)-1 and (R,R)-2 are effective chiral promoters in the enantioselective allylation of α-keto ester N-benzoylhydrazone derivatives 3a-g to generate the corresponding N-benzoylhydrazine derivatives 4a-g, with enantiomeric excesses as high as 98%. Representative hydrazine derivatives 4a-b were subsequently treated with SmI2, and the resulting amino esters 5a-b with LiOH to obtain quaternary α-substituted α-allyl α-amino acids 6a-b, whose absolute configuration was assigned as (S), with fundament on chemical correlation and electronic circular dichroism (ECD) data. © 2013 Wiley Periodicals, Inc.

Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

DTIC Science & Technology

2011-09-28

presented according to [51]. Kdo, 2- keto -3-deoxy-octulosonic acid; Ko, D-glycero-D-talo-oct-2-ulosonic acid; Hep, heptulose (ketoheptose); Glc, glucose...pestis. J Bacteriol 98: 1404–1406. 54. Jawetz E, Meyer KF (1943) Avirulent strains of Pasteurella pestis. J Infect Dis 73: 124– 143 . 55. Burrows TW

Metabolomic Analysis of the Secretome of Human Embryonic Stem Cells Following Methyl Parathion and Methyl Paraoxon Exposure Phase 2: Metabolite Downselection for Structural Confirmation

DTIC Science & Technology

2013-12-01

degradation 2 Pipecolic acid II 2-keto-6- aminocaproate II Pyruvate metabolism 1 Malic acid I Purine metabolism 1 Guanine I Propanoate metabolism 1...acetamidobutanoic acid II cis-4-hydroxy-D-proline II D-arginine and D-ornithine metabolism 4 Ornithine II 5-amino-2-oxopentanoic acid II 2-amino-4-oxo...pentanoic acid II (2R,4S)-2,4-diaminopentanoate II Gly, Ser, and Thr metabolism 3 L-cystathionine II Choline II 5-aminolevulinic acid II Val

FeS/S/FeS2 Redox System and Its Oxidoreductase-like Chemistry in the Iron-Sulfur World

NASA Astrophysics Data System (ADS)

Wang, Wei; Yang, Bin; Qu, Youpeng; Liu, Xiaoyang; Su, Wenhui

2011-06-01

The iron-sulfur world (ISW) theory is an intriguing prediction regarding the origin of life on early Earth. It hypothesizes that life arose as a geochemical process from inorganic starting materials on the surface of sulfide minerals in the vicinity of deep-sea hot springs. During the last two decades, many experimental studies have been carried out on this topic, and some interesting results have been achieved. Among them, however, the processes of carbon/nitrogen fixation and biomolecular assembly on the mineral surface have received an inordinate amount of attention. To the present, an abiotic model for the oxidation-reduction of intermediates participating in metabolic pathways has been ignored. We examined the oxidation-reduction effect of a prebiotic FeS/S/FeS2 redox system on the interconversion between several pairs of ±-hydroxy acids and ±-keto acids (i.e., lactate/pyruvate, malate/oxaloacetate, and glycolate/glyoxylate). We found that, in the absence of FeS, elemental sulfur (S) oxidized ±-hydroxy acids to form corresponding keto acids only at a temperature higher than its melting point (113°C); in the presence of FeS, such reactions occurred more efficiently through a coupled reaction mechanism, even at a temperature below the phase transition point of S. On the other hand, FeS was shown to have the capacity to reversibly reduce the keto acids. Such an oxidoreductase-like chemistry of the FeS/S/FeS2 redox system suggests that it can determine the redox homeostasis of metabolic intermediates in the early evolutionary phase of life. The results provide a possible pathway for the development of primordial redox biochemistry in the iron-sulfur world.

Urinary biomarkers of oxidative damage in Maple syrup urine disease: the L-carnitine role.

PubMed

Guerreiro, Gilian; Mescka, Caroline Paula; Sitta, Angela; Donida, Bruna; Marchetti, Desirèe; Hammerschmidt, Tatiane; Faverzani, Jessica; Coelho, Daniella de Moura; Wajner, Moacir; Dutra-Filho, Carlos Severo; Vargas, Carmen Regla

2015-05-01

Maple syrup urine disease (MSUD) is a disorder of branched-chain amino acids (BCAA). The defect in the branched-chain α-keto acid dehydrogenase complex activity leads to an accumulation of these compounds and their corresponding α-keto-acids and α-hydroxy-acids. Studies have shown that oxidative stress may be involved in neuropathology of MSUD. L-carnitine (L-car), which has demonstrated an important role as antioxidant by reducing and scavenging free radicals formation and by enhancing the activity of antioxidant enzymes, have been used in the treatment of some metabolic rare disorders. This study evaluated the oxidative stress parameters, di-tyrosine, isoprostanes and antioxidant capacity, in urine of MSUD patients under protein-restricted diet supplemented or not with L-car capsules at a dose of 50 mg kg(-1) day(-1). It was also determined urinary α-keto isocaproic acid levels as well as blood free L-car concentrations in blood. It was found a deficiency of carnitine in patients before the L-car supplementation. Significant increases of di-tyrosine and isoprostanes, as well as reduced antioxidant capacity, were observed before the treatment with L-car. The L-car supplementation induced beneficial effects on these parameters reducing the di-tyrosine and isoprostanes levels and increasing the antioxidant capacity. It was also showed a significant increase in urinary of α-ketoisocaproic acid after 2 months of L-car treatment, compared to control group. In conclusion, our results suggest that L-car may have beneficial effects in the treatment of MSUD by preventing oxidative damage to the cells and that urine can be used to monitorize oxidative damage in patients affected by this disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chemoenzymatic synthesis of differentially protected 3-deoxysugars

NASA Astrophysics Data System (ADS)

Gillingham, Dennis G.; Stallforth, Pierre; Adibekian, Alexander; Seeberger, Peter H.; Hilvert, Donald

2010-02-01

3-Deoxysugars are important constituents of complex carbohydrates. For example, 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) is an essential component of lipopolysaccharides in Gram-negative bacteria, 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN) is widely found in carbohydrates of the bacterial cell wall and in lower vertebrates, and sialic acid is a common cap of mammalian glycoproteins. Although ready access to such sugars would benefit the creation of vaccine candidates, antibiotics and small-molecule drugs, their chemical synthesis is difficult. Here we present a simple chemoenzymatic method for preparing differentially protected 3-deoxysugar derivatives from readily available starting materials. It exploits the promiscuous aldolase activity of the enzyme macrophomate synthase (MPS) to add pyruvate enolate diastereoselectively to a wide range of structurally complex aldehydes. A short synthesis of KDN illustrates the utility of this approach. Enzyme promiscuity, which putatively fosters large functional leaps in natural evolution, has great promise as a source of synthetically useful catalytic transformations.

STUDIES ON MAMMALIAN AND HUMAN PYRUVATE AND ALPHA-KETOGLUTARATE DEHYDROGENATION COMPLEXES

DTIC Science & Technology

bound lipoic acid and 17 moles of bound FAD. Alpha -ketoglutarate dehydrogenase complex contains approximately 10 moles of protein-bound lipoic acid , 9...typical metal activators of oxidative decarboxylation reaction of alpha -keto acid . These activating effects were in good agreement with the results of...A coenzyme A- and NAD-linked pyruvate and alpha -ketoglutarate dehydrogenase complexes have been isolated from pig heart muscle as multienzyme units

Evolution of Enzymatic Activities in the Enolase Superfamily: D-Mannonate Dhydratase from Novosphingobium aromaticivorans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rakus,J.; Fedorov, A.; Fedorov, E.

2007-01-01

The d-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteins in the mechanistically diverse enolase superfamily by screening a library of acid sugars. Structures of the wild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence of Mg2+ and also in the presence of Mg2+ and the 2-keto-3-keto-d-gluconate dehydration product; the structure of the catalytically active K271E mutant was determined at pH 5.5 in the presence of the d-mannonate substrate. As previously observed in the structures of other members of the enolase superfamily, ManD contains two domains, an N-terminal a+{beta} capping domain andmore » a ({beta}/a)7{beta}-barrel domain. The barrel domain contains the ligands for the essential Mg2+, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth {beta}-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second {beta}-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth {beta}-strands, respectively, that are found in many members of the superfamily. Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second {beta}-strand and Arg 147 at the end of the second {beta}-strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr 159 and His 212, at the end of the third {beta}-strand, are positioned to facilitate both syn-dehydration and ketonization of the resulting enol intermediate to yield the 2-keto-3-keto-d-gluconate product with the observed retention of configuration. The identities and locations of these acid/base catalysts as well as of cationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategy for catalysis (subgroup) in the mechanistically diverse enolase superfamily. With these differences, we provide additional evidence that the ligands for the essential Mg2+ are the

Dietary supplementation of branched-chain amino acids increases muscle net amino acid fluxes through elevating their substrate availability and intramuscular catabolism in young pigs.

PubMed

Zheng, Liufeng; Zuo, Fangrui; Zhao, Shengjun; He, Pingli; Wei, Hongkui; Xiang, Quanhang; Pang, Jiaman; Peng, Jian

2017-04-01

Branched-chain amino acids (BCAA) have been clearly demonstrated to have anabolic effects on muscle protein synthesis. However, little is known about their roles in the regulation of net AA fluxes across skeletal muscle in vivo. This study was aimed to investigate the effect and related mechanisms of dietary supplementation of BCAA on muscle net amino acid (AA) fluxes using the hindlimb flux model. In all fourteen 4-week-old barrows were fed reduced-protein diets with or without supplemental BCAA for 28 d. Pigs were implanted with carotid arterial, femoral arterial and venous catheters, and fed once hourly with intraarterial infusion of p-amino hippurate. Arterial and venous plasma and muscle samples were obtained for the measurement of AA, branched-chain α-keto acids (BCKA) and 3-methylhistidine (3-MH). Metabolomes of venous plasma were determined by HPLC-quadrupole time-of-flight-MS. BCAA-supplemented group showed elevated muscle net fluxes of total essential AA, non-essential AA and AA. As for individual AA, muscle net fluxes of each BCAA and their metabolites (alanine, glutamate and glutamine), along with those of histidine, methionine and several functional non-essential AA (glycine, proline and serine), were increased by BCAA supplementation. The elevated muscle net AA fluxes were associated with the increase in arterial and intramuscular concentrations of BCAA and venous metabolites including BCKA and free fatty acids, and were also related to the decrease in the intramuscular concentration of 3-MH. Correlation analysis indicated that muscle net AA fluxes are highly and positively correlated with arterial BCAA concentrations and muscle net BCKA production. In conclusion, supplementing BCAA to reduced-protein diet increases the arterial concentrations and intramuscular catabolism of BCAA, both of which would contribute to an increase of muscle net AA fluxes in young pigs.

Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

PubMed Central

Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

2006-01-01

Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C2221 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å. PMID:17142919

Endothelial cell markers in vascular neoplasms: an immunohistochemical study comparing factor VIII-related antigen, blood group specific antigens, 6-keto-PGF1 alpha, and Ulex europaeus 1 lectin.

PubMed

Little, D; Said, J W; Siegel, R J; Fealy, M; Fishbein, M C

1986-06-01

Markers for endothelial cells including Ulex europaeus 1 lectin, blood group A, B, and H, and the prostaglandin metabolite 6-keto-PGF1 alpha were evaluated in paraffin secretions from formalin-fixed benign and malignant vascular neoplasms using a variety of immunohistochemical techniques, and results compared with staining for factor VIII-related antigen. Staining for Ulex appeared more sensitive than factor VIII-related antigen in identifying poorly differentiated neoplasms including haemangiosarcomas and spindle cell proliferations in Kaposi's sarcoma. Staining for blood group related antigens correlated with blood group in all cases. Ulex europaeus 1 lectin was the only marker for endothelial cells in lymphangiomas.

Determination of endrin and δ-keto endrin in five food products of animal origin using GC-μECD: A modified QuEChERS approach to traditional detection.

PubMed

Rahman, Md Musfiqur; Lee, Han Sol; Abd El-Aty, A M; Kabir, Md Humayun; Chung, Hyung Suk; Park, Jong-Hyouk; Kim, Mi-Ra; Kim, Ji-Hyun; Shin, Ho-Chul; Shin, Sung Shik; Shim, Jae-Han

2018-10-15

A simple quick, easy, cheap, effective, rugged, and safe (QuEChERS)-based method was developed for the analysis of endrin and its metabolite, δ-keto endrin, in five animal-derived food products (chicken, pork, beef, egg, and milk) using a gas chromatography-micro electron capture detector (GC-μECD). Samples were extracted with acidified acetonitrile, salted out with magnesium sulfate and sodium acetate, and finally purified with a dual layer solid-phase extraction cartridge (SPE) that contains both Supelclean ENVI-Carb (upper layer) and primary secondary amine (lower layer) SPE sorbents. A seven-point external calibration curve was constructed both for the solvent and matrix for both compounds. Good linearity was achieved for both analytes, with coefficients of determination (R 2 ) ≥ 0.9960. The limits of detection (LODs) were 0.003 mg/kg, whereas the limits of quantification (LOQ) were 0.01 mg/kg, which were 10 times lower than the extraneous maximum residue limit (EMRL) designated by CODEX Alimentarius for the specified matrices. The method was validated via recovery performances in triplicates, with three fortification levels equivalent to LOQ, 2 × LOQ, and 10 × LOQ. The method provided excellent recoveries, ranging between 75.63 and 117.92%, with relative standard deviations (RSD) ≤ 8.52% for both analytes in various matrices. The developed method was successfully applied to monitor market samples collected from 20 different places throughout the Republic of Korea, and none of the tested analytes were found in the analyzed samples. Conclusively, we could propose that the current method can be used for routine analysis of endrin and δ-keto endrin in any type of fatty food matrix. Copyright © 2018 Elsevier Ltd. All rights reserved.

Composition of fatty acids in plasma and erythrocytes and eicosanoids level in patients with metabolic syndrome

PubMed Central

2011-01-01

Background Disturbances of the fatty acids composition in plasma and red blood cells and eicosanoid synthesis play an important role in the metabolic syndrome (MS) formation. Methods The observation group included 61 people with metabolic syndrome (30 patients with MS and normal levels of insulin, 31 people with MS and insulin resistance - IR). The parameters of carbohydrate and lipid metabolism in blood serum were examined. The composition of nonesterified fatty acids (NEFA), fatty acid (FA) of red blood cells lipids was analyzed by gas-liquid chromatography. Eicosanoids level in MS patients blood serum was studied by enzyme immunoassay. Results In MS patients in the absence of glucose-insulin homeostasis disturbances and in patients with IR the accumulation of polyunsaturated fatty acids (18:2 n6, 18:3 n3, 22:4 n6) and lower pool of saturated FA (12:0, 14:0, 16: 0, 17:0) in plasma were discovered. A deficit of polyunsaturated FA (18:3 n3, 20:4 n6) with a predominance of on-saturated FA (14:0, 18:0) in erythrocyte membranes was revealed. In MS patients regardless of the carbohydrate metabolism status high levels of leukotriene B4 and 6-keto-prostaglandin-F1α in serum were found. The development of IR in MS patients leads to increased synthesis of thromboxane A2. Conclusion The results revealed a disturbance in nonesterified fatty acids of plasma lipids and red blood cells, eicosanoid synthesis in MS patients. The breach of the plasma and cell membranes fatty acids compositions, synthesis of vasoactive and proinflammatory eicosanoids is an important pathogenetic part of the MS development. PMID:21595891

Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide

PubMed Central

Boger, Dale L.; Sato, Haruhiko; Lerner, Aaron E.; Hedrick, Michael P.; Fecik, Robert A.; Miyauchi, Hiroshi; Wilkie, Gordon D.; Austin, Bryce J.; Patricelli, Matthew P.; Cravatt, Benjamin F.

2000-01-01

The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features—an optimal C12–C8 chain length, π-unsaturation introduction at the corresponding arachidonoyl Δ8,9/Δ11,12 and oleoyl Δ9,10 location, and an α-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with Kis that drop below 200 pM and are 102–103 times more potent than the corresponding trifluoromethyl ketones. PMID:10805767

Amino acid fermentation at the origin of the genetic code

PubMed Central

2012-01-01

There is evidence that the genetic code was established prior to the existence of proteins, when metabolism was powered by ribozymes. Also, early proto-organisms had to rely on simple anaerobic bioenergetic processes. In this work I propose that amino acid fermentation powered metabolism in the RNA world, and that this was facilitated by proto-adapters, the precursors of the tRNAs. Amino acids were used as carbon sources rather than as catalytic or structural elements. In modern bacteria, amino acid fermentation is known as the Stickland reaction. This pathway involves two amino acids: the first undergoes oxidative deamination, and the second acts as an electron acceptor through reductive deamination. This redox reaction results in two keto acids that are employed to synthesise ATP via substrate-level phosphorylation. The Stickland reaction is the basic bioenergetic pathway of some bacteria of the genus Clostridium. Two other facts support Stickland fermentation in the RNA world. First, several Stickland amino acid pairs are synthesised in abiotic amino acid synthesis. This suggests that amino acids that could be used as an energy substrate were freely available. Second, anticodons that have complementary sequences often correspond to amino acids that form Stickland pairs. The main hypothesis of this paper is that pairs of complementary proto-adapters were assigned to Stickland amino acids pairs. There are signatures of this hypothesis in the genetic code. Furthermore, it is argued that the proto-adapters formed double strands that brought amino acid pairs into proximity to facilitate their mutual redox reaction, structurally constraining the anticodon pairs that are assigned to these amino acid pairs. Significance tests which randomise the code are performed to study the extent of the variability of the energetic (ATP) yield. Random assignments can lead to a substantial yield of ATP and maintain enough variability, thus selection can act and refine the assignments

Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels.

PubMed

Zhong, Linlin; Liu, Ziwen; Yan, Ruilan; Johnson, Stephen; Zhao, Yupei; Fang, Xiubin; Cao, Deliang

2009-09-18

Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 microM, 4-hydroxynonenal (HNE) at 0.10 microM, trans-2-hexanal at 0.10 microM, and trans-2,4-hexadienal at 0.05 microM, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 microM (toxic) by converting to 1,4-dihydroxynonene, protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.

Anomalous regioselective four-member multicomponent Biginelli reaction II: one-pot parallel synthesis of spiro heterobicyclic aliphatic rings.

PubMed

Byk, Gerardo; Kabha, Eihab

2004-01-01

In a previous preliminary study, we found that a cyclic five-member ring beta-keto ester (lactone) reacts with one molecule of urea and two of aldehyde to give a new family of spiro heterobicyclic aliphatic rings in good yields with no traces of the expected dihydropyrimidine (Biginelli) products. The reaction is driven by a regiospecific condensation of two molecules of aldehyde with urea and beta-keto-gamma-lactone to afford only products harboring substitutions exclusively in a syn configuration (Byk, G.; Gottlieb, H. E.; Herscovici, J.; Mirkin, F. J. Comb. Chem. 2000, 2, 732-735). In the present work ((a) Presented in part at ISCT Combitech, October 15, 2002, Israel, and Eurocombi-2, Copenhagen 2003 (oral and poster presentation). (b) Also in American Peptide Society Symposium, Boston, 2003 (poster presentation). (c) Abstract in Biopolymers 2003, 71 (3), 354-355), we report a large and exciting extension of this new reaction utilizing parallel organic synthesis arrays, as demonstrated by the use of chiral beta-keto-gamma-lactams, derived from natural amino acids, instead of tetronic acid (beta-keto-gamma-lactone) and the potential of the spirobicyclic products for generating "libraries from libraries". Interestingly, we note an unusual and important anisotropy effect induced by perpendicular interactions between rigid pi systems and different groups placed at the alpha position of the obtained spirobicyclic system. Stereo/regioselectivity of the aldehyde condensation is driven by the nature of the substitutions on the starting beta-keto-gamma-lactam. Aromatic aldehydes can be used as starting reagents with good yields; however, when aliphatic aldehydes are used, the desired products are obtained in poor yields, as observed in the classical Biginelli reaction. The possible reasons for these poor yields are addressed and clarify, to some extent, the complexity of the Biginelli multicomponent reaction mechanism and, in particular, the mechanism of the present

FeS/S/FeS(2) redox system and its oxidoreductase-like chemistry in the iron-sulfur world.

PubMed

Wang, Wei; Yang, Bin; Qu, Youpeng; Liu, Xiaoyang; Su, Wenhui

2011-06-01

The iron-sulfur world (ISW) theory is an intriguing prediction regarding the origin of life on early Earth. It hypothesizes that life arose as a geochemical process from inorganic starting materials on the surface of sulfide minerals in the vicinity of deep-sea hot springs. During the last two decades, many experimental studies have been carried out on this topic, and some interesting results have been achieved. Among them, however, the processes of carbon/nitrogen fixation and biomolecular assembly on the mineral surface have received an inordinate amount of attention. To the present, an abiotic model for the oxidation-reduction of intermediates participating in metabolic pathways has been ignored. We examined the oxidation-reduction effect of a prebiotic FeS/S/FeS(2) redox system on the interconversion between several pairs of α-hydroxy acids and α-keto acids (i.e., lactate/pyruvate, malate/oxaloacetate, and glycolate/glyoxylate). We found that, in the absence of FeS, elemental sulfur (S) oxidized α-hydroxy acids to form corresponding keto acids only at a temperature higher than its melting point (113°C); in the presence of FeS, such reactions occurred more efficiently through a coupled reaction mechanism, even at a temperature below the phase transition point of S. On the other hand, FeS was shown to have the capacity to reversibly reduce the keto acids. Such an oxidoreductase-like chemistry of the FeS/S/FeS(2) redox system suggests that it can determine the redox homeostasis of metabolic intermediates in the early evolutionary phase of life. The results provide a possible pathway for the development of primordial redox biochemistry in the iron-sulfur world. Key Words: Iron-sulfur world-FeS/S/FeS(2) redox system-Oxidoreductase-like chemistry. Astrobiology 11, 471-476.

Ketoconazole inhibits the in vitro and in vivo metabolism of all-trans-retinoic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Wauwe, J.P.; Coene, M.C.; Goossens, J.

1988-05-01

Ketoconazole, an antifungal agent and inhibitor of certain mammalian cytochrome P-450-dependent enzymes, was studied for its effects on the in vitro and in vivo metabolism of all-trans-retinoic acid (RA). In vitro, ketoconazole (Ki = 0.75 microM) inhibited, in an apparently competitive manner, the cytochrome P-450-mediated metabolism to 4-hydroxy- and 4-keto-retinoic acids by hamster liver microsomes. In vivo, ketoconazole suppressed the formation of polar RA metabolites by normal rats dosed intrajugularly with 200 ng of (/sup 3/H)RA. After p.o. treatment with ketoconazole (2.5-40 mg/kg) given 1 hr before the (/sup 3/H)RA injection, the radioactivity extracted from the liver consisted of 25more » to 50% polar metabolites (control 66 +/- 1%) and 50 to 75% undegraded RA (control 34 +/- 1%) as evidenced by reverse-phase high-performance liquid chromatography. Time course experiments showed that ketoconazole's inhibitory effects lasted for 3 hr. Our data indicate the quantitative importance of the cytochrome P-450 enzymatic pathway in the biotransformation of RA. They also suggest that ketoconazole is capable of prolonging the biological half-life of RA and of improving the tissue levels of this compound.« less

Selenium-mediated synthesis of biaryls through rearrangement.

PubMed

Shahzad, Sohail A; Vivant, Clotilde; Wirth, Thomas

2010-03-19

A new cyclization of beta-keto ester substituted stilbene derivatives using selenium electrophiles in the presence of Lewis acids is described. Substituted naphthols are obtained through cyclization and subsequent 1,2-rearrangement of aryl groups under very mild reaction conditions.

Consolidated conversion of protein waste into biofuels and ammonia using Bacillus subtilis.

PubMed

Choi, Kwon-Young; Wernick, David G; Tat, Christine A; Liao, James C

2014-05-01

The non-recyclable use of nitrogen fertilizers in microbial production of fuels and chemicals remains environmentally detrimental. Conversion of protein wastes into biofuels and ammonia by engineering nitrogen flux in Escherichia coli has been demonstrated as a method to reclaim reduced-nitrogen and curb its environmental deposition. However, protein biomass requires a proteolysis process before it can be taken up and converted by any microbe. Here, we metabolically engineered Bacillus subtilis to hydrolyze polypeptides through its secreted proteases and to convert amino acids into advanced biofuels and ammonia fertilizer. Redirection of B. subtilis metabolism for amino-acid conversion required inactivation of the branched-chain amino-acid (BCAA) global regulator CodY. Additionally, the lipoamide acyltransferase (bkdB) was deleted to prevent conversion of branched-chain 2-keto acids into their acyl-CoA derivatives. With these deletions and heterologous expression of a keto-acid decarboxylase and an alcohol dehydrogenase, the final strain produced biofuels and ammonia from an amino-acid media with 18.9% and 46.6% of the maximum theoretical yield. The process was also demonstrated on several waste proteins. The results demonstrate the feasibility of direct microbial conversion of polypeptides into sustainable products. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Fine tuning of the spectral properties of LH2 by single amino acid residues.

PubMed

Silber, Martina V; Gabriel, Günther; Strohmann, Brigitte; Garcia-Martin, Adela; Robert, Bruno; Braun, Paula

2008-05-01

The peripheral light-harvesting complex, LH2, of Rhodobacter sphaeroides consists of an assembly of membrane-spanning alpha and beta polypeptides which assemble the photoactive bacteriochlorophyll and carotenoid molecules. In this study we systematically investigated bacteriochlorophyll-protein interactions and their effect on functional bacteriochlorophyll assembly by site-directed mutations of the LH2 alpha-subunit. The amino acid residues, isoleucine at position -1 and serine at position -4 were replaced by 12 and 13 other residues, respectively. All residues replacing isoleucine at position -1 supported the functional assembly of LH2. The replacement of isoleucine by glycine, glutamine or asparagine, however, produced LH2 complex with significantly altered spectral properties in comparison to LH2 WT. As indicated by resonance Raman spectroscopy extensive rearrangement of the bacteriochlorophyll-B850 macrocycle(s) took place in LH2 in which isoleucine -1 was replaced by glycine. The replacement results in disruption of the H-bond between the C3 acetyl groups and the aromatic residues +13/+14 without affecting the H-bond involving the C13(1) keto group. In contrast, nearly all amino acid replacements of serine at position -4 resulted in shifting of the bacteriochlorophyll-B850 red most absorption maximum. Interestingly, the extent of shifting closely correlated with the volume of the residue at position -4. These results illustrate that fine tuning of the spectral properties of the bacteriochlorophyll-B850 molecules depend on their packing with single amino acid residues at distinct positions.

Identification and semi-quantification of biogenic organic nitrates in ambient particulate matters by UHPLC/ESI-MS

NASA Astrophysics Data System (ADS)

Li, Rui; Wang, Xinfeng; Gu, Rongrong; Lu, Chunying; Zhu, Fanping; Xue, Likun; Xie, Huijun; Du, Lin; Chen, Jianmin; Wang, Wenxing

2018-03-01

Particulate biogenic organic nitrates (PBONs) are important components of secondary organic aerosols and play an important role in the tropospheric atmosphere chemistry. However, the concentrations and the chemistry of PBONs remain poorly understood due to the lack of accurate measurement techniques on specific organic nitrates. In this study, ultra high performance liquid chromatography/electrospray mass spectrometry was applied in detection of individual PBONs in ambient atmosphere. Total five kinds of PBONs were identified in PM2.5 samples collected in urban Ji'nan in spring according to characteristic fragments of NO2, NO3, HNO3, CO2, and H2O, including monoterpene hydroxyl nitrate (MW = 215, MHN215), pinene keto nitrate (MW = 229, PKN229), limonene di-keto nitrate (MW = 247, LDKN247), oleic acid keto nitrate (MW = 359, OAKN359), and oleic acid hydroxyl nitrate (MW = 361, OAHN361). Among them, three kinds of PBONs originated from biogenic volatile organic compounds of pinene and limonene and two kinds of PBONs came from chemical conversion of oleic acid. The concentrations of these PBONs were roughly quantified with surrogate standards of (1R,2R,5R)-(+)-2-hydroxy-3-pinanone and ricinoleic acid. The average concentrations of MHN215, PKN229, LDKN247, OAKN359, and OAHN361 were 111.6 ± 23.0, 93.1 ± 49.6, 55.3 ± 7.4, 23.4 ± 14.5, 36.8 ± 18.3 ng m-3, respectively. The total concentration of these PBONs was 325.4 ± 116.7 ng m-3, contributing to 1.64 ± 0.34‰ of PM2.5.

Isolation and primary structural analysis of two conjugated polyketone reductases from Candida parapsilosis.

PubMed

Hidalgo, A R; Akond, M A; Kita, K; Kataoka, M; Shimizu, S

2001-12-01

Two conjugated polyketone reductases (CPRs) were isolated from Candida parapsilosis IFO 0708. The primary structures of CPRs (C1 and C2) were analyzed by amino acid sequencing. The amino acid sequences of both enzymes had high similarity to those of several proteins of the aldo-keto-reductase (AKR) superfamily. However, several amino acid residues in the putative active sites of AKRs were not conserved in CPRs-C1 and -C2.

The Tautomeric Half-reaction of BphD, a C-C Bond Hydrolase Kinetic and Structural Evidence Supporting a Key Role for Histidine 265 of the Catalytic triad

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horsman, Geoff P.; Bhowmik, Shiva; Seah, Stephen Y.K.

2010-01-07

BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem-diol intermediate. The role of the canonical catalytic triad (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA ({lambda}{sub max} is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum ({lambda}{sub max} is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. T.,more » and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate ({lambda}{sub max} is 506 nm) with a similar rate, 1/{tau} {approx} 500 s{sup -1}. The crystal structure of the S112A:HOPDA complex at 1.8-{angstrom} resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7{angstrom} away.« less

Acetobacter fabarum sp. nov., an acetic acid bacterium from a Ghanaian cocoa bean heap fermentation.

PubMed

Cleenwerck, Ilse; Gonzalez, Angel; Camu, Nicholas; Engelbeen, Katrien; De Vos, Paul; De Vuyst, Luc

2008-09-01

Six acetic acid bacterial isolates, obtained during a study of the microbial diversity of spontaneous fermentations of Ghanaian cocoa beans, were subjected to a polyphasic taxonomic study. (GTG)(5)-PCR fingerprinting grouped the isolates together, but they could not be identified using this method. Phylogenetic analysis based on 16S rRNA gene sequences allocated the isolates to the genus Acetobacter and revealed Acetobacter lovaniensis, Acetobacter ghanensis and Acetobacter syzygii to be nearest neighbours. DNA-DNA hybridizations demonstrated that the isolates belonged to a single novel genospecies that could be differentiated from its phylogenetically nearest neighbours by the following phenotypic characteristics: no production of 2-keto-D-gluconic acid from D-glucose; growth on methanol and D-xylose, but not on maltose, as sole carbon sources; no growth on yeast extract with 30% D-glucose; and weak growth at 37 degrees C. The DNA G+C contents of four selected strains were 56.8-58.0 mol%. The results obtained prove that the isolates should be classified as representatives of a novel Acetobacter species, for which the name Acetobacter fabarum sp. nov. is proposed. The type strain is strain 985(T) (=R-36330(T) =LMG 24244(T) =DSM 19596(T)).

An oxidized metabolite of linoleic acid increases intracellular calcium in rat adrenal glomerulosa cells.

PubMed

Payet, Marcel D; Goodfriend, Theodore L; Bilodeau, Lyne; Mackendale, Cherilu; Chouinard, Lucie; Gallo-Payet, Nicole

2006-12-01

EKODE, an epoxy-keto derivative of linoleic acid, was previously shown to stimulate aldosterone secretion in rat adrenal glomerulosa cells. In the present study, we investigated the effect of exogenous EKODE on cytosolic [Ca(2+)] increase and aimed to elucidate the mechanism involved in this process. Through the use of the fluorescent Ca(2+)-sensitive dye Fluo-4, EKODE was shown to rapidly increase intracellular [Ca(2+)] ([Ca(2+)](i)) along a bell-shaped dose-response relationship with a maximum peak at 5 microM. Experiments performed in the presence or absence of Ca(2+) revealed that this increase in [Ca(2+)](i) originated exclusively from intracellular pools. EKODE-induced [Ca(2+)](i) increase was blunted by prior application of angiotensin II, Xestospongin C, and cyclopiazonic acid, indicating that inositol trisphosphate (InsP(3))-sensitive Ca(2+) stores can be mobilized by EKODE despite the absence of InsP(3) production. Accordingly, EKODE response was not sensitive to the phospholipase C inhibitor U-73122. EKODE mobilized a Ca(2+) store included in the thapsigargin (TG)-sensitive stores, although the interaction between EKODE and TG appears complex, since EKODE added at the plateau response of TG induced a rapid drop in [Ca(2+)](i). 9-oxo-octadecadienoic acid, another oxidized derivative of linoleic acid, also increases [Ca(2+)](i), with a dose-response curve similar to EKODE. However, arachidonic and linoleic acids at 10 microM failed to increase [Ca(2+)](i) but did reduce the amplitude of the response to EKODE. It is concluded that EKODE mobilizes Ca(2+) from an InsP(3)-sensitive store and that this [Ca(2+)](i) increase is responsible for aldosterone secretion by glomerulosa cells. Similar bell-shaped dose-response curves for aldosterone and [Ca(2+)](i) increases reinforce this hypothesis.

Mechanism of MenE Inhibition by Acyl-Adenylate Analogues and Discovery of Novel Antibacterial Agents

PubMed Central

Sharma, Indrajeet; Lavaud, Lubens J.; Ngo, Stephen C.; Shek, Roger; Rajashankar, Kanagalaghatta R.; French, Jarrod B.; Tan, Derek S.; Tonge, Peter J.

2015-01-01

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1) which has an IC50 value of ≤ 25 nM for the Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in S. aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ~1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure–activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto-acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively-charged keto-acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future. PMID:26394156

Mechanism of MenE inhibition by acyl-adenylate analogues and discovery of novel antibacterial agents.

PubMed

Matarlo, Joe S; Evans, Christopher E; Sharma, Indrajeet; Lavaud, Lubens J; Ngo, Stephen C; Shek, Roger; Rajashankar, Kanagalaghatta R; French, Jarrod B; Tan, Derek S; Tonge, Peter J

2015-10-27

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1), which has an IC50 value of ≤25 nM for Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in Staphylococcus aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ∼1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure-activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future.

Characterization of alpha-ketobutyrate metabolism in rat tissues: effects of dietary protein and fasting.

PubMed

Steele, R D; Weber, H; Patterson, J I

1984-04-01

The oxidative decarboxylation of alpha-ketobutyrate was studied in rat tissue preparations. Decarboxylation was confined to the mitochondrial fraction and required coenzyme A, NAD, TPP and FAD for optimal activity in solubilized preparations. The pH optimum for this reaction in liver was 7.8, somewhat higher than that reported for other alpha-keto acid dehydrogenases. An apparent Km of 0.63 mM for alpha-ketobutyrate was determined for the rat liver system. Competition by other alpha-keto acids at 10 mM concentrations inhibited enzyme activity up to 75%. Tissue distribution of alpha-ketobutyrate dehydrogenase activity relative to liver activity was (in percent): liver, 100; heart, 127; brain, 63; kidney, 57; skeletal muscle, 38; and small intestine, 7. Total liver alpha-ketobutyrate dehydrogenase was decreased by 40% after a 24-hour fast. Similar results were found for kidney and heart activity. alpha-Aminobutyrate-pyruvate aminotransferase activity in liver or kidney was not affected by fasting; however, it was induced in liver by 50% after feeding a 40% casein diet for 10 days compared to rats fed a 20% casein diet. Increasing the dietary casein content from 6 through 40% of the diet resulted in about a fivefold increase in liver alpha-ketobutyrate dehydrogenase activity. The substantial extrahepatic capacity for alpha-ketobutyrate metabolism makes it unlikely that a loss of liver function results in an inability to metabolize alpha-ketobutyrate. Whether alpha-ketobutyrate is decarboxylated by a specific enzyme or by an already characterized complex such as pyruvate dehydrogenase or the branched-chain keto acid dehydrogenase remains to be established.

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna[C][W

PubMed Central

Bedewitz, Matthew A.; Góngora-Castillo, Elsa; Uebler, Joseph B.; Gonzales-Vigil, Eliana; Wiegert-Rininger, Krystle E.; Childs, Kevin L.; Hamilton, John P.; Vaillancourt, Brieanne; Yeo, Yun-Soo; Chappell, Joseph; DellaPenna, Dean; Jones, A. Daniel; Buell, C. Robin; Barry, Cornelius S.

2014-01-01

The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine. PMID:25228340

PGC-1α-mediated branched-chain amino acid metabolism in the skeletal muscle.

PubMed

Hatazawa, Yukino; Tadaishi, Miki; Nagaike, Yuta; Morita, Akihito; Ogawa, Yoshihiro; Ezaki, Osamu; Takai-Igarashi, Takako; Kitaura, Yasuyuki; Shimomura, Yoshiharu; Kamei, Yasutomi; Miura, Shinji

2014-01-01

Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) is a coactivator of various nuclear receptors and other transcription factors, which is involved in the regulation of energy metabolism, thermogenesis, and other biological processes that control phenotypic characteristics of various organ systems including skeletal muscle. PGC-1α in skeletal muscle is considered to be involved in contractile protein function, mitochondrial function, metabolic regulation, intracellular signaling, and transcriptional responses. Branched-chain amino acid (BCAA) metabolism mainly occurs in skeletal muscle mitochondria, and enzymes related to BCAA metabolism are increased by exercise. Using murine skeletal muscle overexpressing PGC-1α and cultured cells, we investigated whether PGC-1α stimulates BCAA metabolism by increasing the expression of enzymes involved in BCAA metabolism. Transgenic mice overexpressing PGC-1α specifically in the skeletal muscle had increased the expression of branched-chain aminotransferase (BCAT) 2, branched-chain α-keto acid dehydrogenase (BCKDH), which catabolize BCAA. The expression of BCKDH kinase (BCKDK), which phosphorylates BCKDH and suppresses its enzymatic activity, was unchanged. The amount of BCAA in the skeletal muscle was significantly decreased in the transgenic mice compared with that in the wild-type mice. The amount of glutamic acid, a metabolite of BCAA catabolism, was increased in the transgenic mice, suggesting the activation of muscle BCAA metabolism by PGC-1α. In C2C12 cells, the overexpression of PGC-1α significantly increased the expression of BCAT2 and BCKDH but not BCKDK. Thus, PGC-1α in the skeletal muscle is considered to significantly contribute to BCAA metabolism.

PGC-1α-Mediated Branched-Chain Amino Acid Metabolism in the Skeletal Muscle

PubMed Central

Nagaike, Yuta; Morita, Akihito; Ogawa, Yoshihiro; Ezaki, Osamu; Takai-Igarashi, Takako; Kitaura, Yasuyuki; Shimomura, Yoshiharu; Kamei, Yasutomi; Miura, Shinji

2014-01-01

Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) is a coactivator of various nuclear receptors and other transcription factors, which is involved in the regulation of energy metabolism, thermogenesis, and other biological processes that control phenotypic characteristics of various organ systems including skeletal muscle. PGC-1α in skeletal muscle is considered to be involved in contractile protein function, mitochondrial function, metabolic regulation, intracellular signaling, and transcriptional responses. Branched-chain amino acid (BCAA) metabolism mainly occurs in skeletal muscle mitochondria, and enzymes related to BCAA metabolism are increased by exercise. Using murine skeletal muscle overexpressing PGC-1α and cultured cells, we investigated whether PGC-1α stimulates BCAA metabolism by increasing the expression of enzymes involved in BCAA metabolism. Transgenic mice overexpressing PGC-1α specifically in the skeletal muscle had increased the expression of branched-chain aminotransferase (BCAT) 2, branched-chain α-keto acid dehydrogenase (BCKDH), which catabolize BCAA. The expression of BCKDH kinase (BCKDK), which phosphorylates BCKDH and suppresses its enzymatic activity, was unchanged. The amount of BCAA in the skeletal muscle was significantly decreased in the transgenic mice compared with that in the wild-type mice. The amount of glutamic acid, a metabolite of BCAA catabolism, was increased in the transgenic mice, suggesting the activation of muscle BCAA metabolism by PGC-1α. In C2C12 cells, the overexpression of PGC-1α significantly increased the expression of BCAT2 and BCKDH but not BCKDK. Thus, PGC-1α in the skeletal muscle is considered to significantly contribute to BCAA metabolism. PMID:24638054

Synthetic study toward ecteinascidin 743: concise construction of the diazabicyclo[3.3.1]nonane skeleton and assembly of the pentacyclic core.

PubMed

Enomoto, Taro; Yasui, Yoshizumi; Takemoto, Yoshiji

2010-07-16

Synthesis of the pentacyclic core of ecteinascidin 743 is described. This synthesis features concise construction of the diazabicyclo[3.3.1]nonane skeleton using gold(I)-catalyzed one-pot keto amide formation, acid-promoted enamide formation, and oxidative Friedel-Crafts cyclization as the key steps.

Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Linlin; Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001; Liu, Ziwen

2009-09-18

Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 {mu}M, 4-hydroxynonenal (HNE) at 0.10 {mu}M, trans-2-hexanal at 0.10 {mu}M, and trans-2,4-hexadienal at 0.05 {mu}M, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 {mu}M (toxic) by converting to 1,4-dihydroxynonene,more » protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.« less

Interactions of Enolizable Barbiturate Dyes.

PubMed

Schade, Alexander; Schreiter, Katja; Rüffer, Tobias; Lang, Heinrich; Spange, Stefan

2016-04-11

The specific barbituric acid dyes 1-n-butyl-5-(2,4-dinitro-phenyl) barbituric acid and 1-n-butyl-5-{4-[(1,3-dioxo-1H-inden-(3 H)-ylidene)methyl]phenyl}barbituric acid were used to study complex formation with nucleobase derivatives and related model compounds. The enol form of both compounds shows a strong bathochromic shift of the UV/Vis absorption band compared to the rarely coloured keto form. The keto-enol equilibria of the five studied dyes are strongly dependent on the properties of the environment as shown by solvatochromic studies in ionic liquids and a set of organic solvents. Enol form development of the barbituric acid dyes is also associated with alteration of the hydrogen bonding pattern from the ADA to the DDA type (A=hydrogen bond acceptor site, D=donor site). Receptor-induced altering of ADA towards DDA hydrogen bonding patterns of the chromophores are utilised to study supramolecular complex formation. As complementary receptors 9-ethyladenine, 1-n-butylcytosine, 1-n-butylthymine, 9-ethylguanidine and 2,6-diacetamidopiridine were used. The UV/Vis spectroscopic response of acid-base reaction compared to supramolecular complex formation is evaluated by (1)H NMR titration experiments and X-ray crystal structure analyses. An increased acidity of the barbituric acid derivative promotes genuine salt formation. In contrast, supramolecular complex formation is preferred for the weaker acidic barbituric acid. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prostacyclin production in rabbit arteries in situ: inhibition by arachidonic acid-induced endothelial cell damage or by low-dose aspirin.

PubMed

Ingerman-Wojenski, C; Silver, M J; Smith, J B; Nissenbaum, M; Sedar, A W

1981-04-01

The central artery of the rabbit ear was perfused in situ and effluent fractions from the artery were assayed for 6-keto-prostaglandin F1 alpha (6-K-PGF1 alpha) and thromboxane B2 (TxB2), the stable metabolites of prostacyclin (PGI2) and TxA2, using specific radioimmunoassays. These metabolites of arachidonic acid (AA) were not detected in the effluent during infusion of Tyrode's solution but both metabolites were detected when small amounts of AA were infused into the artery. Examination of the arteries by scanning electron microscopy revealed that high concentrations of AA which caused a short burst of 6-K-PGF1 alpha and TxB2 production damaged the endothelial cells while lower concentrations which stimulated continuous production did not cause damage. When a non-damaging concentration of AA was infused into an artery that had previously received a damaging concentration, PG production was greatly reduced. Pretreatment of the rabbits with 4 mg/kg acetyl-salicylic acid (ASA) inhibited 6-K-PGF1 alpha production by the rabbit ear artery in response to AA and 70% inhibition was still evident 18 hours after ASA.

The Yeast Eukaryotic Translation Initiation Factor 2B Translation Initiation Complex Interacts with the Fatty Acid Synthesis Enzyme YBR159W and Endoplasmic Reticulum Membranes

PubMed Central

Browne, Christopher M.; Samir, Parimal; Fites, J. Scott; Villarreal, Seth A.

2013-01-01

Using affinity purifications coupled with mass spectrometry and yeast two-hybrid assays, we show the Saccharomyces cerevisiae translation initiation factor complex eukaryotic translation initiation factor 2B (eIF2B) and the very-long-chain fatty acid (VLCFA) synthesis keto-reductase enzyme YBR159W physically interact. The data show that the interaction is specifically between YBR159W and eIF2B and not between other members of the translation initiation or VLCFA pathways. A ybr159wΔ null strain has a slow-growth phenotype and a reduced translation rate but a normal GCN4 response to amino acid starvation. Although YBR159W localizes to the endoplasmic reticulum membrane, subcellular fractionation experiments show that a fraction of eIF2B cofractionates with lipid membranes in a YBR159W-independent manner. We show that a ybr159wΔ yeast strain and other strains with null mutations in the VLCFA pathway cause eIF2B to appear as numerous foci throughout the cytoplasm. PMID:23263984

Quantification of the Keto-Hydroperoxide (HOOCH2OCHO) and Other Elusive Intermediates during Low-Temperature Oxidation of Dimethyl Ether.

PubMed

Moshammer, Kai; Jasper, Ahren W; Popolan-Vaida, Denisia M; Wang, Zhandong; Bhavani Shankar, Vijai Shankar; Ruwe, Lena; Taatjes, Craig A; Dagaut, Philippe; Hansen, Nils

2016-10-04

This work provides new temperature-dependent mole fractions of elusive intermediates relevant to the low-temperature oxidation of dimethyl ether (DME). It extends the previous study of Moshammer et al. [ J. Phys. Chem. A 2015 , 119 , 7361 - 7374 ] in which a combination of a jet-stirred reactor and molecular beam mass spectrometry with single-photon ionization via tunable synchrotron-generated vacuum-ultraviolet radiation was used to identify (but not quantify) several highly oxygenated species. Here, temperature-dependent concentration profiles of 17 components were determined in the range of 450-1000 K and compared to up-to-date kinetic modeling results. Special emphasis is paid toward the validation and application of a theoretical method for predicting photoionization cross sections that are hard to obtain experimentally but essential to turn mass spectral data into mole fraction profiles. The presented approach enabled the quantification of the hydroperoxymethyl formate (HOOCH 2 OCH 2 O), which is a key intermediate in the low-temperature oxidation of DME. The quantification of this keto-hydroperoxide together with the temperature-dependent concentration profiles of other intermediates including H 2 O 2 , HCOOH, CH 3 OCHO, and CH 3 OOH reveals new opportunities for the development of a next-generation DME combustion chemistry mechanism.

Oxidation of C18 Hydroxy-Polyunsaturated Fatty Acids to Epoxide or Ketone by Catalase-Related Hemoproteins Activated with Iodosylbenzene.

PubMed

Teder, Tarvi; Boeglin, William E; Brash, Alan R

2017-07-01

Small catalase-related hemoproteins with a facility to react with fatty acid hydroperoxides were examined for their potential mono-oxygenase activity when activated using iodosylbenzene. The proteins tested were a Fusarium graminearum 41 kD catalase hemoprotein (Fg-cat, gene FGSG_02217), a Pseudomonas fluorescens Pfl01 catalase (37.5 kD, accession number WP_011333788.1), and a Mycobacterium avium ssp. paratuberculosis 33 kD catalase (gene MAP-2744c). 13-Hydroxy-octadecenoic acids (which are normally unreactive) were selected as substrates because these enzymes react specifically with the corresponding 13S-hydroperoxides (Pakhomova et al. 18:2559-2568, 5; Teder et al. 1862:706-715, 14). In the presence of iodosylbenzene Fg-cat converted 13S-hydroxy-fatty acids to two products: the 15,16-double bond of 13S-hydroxy α-linolenic acid was oxidized stereospecifically to the 15S,16R-cis-epoxide or the 13-hydroxyl was oxidized to the 13-ketone. Products were identified by UV, HPLC, LC-MS, NMR and by comparison with authentic standards prepared for this study. The Pfl01-cat displayed similar activity. MAP-2744c oxidized 13S-hydroxy-linoleic acid to the 13-ketone, and epoxidized the double bonds to form the 9,10-epoxy-13-hydroxy, 11,12-epoxy-13-hydroxy, and 9,10-epoxy-13-keto derivatives; equivalent transformations occurred with 9S-hydroxy-linoleic acid as substrate. In parallel incubations in the presence of iodosylbenzene, human catalase displayed no activity towards 13S-hydroxy-linoleic acid, as expected from the highly restricted access to its active site. The results indicated that with suitable transformation to Compound I, monooxygenase activity can be demonstrated by these catalase-related hemoproteins with tyrosine as the proximal heme ligand.

Industrial Organic Electrosynthesis.

ERIC Educational Resources Information Center

Wagenknecht, John H.

1983-01-01

Four examples of industrial electrochemical synthesis of organic compounds are described. These include acrylonitrile dimerization, tetramethyl lead, electrochemical fluorination, and production of diacetone-2-keto-L-gulonic acid. Additional examples are also cited, including the production of several compounds by the BASF company of Germany. (JN)

The Role of Human Aldo-Keto Reductases in the Metabolic Activation and Detoxication of Polycyclic Aromatic Hydrocarbons: Interconversion of PAH Catechols and PAH o-Quinones

PubMed Central

Zhang, Li; Jin, Yi; Huang, Meng; Penning, Trevor M.

2012-01-01

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants. They are procarcinogens requiring metabolic activation to elicit their deleterious effects. Aldo-keto reductases (AKR) catalyze the oxidation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active PAH o-quinones. AKRs are also found to be capable of reducing PAH o-quinones to form PAH catechols. The interconversion of o-quinones and catechols results in the redox-cycling of PAH o-quinones to give rise to the generation of reactive oxygen species and subsequent oxidative DNA damage. On the other hand, PAH catechols can be intercepted through phase II metabolism by which PAH o-quinones could be detoxified and eliminated. The aim of the present review is to summarize the role of human AKRs in the metabolic activation/detoxication of PAH and the relevance of phase II conjugation reactions to human lung carcinogenesis. PMID:23162467

Ultra-pressure liquid chromatography/tandem mass spectrometry targeted profiling of arachidonic acid and eicosanoids in human colorectal cancer.

PubMed

Mal, Mainak; Koh, Poh Koon; Cheah, Peh Yean; Chan, Eric Chun Yong

2011-03-30

Cumulative evidence shows that eicosanoids such as prostaglandins, leukotrienes, thromboxanes and hydroxy eicosatetraenoic acids play an important role in associating inflammation with human colorectal cancer (CRC). In this study an ultra-pressure liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the targeted profiling of eight relevant eicosanoids and the major metabolic precursor, arachidonic acid (AA), in human colon. Multiple reaction monitoring (MRM) experiments were performed in negative electrospray ionization mode. The metabolites were separated using a C(18) column consisting of 1.7 µm ethylene-bridged hybrid particles (100 × 2.1 mm i.d.) and gradient elution (50 to 95% of solvent B) with a mobile phase comprising water (0.1% formic acid) [solvent A] and acetonitrile (0.1% formic acid) [solvent B] at a flow rate of 0.4 mL/min. The analysis time for each sample was 5.5 min. Our UPLC/MS/MS method demonstrated satisfactory validation results in terms of selectivity, sensitivity, matrix effect, linearity, extraction efficiency, intra- and inter-day precision, accuracy and autosampler stability. The method was applied for the clinical profiling of matched pairs of cancerous and normal colon mucosae obtained from eight colorectal cancer patients. Endogenous levels of AA and selected eicosanoids such as prostaglandin E(2) (PGE(2)), prostacyclin (PGI(2)) [assayed as its stable hydrolytic product 6-keto-prostaglandin(1α) (6-k PGF(1α))] and 12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HETE) were found to be significantly different (p <0.05; paired t-test) between cancerous and normal mucosae. Copyright © 2011 John Wiley & Sons, Ltd.

Structural characterization of the Mycobacterium tuberculosis biotin biosynthesis enzymes 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase .

PubMed

Dey, Sanghamitra; Lane, James M; Lee, Richard E; Rubin, Eric J; Sacchettini, James C

2010-08-10

Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 and 1.85 A. Superimposition of the DAPAS structures bound either to the SAM analogue sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 A, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb.

Specific tritium labeling of gangliosides at the 3-position of sphingosines.

PubMed

Ghidoni, R; Sonnino, S; Masserini, M; Orlando, P; Tettamanti, G

1981-11-01

GM1 and GD1a gangliosides, treated with 2,3-dichloro-5,6-dicyano benzoquinone (DDQ) in the presence of Triton X-100 and in a toluene medium were specifically oxidized at the 3-position of sphingosine. The maximum reaction yield (65%) was obtained after 40 hours at 37 degrees C with the following molar ratio of reactants: ganglioside-Triton X-100-DDQ 1:70:125. The formation of the 3-keto derivatives of GM1 and GD1a was demonstrated by: a) the appearance of a sharp peak at 1700 cm-1 and of a broad band at 1250 cm-1 (typical of allylic ketones and of carbonyl groups, respectively) in the infra-red spectrum; b) the appearance of an absorption maximum at 230 nm, identical to that featured by 3-keto-cerebrosides, in the ultraviolet spectrum; c) the degradation of long chain bases during the process of release from gangliosides and derivatization for analysis by gas-liquid chromatography (expected for long chain bases carrying a keto group in the 3-position); and d) the quantitative transformation of 3-keto-GM1 and 3-keto-GD1a to GM1 and GD1a, respectively, upon NaBH4 reduction. Reduction of 3-keto-GM1 and 3-keto-GD1a with [3H]-NaBH4 produced 3H-labeled GM1 and GD1a. [3H]GM1 and [3H]GD1a maintained the same carbohydrate and fatty acid composition of the original GM1 and GD1a, and did not contain any saturated long chain bases. Direct proof that the label was at C-3 of long chain bases was given by reoxidation with DDQ, which completely removed the label, and by ozonolysis, after which label was retained on the oligosaccharide-containing fragment. More than 99% of incorporated radioactivity was carried by the long chain bases. The radiochemical purity of labeled gangliosides was greater than 95% and the specific radioactivity was 1.25 and 1.28 Ci/m mol for [3H]GM1 and [3H]GD1a, respectively.

Molecular distributions of water soluble dicarboxylic acids in marine aerosols over the Pacific Ocean including tropics

NASA Astrophysics Data System (ADS)

Kawamura, Kimitaka; Sakaguchi, Futoshi

1999-02-01

Remote marine aerosols collected over the western North to equatorial Pacific (34°N-14°S, 140°E-150°W) were studied for low molecular weight dicarboxylic acids using a capillary gas chromatography (GC) and GC/mass spectrometer, and for total carbon and nitrogen contents. Homologous series of dicarboxylic acids (C2-C10) including keto- and hydroxy-dicarboxylic acids were detected in the samples with a concentration range of 10-250 ng m-3 (average 63 ng m-3 and median 44 ng m-3). Their molecular distributions showed a predominance of oxalic acid (C2), followed by malonic acid (C3). The smallest diacid (C2, 6.5-161 ng m-3 with average 40 ng m-3 and median 17 ng m-3) composed 45-75% (average 65%) of the total diacids. The diacids showed higher concentrations in the western Pacific rim near Japanese islands and showed lower concentrations in the central and tropical Pacific. However, relative abundances of the diacid-carbon in the total aerosol carbon (1.1-15.8%) were found to be higher in the equatorial central Pacific. These diacids are probably in situ produced in the Pacific atmosphere by photochemical oxidation of gaseous and particulate precursors. Results of principal component analysis of individual diacid, coupled with an information on photochemical reactions, further support that C2 and C3 diacids are likely produced by the oxidation of C4 and longer-chain diacids, whereas longer-chain (C5-C10) diacids are produced through the oxidation of semivolatile fatty acids which are also oxidation products of unsaturated fatty acids. Concentrations of total C (0.069-5.27 μg m-3 with average 0.39 μg m-3 and median 0.15 μg m-3) and total N (0.026-1.44 μg m-3 with average 0.12 μg m-3 and median 0.077 μg m-3) were generally higher over the western Pacific.

A root-expressed L-phenylalanine:4-hydroxyphenylpyruvate aminotransferase is required for tropane alkaloid biosynthesis in Atropa belladonna.

PubMed

Bedewitz, Matthew A; Góngora-Castillo, Elsa; Uebler, Joseph B; Gonzales-Vigil, Eliana; Wiegert-Rininger, Krystle E; Childs, Kevin L; Hamilton, John P; Vaillancourt, Brieanne; Yeo, Yun-Soo; Chappell, Joseph; DellaPenna, Dean; Jones, A Daniel; Buell, C Robin; Barry, Cornelius S

2014-09-01

The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine. © 2014 American Society of Plant Biologists. All rights reserved.

[Catalytic properties of enzymes from Erwinia carotovora involved in transamination of phenylpyruvate].

PubMed

Paloian, A M; Stepanian, L A; Dadaian, S A; Ambartsumian, A A; Alebian, G P; Sagian, A S

2013-01-01

Km for L-phenylalanine, L-glutamic acid, L-aspartic acid, and the corresponding keto acids were calculated, as well as Vmax, was measured for the following pairs of substrates: L-phenylalanine-2-ketoglutarate, L-phenylalanine-oxaloacetate, L-glutamic acid-phenylpyruvate, and L-aspartic acid-phenylpyruvate for aminotransferases PATI, PAT2, and PAT3 from Erwinia carotovora catalyzing transamination of phenylpyruvate. The ping-pong bi-bi mechanism was shown for the studied aminotransferases. The substrate inhibition (Ks) of PAT3 with 2-ketoglutarate and oxaloacetate was 10.23 +/- 3.20 and 3.73 +/- 1.99 mM, respectively.

Synthesis of 3 alpha,7 alpha,12 alpha,25-tetrahydroxy-5 beta-cholestan-24-one, an intermediate in the 25-hydroxylation pathway of cholic acid biosynthesis from cholesterol.

PubMed

Dayal, B; Tint, G S; Batta, A K; Shefer, S; Salen, G; Bose, A K; Pramanik, B N

1983-02-01

This paper describes the chemical synthesis of 3 alpha,7 alpha,12 alpha,25-tetrahydroxy-5 beta-cholestan-24-one via selective oxidation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 24 xi,25-pentol with silver carbonate on celite. The structure of this 24-keto bile alcohol was confirmed by gas-liquid chromatography and mass spectrometry. Synthesis of this compound via pyridinium chlorochromate oxidation of the triacetoxy derivative of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,25-pentol followed by saponification further established its structure. 3 alpha,7 alpha,12 alpha,25-Tetrahydroxy-5 beta-cholestan-24-one was required for the in vivo and in vitro studies of side-chain oxidation and cleavage in the 25-hydroxylation pathway of cholic acid biosynthesis.

Aldo-keto reductase 1B10 promotes development of cisplatin resistance in gastrointestinal cancer cells through down-regulating peroxisome proliferator-activated receptor-γ-dependent mechanism.

PubMed

Matsunaga, Toshiyuki; Suzuki, Ayaka; Kezuka, Chihiro; Okumura, Naoko; Iguchi, Kazuhiro; Inoue, Ikuo; Soda, Midori; Endo, Satoshi; El-Kabbani, Ossama; Hara, Akira; Ikari, Akira

2016-08-25

Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most effective chemotherapeutic drugs that are used for treatment of patients with gastrointestinal cancer cells, but its continuous administration often evokes the development of chemoresistance. In this study, we investigated alterations in antioxidant molecules and functions using a newly established CDDP-resistant variant of gastric cancer MKN45 cells, and found that aldo-keto reductase 1B10 (AKR1B10) is significantly up-regulated with acquisition of the CDDP resistance. In the nonresistant MKN45 cells, the sensitivity to cytotoxic effect of CDDP was decreased and increased by overexpression and silencing of AKR1B10, respectively. In addition, the AKR1B10 overexpression markedly suppressed accumulation and cytotoxicity of 4-hydroxy-2-nonenal that is produced during lipid peroxidation by CDDP treatment, suggesting that the enzyme acts as a crucial factor for facilitation of the CDDP resistance through inhibiting induction of oxidative stress by the drug. Transient exposure to CDDP and induction of the CDDP resistance decreased expression of peroxisome proliferator-activated receptor-γ (PPARγ) in MKN45 and colon cancer LoVo cells. Additionally, overexpression of PPARγ in the cells elevated the sensitivity to the CDDP toxicity, which was further augmented by concomitant treatment with a PPARγ ligand rosiglitazone. Intriguingly, overexpression of AKR1B10 in the cells resulted in a decrease in PPARγ expression, which was recovered by addition of an AKR1B10 inhibitor oleanolic acid, inferring that PPARγ is a downstream target of AKR1B10-dependent mechanism underlying the CDDP resistance. Combined treatment with the AKR1B10 inhibitor and PPARγ ligand elevated the CDDP sensitivity, which was almost the same level as that in the parental cells. These results suggest that combined treatment with the AKR1B10 inhibitor and PPARγ ligand is an effective adjuvant therapy for overcoming CDDP resistance of

Endurance performance and energy metabolism during exercise in mice with a muscle-specific defect in the control of branched-chain amino acid catabolism.

PubMed

Xu, Minjun; Kitaura, Yasuyuki; Ishikawa, Takuya; Kadota, Yoshihiro; Terai, Chihaya; Shindo, Daichi; Morioka, Takashi; Ota, Miki; Morishita, Yukako; Ishihara, Kengo; Shimomura, Yoshiharu

2017-01-01

It is known that the catabolism of branched-chain amino acids (BCAAs) in skeletal muscle is suppressed under normal and sedentary conditions but is promoted by exercise. BCAA catabolism in muscle tissues is regulated by the branched-chain α-keto acid (BCKA) dehydrogenase complex, which is inactivated by phosphorylation by BCKA dehydrogenase kinase (BDK). In the present study, we used muscle-specific BDK deficient mice (BDK-mKO mice) to examine the effect of uncontrolled BCAA catabolism on endurance exercise performance and skeletal muscle energy metabolism. Untrained control and BDK-mKO mice showed the same performance; however, the endurance performance enhanced by 2 weeks of running training was somewhat, but significantly less in BDK-mKO mice than in control mice. Skeletal muscle of BDK-mKO mice had low levels of glycogen. Metabolome analysis showed that BCAA catabolism was greatly enhanced in the muscle of BDK-mKO mice and produced branched-chain acyl-carnitine, which induced perturbation of energy metabolism in the muscle. These results suggest that the tight regulation of BCAA catabolism in muscles is important for homeostasis of muscle energy metabolism and, at least in part, for adaptation to exercise training.

Engineering the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum by site saturation mutagenesis for D-phenylalanine synthesis.

PubMed

Gao, Xiuzhen; Huang, Fang; Feng, Jinhui; Chen, Xi; Zhang, Hailing; Wang, Zhixiang; Wu, Qiaqing; Zhu, Dunming

2013-08-01

In order to enlarge the substrate binding pocket of the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum to accommodate larger 2-keto acids, four amino acid residues (Phe146, Thr171, Arg181, and His227) were targeted for site saturation mutagenesis. Among all mutants, the single mutant H227V had a specific activity of 2.39 ± 0.06 U · mg(-1), which was 35.1-fold enhancement over the wild-type enzyme.

Ligand complex structures of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change.

PubMed

Im, Dohyun; Matsui, Daisuke; Arakawa, Takatoshi; Isobe, Kimiyasu; Asano, Yasuhisa; Fushinobu, Shinya

2018-03-01

l-Amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 (l-AAO/MOG) catalyzes both the oxidative deamination and oxidative decarboxylation of the α-group of l-Lys to produce a keto acid and amide, respectively. l-AAO/MOG exhibits limited specificity for l-amino acid substrates with a basic side chain. We previously determined its ligand-free crystal structure and identified a key residue for maintaining the dual activities. Here, we determined the structures of l-AAO/MOG complexed with l-Lys, l-ornithine, and l-Arg and revealed its substrate recognition. Asp238 is located at the ceiling of a long hydrophobic pocket and forms a strong interaction with the terminal, positively charged group of the substrates. A mutational analysis on the D238A mutant indicated that the interaction is critical for substrate binding but not for catalytic control between the oxidase/monooxygenase activities. The catalytic activities of the D238E mutant unexpectedly increased, while the D238F mutant exhibited altered substrate specificity to long hydrophobic substrates. In the ligand-free structure, there are two channels connecting the active site and solvent, and a short region located at the dimer interface is disordered. In the l-Lys complex structure, a loop region is displaced to plug the channels. Moreover, the disordered region in the ligand-free structure forms a short helix in the substrate complex structures and creates the second binding site for the substrate. It is assumed that the amino acid substrate enters the active site of l-AAO/MOG through this route. The atomic coordinates and structure factors (codes 5YB6, 5YB7, and 5YB8) have been deposited in the Protein Data Bank (http://wwpdb.org/). 1.4.3.2 (l-amino acid oxidase), 1.13.12.2 (lysine 2-monooxygenase).

Specific Inhibitors of Histone Demethylases: Novel Chemical Agents for Breast Cancer Therapy

DTIC Science & Technology

2012-08-01

number of substrate analogs bearing either di- and tri- methylammonium groups or - keto -acid fragments (Chart 1) to be tested for their inhibition...ution ( 143 mg, 0.4 mmol). The solution was heated at 70 °C and stirred for 1 h. The resulting dark-purple solution was cooled to room temperature

Cloning and Characterization of a Novel β-Transaminase from Mesorhizobium sp. Strain LUK: a New Biocatalyst for the Synthesis of Enantiomerically Pure β-Amino Acids▿

PubMed Central

Kim, Juhan; Kyung, Dohyun; Yun, Hyungdon; Cho, Byung-Kwan; Seo, Joo-Hyun; Cha, Minho; Kim, Byung-Gee

2007-01-01

A novel β-transaminase gene was cloned from Mesorhizobium sp. strain LUK. By using N-terminal sequence and an internal protein sequence, a digoxigenin-labeled probe was made for nonradioactive hybridization, and a 2.5-kb gene fragment was obtained by colony hybridization of a cosmid library. Through Southern blotting and sequence analysis of the selected cosmid clone, the structural gene of the enzyme (1,335 bp) was identified, which encodes a protein of 47,244 Da with a theoretical pI of 6.2. The deduced amino acid sequence of the β-transaminase showed the highest sequence similarity with glutamate-1-semialdehyde aminomutase of transaminase subgroup II. The β-transaminase showed higher activities toward d-β-aminocarboxylic acids such as 3-aminobutyric acid, 3-amino-5-methylhexanoic acid, and 3-amino-3-phenylpropionic acid. The β-transaminase has an unusually broad specificity for amino acceptors such as pyruvate and α-ketoglutarate/oxaloacetate. The enantioselectivity of the enzyme suggested that the recognition mode of β-aminocarboxylic acids in the active site is reversed relative to that of α-amino acids. After comparison of its primary structure with transaminase subgroup II enzymes, it was proposed that R43 interacts with the carboxylate group of the β-aminocarboxylic acids and the carboxylate group on the side chain of dicarboxylic α-keto acids such as α-ketoglutarate and oxaloacetate. R404 is another conserved residue, which interacts with the α-carboxylate group of the α-amino acids and α-keto acids. The β-transaminase was used for the asymmetric synthesis of enantiomerically pure β-aminocarboxylic acids. (3S)-Amino-3-phenylpropionic acid was produced from the ketocarboxylic acid ester substrate by coupled reaction with a lipase using 3-aminobutyric acid as amino donor. PMID:17259358

PROLINE OXIDASES IN HANSENULA SUBPELLICULOSA

PubMed Central

Ling, Chung-Mei; Hedrick, L. R.

1964-01-01

Ling, Chung-Mei (Illinois Institute of Technology, Chicago), and L. R. Hedrick. Proline oxidases in Hansenula subpelliculosa. J. Bacteriol. 87:1462–1470. 1964—Cells of Hansenula subpelliculosa can use l-proline as a carbon and a nitrogen source after a 6- to 8-hr induction period. However, they cannot use l-glutamate as both nitrogen and carbon sources unless the induction period is of several days' duration. Two l-proline oxidases were demonstrated in the mitochondrial preparation of this yeast. One forms the product Δ′-pyrroline-2-carboxylic acid (P2C), which is in equilibrium with α-keto-δ-amino-valeric acid; the other forms the product Δ′-pyrroline-5-carboxylic acid (P5C), which is in equilibrium with glutamic-γ-semialdehyde. The first-mentioned enzyme is induced when l-proline is the carbon source; the second appears to be constitutive, and is probably associated with the use of l-proline as a nitrogen source. The P2C-forming enzyme is specific for the l isomer of proline, and is inactive against l-hydroxyproline. The enzyme activity is at its peak when the mitochondria are prepared from logarithmically grown cells, and is rapidly reduced after cells reach the stationary phase of growth. Kinetic studies with varying concentrations of substrate indicate a Michaelis-Menten constant of 2.45 × 10−2m. Paper chromatographic studies, chemical tests with H2O2, sensitivity to freezing, and spectral measurements indicate that proline oxidase from H. subpelliculosa mitochondria forms a product from l-proline which is like, if not identical to, P2C formed by the action of sheep kidney d-proline oxidase upon dl-proline. The soluble portion of the cell extract contains NAD+ enzymes which use either P2C (α-keto-δ-amino-valeric acid) or P5C (glutamic-γ-semialdehyde) as substrates. No glutamic dehydrogenase activity could be detected when l-glutamic acid and the nicotinamide adenine dinucleotide (NAD+) cofactor were added to the supernatant solution with the

Engineering the meso-Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum by Site Saturation Mutagenesis for d-Phenylalanine Synthesis

PubMed Central

Gao, Xiuzhen; Huang, Fang; Feng, Jinhui; Chen, Xi; Zhang, Hailing; Wang, Zhixiang; Wu, Qiaqing

2013-01-01

In order to enlarge the substrate binding pocket of the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum to accommodate larger 2-keto acids, four amino acid residues (Phe146, Thr171, Arg181, and His227) were targeted for site saturation mutagenesis. Among all mutants, the single mutant H227V had a specific activity of 2.39 ± 0.06 U · mg−1, which was 35.1-fold enhancement over the wild-type enzyme. PMID:23728814

[The role of balanced low-protein diet in inhibition of predialysis chronic kidney disease progression in patients with systemic diseases].

PubMed

Milovanov, Iu S; Lysenko, L V; Milovanova, L Iu; Dobrosmyslov, I A

2009-01-01

To evaluate the effects of low-protein diet (LPD) balanced by addition of highly energetic mix and essential keto/amino acids on inhibition of renal failure in patients with systemic diseases with predialysis stages of chronic disease of the kidney (CDK). Forty six patients with stage III--IV of CDK in systemic diseases (33 SLE patients and 13 with systemic vasculitis) were randomized into three groups. Group 1 consisted of 18 patients with CDK (10 with stage III and 8 with stage IV). They received LPD (0.6 g/kg/day) with addition of essential keto/amino acids for 24-48 months. Group 2 of 18 CDK patients with the same stages received the same diet but greater amount of vegetable protein (highly purified soya protein) to 0.3 g/kg/day in highly energetic nutrient mixture. Group 3--10 CDK patients (7 with stage III and 3 with stage IV) received free diet. Group 1 and 2 patients received LPD irrespective of the nutrient status assessed basing on anthropometric and other data. Protein consumption and caloric value were estimated by 3-day food diary. Before diet therapy, out of 46 examinees nutrient status was abnormal in 45.7% patients. Both variants of LPD were well tolerated and nutrient status was corrected while the rate of nutritive disorders in group 3 increased 1.5-fold (from 40 to 60%) with progression of renal failure. Intake of LPD diet for at least a year reduced glomerular filtration rate inhibition, especially in addition of highly energetic mixture. Early (predialysis) restriction of diet protein (0.6 g/kg/day) with addition of highly energetic mixture and essential keto/amino acids improves a nutritive status of CDK patients and inhibits GFR decline.

Metabolism of the Synthetic Progestogen Norethynodrel by Human Ketosteroid Reductases of the Aldo-Keto Reductase Superfamily

PubMed Central

Jin, Yi; Duan, Ling; Chen, Mo; Penning, Trevor M; Kloosterboer, Helenius J.

2012-01-01

Human ketosteroid reductases of the aldo-keto reductase (AKR) superfamily, i.e. AKR1C1-4, are implicated in the biotransformation of synthetic steroid hormones. Norethynodrel (NOR, 17α-ethynyl-17β-hydroxy-estra-5(10)-en-3-one), the progestin component of the first marketed oral contraceptive, is known to undergo rapid and extensive metabolism to 3α- and 3β-hydroxy metabolites. The ability of the four human AKR1C enzymes to catalyze the metabolism of NOR has now been characterized. AKR1C1 and AKR1C2 almost exclusively converted NOR to 3β-hydroxy NOR, while AKR1C3 gave 3β-hydroxy NOR as the main product and AKR1C4 predominantly formed 3α-hydroxy NOR. Individual AKR1C enzymes also displayed distinct kinetic properties in the reaction of NOR. In contrast, norethindrone (NET), the Δ4-isomer of NOR and the most commonly used synthetic progestin, was not a substrate for the AKR1C enzymes. NOR is also structurally identical to the hormone replacement therapeutic tibolone (TIB), except TIB has a methyl group at the 7α-position. Product profiles and kinetic parameters for the reduction of NOR catalyzed by each individual AKR1C isoform were identical to those for the reduction of TIB catalyzed by the respective isoform. These data suggest that the presence of the 7α-methyl group has a minimal effect on the stereochemical outcome of the reaction and kinetic behavior of each enzyme. Results indicate a role of AKR1C in the hepatic and peripheral metabolism of NOR to 3α- and 3β-hydroxy NOR and provide insights into the differential pharmacological properties of NOR, NET and TIB. PMID:22210085

Efficient production of hyperpolarized bicarbonate by chemical reaction on a DNP precursor to measure pH.

PubMed

Ghosh, Rajat K; Kadlecek, Stephen J; Pourfathi, Mehrdad; Rizi, Rahim R

2015-11-01

To produce hyperpolarized bicarbonate indirectly via chemical reaction from a hyperpolarized precursor and utilize it for the simultaneous regional measurement of metabolism and pH. Alpha keto carboxylic acids are first hyperpolarized by dissolution dynamic nuclear polarization (DNP). These precursor molecules are rapidly reacted with hydrogen peroxide (H2O2) to decarboxylate the species, resulting in new target molecules. Unreacted H2O2 is removed from the system by reaction with sulfite. Interrogation of the ratio of dissolved carbon dioxide (CO2) to bicarbonate can be used to determine pH. Conversion of hyperpolarized alpha keto acids to bicarbonate and CO2 results in a minimal loss of the spin order. The reaction can be conducted to completion within seconds and preserves the nuclear spin polarization. Through a rapid chemical reaction, we can conserve the nuclear spin order of a DNP precursor to generate multiple hyperpolarized bioprobes otherwise unamenable to polarization. This indirect technique for the production of hyperpolarized agents can be applied to different precursor compounds to generate additional novel probes. © 2014 Wiley Periodicals, Inc.

Interaction of hydrated electron with dietary flavonoids and phenolic acids: rate constants and transient spectra studied by pulse radiolysis.

PubMed

Cai, Z; Li, X; Katsumura, Y

1999-10-01

The reaction rate constants and transient spectra of 11 flavonoids and 4 phenolic acids reacting with e(aq)- at neutral pH were measured. Absorption bands of the transients of e(aq)- reacting with the above compounds all located at a wavelength shorter than 400 nm. The e(aq)- scavenging abilities were divided into three groups: (+)catechin ((1.2 +/-0.1) x 10(8) M(-1)s(-1)) < 4-chromanol ((4.4 +/- 0.4) x 10(8) M(-1)s(-1)) < genistein ((6.2+/-0.4) x 10(9) M (-1) s(-1) approximately genistin ((8 +/- 1) x 10(9) M(-1)s(-1)) approximately rutin ((7.6 +/- 0.4) x M(-1)s(-1) approximately caffeic acid ((8.3 +/- 0.5) x 10(9)M(-1)s(-1)) < transcinnamic acid((1.1 +/- 0.1) x 10(10) M(-1)s(-1)) approximately p-coumaric acid ((1.1 +/- 0.1) x 10(10) M(-1)s(-1) approximately 2,4,6-trihydroxylbenzoic acid((1.1 +/- 0.1) x 10(10) M(-1)s(-1)) approximately baicalein ((1.1 +/- 0.5) x 10(10) M(-1)s(-1)) approximately baicalin((1.3 + 0.1) X 10(10) M(-1)s(-1)) approximately naringenin ((1.2 +/- 0.1) x 10(10) M(-1)s(-1)) approximately naringin ((1.0 +/- 0.1) x 10(10) M(-1)s(-1)) approximately gossypin((1.2 +/- 0.1) x 10(10) M(-1)s(-1)) approximately quercetin((1.3 +/- 0.5) x 10(10) M(-1)s(-1)). These results suggested that C4 keto group is the active site for e(aq)- to attack on flavonoids and phenolic acids, whereas the o-dihydroxy structure in B ring, the C2,3 double bond, the C3-OH group, and glucosylation, which are key structures that influence the antioxidant activities of flavonoids and phenolic acids, have little effects on the e(aq)- scavenging activities.

Field observation on secondary organic aerosols during Asian dust storm periods: Formation mechanism of oxalic acid and related compounds on dust surface

NASA Astrophysics Data System (ADS)

Wang, Gehui; Cheng, Chunlei; Meng, Jingjing; Huang, Yao; Li, Jianjun; Ren, Yanqin

2015-07-01

Chemical evolution of East Asian dust during transpacific transport has been given much attention for inorganic species such as sulfate, nitrate and ammonium. However, the role of organic species during the transport has almost entirely been ignored. To understand the formation mechanism of secondary organic aerosols (SOA) on dust surfaces, this study investigated the concentrations and compositions of dicarboxylic acids, keto-carboxylic acids, α-dicarbonyls and inorganic ions in size-segregated aerosols (9-stages) collected in Xi'an, central China during the two dust storm episodes in the springs of 2009 and 2011 and compared with those in nondust storm periods. During the events the ambient particulate dicarboxylic acids were 932-2240 ng m-3, which are comparable and even higher than those in nondust periods. Molecular compositions of the above SOA are similar to those in nondust periods with oxalic acid being the leading species. In the presence of the dust storms, all the above mentioned SOA species in Xi'an were predominantly enriched on the coarse particles (>2.1 μm), and oxalic acid well correlated with NO3- (R2 = 0.72, p < 0.001) rather than SO42-. This phenomenon differs greatly from the SOA in any other nondust period that is usually characterized by an enrichment of oxalic acid in fine mode and a strong correlation of oxalic acid with SO42-. We propose a formation pathway to explain these observations, in which nitric acid and/or nitrogen oxides react with dust to produce Ca(NO3)2 and form a liquid phase on the surface of dust aerosols via water vapor-absorption of Ca(NO3)2, followed by a partitioning of the gas-phase water-soluble organic precursors (e.g.,glyoxal and methylglyoxal) into the aqueous-phase and a subsequent oxidation into oxalic acid. To the best of our knowledge, we found for the first time the enrichment of glyoxal and methylglyoxal on dust surface. Our data suggest an important role of nitrate in the heterogeneous formation process of

[Clinical characteristics and analysis of mass spectrometric data in 33 patients with maple syrup urine disease].

PubMed

Yang, Nan; Han, Lian-shu; Ye, Jun; Qiu, Wen-juan; Zhang, Hui-wen; Gao, Xiao-lan; Wang, Yu; Li, Xiao-yan; Xu, Hao; Gu, Xue-fan

2012-10-30

To explore the clinical characteristics and the diagnostic method of maple syrup urine disease (MSUD). From January 2003 to December 2011, a total of 14 000 patients with suspected inherited metabolism diseases were tested. The blood levels of leucine and valine of these patients were detected by tandem mass spectrometry. The urinary level of branched-chain α-ketoacids was tested by gas chromatography-mass spectrometry. And the diagnosis was based on the elevated levels of leucine and valine in blood and branched-chain α-ketoacids in urine. Thirty-three MSUD patients were confirmed. Their median age of initial visit was 0.17 years old (range: 7 days to 30 years old). The peak onset age of them was 2-30 days old, including 28 cases of neonatal onset (84.8%). The presenting symptoms of 28 cases were feeding difficulties (n=14), poor response, lethargy and seizures. Their median blood levels of leucine and valine (1901 (458-5804) and 600 (315-1617) µmol/L) were significantly higher than their normal levels ((50-300) and (60-250) µmol/L, both P<0.01). Their urinary levels of 2-OH-isovaleric acid, 2-keto-isovaleric acid, 2-keto-3-methylvaleric acid, 2-keto-isocaproic and acetylglycine (262.5 (5.4-624.3), 35.8 (1.9-156.0), 133.8 (7.4-611.5), 518.7 (17.2-2121.2) and 280.5 (11.0-1087.9) respectively) significantly higher than their normal levels (0, <0.1, 0, 0, <0.1 respectively, all P<0.01). In 5 intermittent MSUD patients, their blood levels of leucine and valine (402 (348-958) and 556 (322-808) µmol/L) were significantly higher than their normal levels (both P<0.01). The urinary level of 2-OH-isovaleric acid was significantly higher than its normal levels (P<0.01) while the urinary levels of other α-ketoacids were normal. The confirmation of MSUD remains difficult because of a lack of specific clinical features. The detections of tandem mass spectrometry and gas chromatography-mass spectrometry may aid its early diagnosis.

Cyclization of arylacetoacetates to indene and dihydronaphthalene derivatives in strong acids. Evidence for involvement of further protonation of O,O-diprotonated beta-ketoester, leading to enhancement of cyclization.

PubMed

Kurouchi, Hiroaki; Sugimoto, Hiromichi; Otani, Yuko; Ohwada, Tomohiko

2010-01-20

The chemical features, such as substrate stability, product distribution, and substrate generality, and the reaction mechanism of Brønsted superacid-catalyzed cyclization reactions of aromatic ring-containing acetoacetates (beta-ketoesters) were examined in detail. While two types of carbonyl cyclization are possible, i.e., keto cyclization and ester cyclization, the former was found to take place exclusively. The reaction constitutes an efficient method to synthesize indene and 3,4-dihydronapthalene derivatives. Acid-base titration monitored with (13)C NMR spectroscopy showed that the acetoacetates are fully O(1),O(3)-diprotonated at H(0) = -11. While the five-membered ring cyclization of the arylacetoacetates proceeded slowly at H(0) = -11, a linear increase in the rate of the cyclization was found with increasing acidity in the high acidity region of H(0) = -11.8 to -13.3. Therefore, the O(1),O(3)-diprotonated acetoacetates exhibited some cyclizing reactivity, but they are not the reactive intermediates responsible for the acceleration of the cyclization in the high acidity region. The reactive cationic species might be formed by further protonation (or protosolvation) of the O(1),O(3)-diprotonated acetoacetates; i.e., they may be tricationic species. Thermochemical data on the acid-catalyzed cyclization of the arylacetoacetates showed that the activation energy is decreased significantly as compared with that of the related acid-catalyzed cyclization reaction of a compound bearing a single functional group, such as a ketone. These findings indicate that intervention of the trication contributes to the activation of the cyclization of arylacetoacetates in strong acid, and the electron-withdrawing nature of the O-protonated ester functionality significantly increases the electrophilicity of the ketone moiety.

Early-onset and classical forms of type 2 diabetes show impaired expression of genes involved in muscle branched-chain amino acids metabolism.

PubMed

Hernández-Alvarez, María Isabel; Díaz-Ramos, Angels; Berdasco, María; Cobb, Jeff; Planet, Evarist; Cooper, Diane; Pazderska, Agnieszka; Wanic, Krzystof; O'Hanlon, Declan; Gomez, Antonio; de la Ballina, Laura R; Esteller, Manel; Palacin, Manuel; O'Gorman, Donal J; Nolan, John J; Zorzano, Antonio

2017-10-23

The molecular mechanisms responsible for the pathophysiological traits of type 2 diabetes are incompletely understood. Here we have performed transcriptomic analysis in skeletal muscle, and plasma metabolomics from subjects with classical and early-onset forms of type 2 diabetes (T2D). Focused studies were also performed in tissues from ob/ob and db/db mice. We document that T2D, both early and late onset, are characterized by reduced muscle expression of genes involved in branched-chain amino acids (BCAA) metabolism. Weighted Co-expression Networks Analysis provided support to idea that the BCAA genes are relevant in the pathophysiology of type 2 diabetes, and that mitochondrial BCAA management is impaired in skeletal muscle from T2D patients. In diabetic mice model we detected alterations in skeletal muscle proteins involved in BCAA metabolism but not in obese mice. Metabolomic analysis revealed increased levels of branched-chain keto acids (BCKA), and BCAA in plasma of T2D patients, which may result from the disruption of muscle BCAA management. Our data support the view that inhibition of genes involved in BCAA handling in skeletal muscle takes place as part of the pathophysiology of type 2 diabetes, and this occurs both in early-onset and in classical type 2 diabetes.

A DIRECT ROUTE TO ACYLHYDROQUINONES FROM ALPHA-KETO ACIDS AND ALPHA-CARBOXAMIDO ACIDS. (R825330)

EPA Science Inventory

Abstract

The reaction of quinones with in situ generated acyl- or carboxamido radicals provides a direct route to the synthesis of acylhydroquinones not accessible by the photochemical reaction of quinones with aldehydes.

Ethacrynic acid improves the antitumor effects of irreversible epidermal growth factor receptor tyrosine kinase inhibitors in breast cancer

PubMed Central

Hu, YunLong; Chen, TingTing; Peng, BoYa; Gao, NingNing; Jin, ZhenChao; Jia, TieLiu; Zhang, Na; Wang, ZhuLin; Jin, GuangYi

2016-01-01

Prolonged treatment of breast cancer with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) often results in acquired resistance and a narrow therapeutic index. One strategy to improve the therapeutic effects of EGFR TKIs is to combine them with drugs used for other clinical indications. Ethacrynic acid (EA) is an FDA approved drug that may have antitumor effects and may enhance the cytotoxicity of chemotherapeutic agents by binding to glutathione and inhibiting WNT signaling. While the α,β-unsaturated-keto structure of EA is similar to that of irreversible TKIs, the mechanism of action of EA when combined with irreversible EGFR TKIs in breast cancer remains unknown. We therefore investigated the combination of irreversible EGFR TKIs and EA. We found that irreversible EGFR TKIs and EA synergistically inhibit breast cancer both in vitro and in vivo. The combination of EGFR TKIs and EA induces necrosis and cell cycle arrest and represses WNT/β-catenin signaling as well as MAPK-ERK1/2 signaling. We conclude that EA synergistically enhances the antitumor effects of irreversible EGFR TKIs in breast cancer. PMID:27487128

Ethacrynic acid improves the antitumor effects of irreversible epidermal growth factor receptor tyrosine kinase inhibitors in breast cancer.

PubMed

Liu, Bing; Huang, XinPing; Hu, YunLong; Chen, TingTing; Peng, BoYa; Gao, NingNing; Jin, ZhenChao; Jia, TieLiu; Zhang, Na; Wang, ZhuLin; Jin, GuangYi

2016-09-06

Prolonged treatment of breast cancer with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) often results in acquired resistance and a narrow therapeutic index. One strategy to improve the therapeutic effects of EGFR TKIs is to combine them with drugs used for other clinical indications. Ethacrynic acid (EA) is an FDA approved drug that may have antitumor effects and may enhance the cytotoxicity of chemotherapeutic agents by binding to glutathione and inhibiting WNT signaling. While the α,β-unsaturated-keto structure of EA is similar to that of irreversible TKIs, the mechanism of action of EA when combined with irreversible EGFR TKIs in breast cancer remains unknown. We therefore investigated the combination of irreversible EGFR TKIs and EA. We found that irreversible EGFR TKIs and EA synergistically inhibit breast cancer both in vitro and in vivo. The combination of EGFR TKIs and EA induces necrosis and cell cycle arrest and represses WNT/β-catenin signaling as well as MAPK-ERK1/2 signaling. We conclude that EA synergistically enhances the antitumor effects of irreversible EGFR TKIs in breast cancer.

Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsunaga, Toshiyuki, E-mail: matsunagat@gifu-pu.ac.jp; Morikawa, Yoshifumi; Haga, Mariko

2014-07-15

Inhalation of 9,10-phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust, exerts fatal damage against a variety of cells involved in respiratory function. Here, we show that treatment with high concentrations of 9,10-PQ evokes apoptosis of lung cancer A549 cells through production of reactive oxygen species (ROS). In contrast, 9,10-PQ at its concentrations of 2 and 5 μM elevated the potentials for proliferation, invasion, metastasis and tumorigenesis, all of which were almost completely inhibited by addition of an antioxidant N-acetyl-L-cysteine, inferring a crucial role of ROS in the overgrowth and malignant progression of lung cancer cells. Comparison of mRNA expression levelsmore » of six aldo-keto reductases (AKRs) in the 9,10-PQ-treated cells advocated up-regulation of AKR1B10 as a major cause contributing to the lung cancer malignancy. In support of this, the elevation of invasive, metastatic and tumorigenic activities in the 9,10-PQ-treated cells was significantly abolished by the addition of a selective AKR1B10 inhibitor oleanolic acid. Intriguingly, zymographic and real-time PCR analyses revealed remarkable increases in secretion and expression, respectively, of matrix metalloproteinase 2 during the 9,10-PQ treatment, and suggested that the AKR1B10 up-regulation and resultant activation of mitogen-activated protein kinase cascade are predominant mechanisms underlying the metalloproteinase induction. In addition, HPLC analysis and cytochrome c reduction assay in in vitro 9,10-PQ reduction by AKR1B10 demonstrated that the enzyme catalyzes redox-cycling of this quinone, by which ROS are produced. Collectively, these results suggest that AKR1B10 is a key regulator involved in overgrowth and malignant progression of the lung cancer cells through ROS production due to 9,10-PQ redox-cycling. - Highlights: • 9,10-PQ promotes invasion, metastasis and tumorigenicity in lung cancer cells. • The 9,10-PQ-elicited promotion is possibly due to AKR1B10

Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes.

PubMed

Kasparyan, Elena; Richter, Michael; Dresen, Carola; Walter, Lydia S; Fuchs, Georg; Leeper, Finian J; Wacker, Tobias; Andrade, Susana L A; Kolter, Geraldine; Pohl, Martina; Müller, Michael

2014-12-01

The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,β-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon-carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.

Altered macrophage arachidonic acid metabolism induced by endotoxin tolerance: characterization and mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rogers, T.S.

Altered macrophage arachidonic acid (AA) metabolism may play a role in endotoxic shock and the phenomenon of endotoxin tolerance induced by repeated injections of endotoxin. Studies were initiated to characterize both lipoxygenase and cyclooxygenase metabolite formation by endotoxin tolerant and non-tolerant macrophages in response to 4 different stimuli, i.e., endotoxin, glucan, zymosan, and the calcium ionophore A23187. In contrast to previous reports of decreased prostaglandin synthesis by tolerant macrophages, A23187-stimulated immunoreactive (i) leukotriene (LT) C/sub 4/D/sub 4/ and prostaglandin (PG) E/sub 2/ production by tolerant cells was greater than that by non-tolerant controls (p <0.001). However, A23187-stimulated i6-keto PGF/sub 1a/more » levels were lower in tolerant macrophages compared to controls (P < 0.05). iL TC/sub 4/D/sub 4/ production was not significantly stimulated by endotoxin or glucan, but was stimulated by zymosan in non-tolerant cells. Synthesis of iLTB/sub 4/ by control macrophages was stimulated by endotoxin (p <0.01). The effect of tolerance on factors that affect AA release was investigated by measuring /sup 14/C-AA incorporation and release and phospholipase A/sub 2/ activity« less

Regio- and stereo-chemical oxidation of linoleic acid by human myoglobin and hydrogen peroxide: Tyr103 affects rate and product distribution

PubMed Central

2004-01-01

Mb (myoglobin) plus H2O2 catalyses the oxidation of various substrates via a peroxidase-like activity. A Y103F (Tyr103→Phe) variant of human Mb has been constructed to assess the effect of exchanging an electron-rich oxidizable amino acid on the peroxidase activity of human Mb. Steady-state analyses of reaction mixtures containing Y103F Mb, purified linoleic acid and H2O2 revealed a lower total yield of lipid oxidation products than mixtures containing the wild-type protein, consistent with the reported decrease in the rate constant for reaction of Y103F Mb with H2O2 [Witting, Mauk and Lay (2002) Biochemistry 41, 11495–11503]. Irrespective of the Mb employed, lipid oxidation yielded 9(R/S)-HODE [9(R,S)-hydroxy-10E,12Z-octadecadienoic acid] in preference to 13(R/S)-HODE [13(R,S)-hydroxy-9Z,11E-octadecadienoic acid], while 9- and 13-keto-octadecadienoic acid were formed in trace amounts. However, lipid oxidation by the Y103F variant of Mb proceeded with a lower Vmax value and an increased Km value relative to the wild-type control. Consistent with the increased Km, the product distribution from reactions with Y103F Mb showed decreased selectivity compared with the wild-type protein, as judged by the decreased yield of 9(S)-relative to 9(R)-HODE. Together, these data verify that Tyr103 plays a significant role in substrate binding and orientation in the haem pocket of human Mb. Also, the midpoint potential for the Fe(III)/(II) one-electron reduction was shifted slightly, but significantly, to a higher potential, confirming the importance of Tyr103 to the hydrogen-bonding network involving residues that line the haem crevice of human Mb. PMID:15035657

An evaluation of beta-hydroxybutyrate in milk and blood for prediction of subclinical ketosis in dairy cows.

PubMed

Samiei, A; Liang, J B; Ghorbani, G R; Hirooka, H; Yaakub, H; Tabatabaei, M

2010-01-01

The first objective of this study was to investigate the relationship between concentrations of beta-hydroxybutyrate (BHBA) in milk and blood to assess the reliability of the BHBA concentrations in milk measured by a semi quantitative keto-test paper to detect subclinical ketosis (SCK) in 50 fresh high-producing Iranian Holstein cows in Golestan Province, Iran. The second objective was the effects of SCK on milk yield and components. Concentrations of nonesterified fatty acids (NEFA) and BHBA were analyzed quantitatively in blood plasma and commercial keto-test paper was used for semi quantitative determination of BHBA concentration in milk. Milk yield was measured until 60 d after calving but milk compositions were measured until 30 d after calving. The mean plasma BHBA, milk BHBA, plasma NEFA, milk yield, milk fat percentage and milk fat: protein ratio were 1,234 micromol/L, 145 micromol/L, 0.482 mEq/L, 29.5 kg, 3.9% and 1.4, respectively. Fifty eight percent of the cows had SCK during the first month of lactation. High correlation coefficients were observed between blood BHBA and blood NEFA, and between blood and milk BHBA. The milk yield of cattle with SCK decreased (P < 0.01) but the fat percentage and milk fat: protein ratio increased (P < 0.01). The commercial keto-test paper used had a low false positive result at a cut-off point of 200 fmol of BHBA/L of milk. The results showed that the best time to assess SCK using the commercial keto-test paper was d 10, 14 and 17 after calving.

The molecular architecture of QdtA, a sugar 3,4-ketoisomerase from Thermoanaerobacterium thermosaccharolyticum.

PubMed

Thoden, James B; Holden, Hazel M

2014-06-01

Unusual di- and trideoxysugars are often found on the O-antigens of Gram-negative bacteria, on the S-layers of Gram-positive bacteria, and on various natural products. One such sugar is 3-acetamido-3,6-dideoxy-D-glucose. A key step in its biosynthesis, catalyzed by a 3,4-ketoisomerase, is the conversion of thymidine diphosphate (dTDP)-4-keto-6-deoxyglucose to dTDP-3-keto-6-deoxyglucose. Here we report an X-ray analysis of a 3,4-ketoisomerase from Thermoanaerobacterium thermosaccharolyticum. For this investigation, the wild-type enzyme, referred to as QdtA, was crystallized in the presence of dTDP and its structure solved to 2.0-Å resolution. The dimeric enzyme adopts a three-dimensional architecture that is characteristic for proteins belonging to the cupin superfamily. In order to trap the dTDP-4-keto-6-deoxyglucose substrate into the active site, a mutant protein, H51N, was subsequently constructed, and the structure of this protein in complex with the dTDP-sugar ligand was solved to 1.9-Å resolution. Taken together, the structures suggest that His 51 serves as a catalytic base, that Tyr 37 likely functions as a catalytic acid, and that His 53 provides a proton shuttle between the C-3' hydroxyl and the C-4' keto group of the hexose. This study reports the first three-dimensional structure of a 3,4-ketoisomerase in complex with its dTDP-sugar substrate and thus sheds new molecular insight into this fascinating class of enzymes. © 2014 The Protein Society.

Isoenzyme-specific up-regulation of glutathione transferase and aldo-keto reductase mRNA expression by dietary quercetin in rat liver.

PubMed

Odbayar, Tseye-Oidov; Kimura, Toshinori; Tsushida, Tojiro; Ide, Takashi

2009-05-01

The impact of quercetin on the mRNA expression of hepatic enzymes involved in drug metabolism was evaluated with a DNA microarray and real-time PCR. Male Sprague-Dawley rats were fed an experimental diet containing either 0, 2.5, 5, 10, or 20 g/kg of quercetin for 15 days. The DNA microarray analysis of the gene expression profile in pooled RNA samples from rats fed diets containing 0, 5, and 20 g/kg of quercetin revealed genes of some isoenzymes of glutathione transferase (Gst) and aldo-keto reductase (Akr) to be activated by this flavonoid. Real-time PCR conducted with RNA samples from individual rats fed varying amounts of quercetin together with the microarray analysis showed that quercetin caused marked dose-dependent increases in the mRNA expression of Gsta3, Gstp1, and Gstt3. Some moderate increases were also noted in the mRNA expression of isoenzymes belonging to the Gstm class. Quercetin also dose-dependently increased the mRNA expression of Akr1b8 and Akr7a3. However, it did not affect the parameters of the other Gst and Akr isoenzymes. It is apparent that quercetin increases the mRNA expression of Gst and Akr involved in drug metabolism in an isoenzyme-specific manner. Inasmuch as Gst and Akr isoenzymes up-regulated in their gene expression are involved in the prevention and attenuation of cancer development, this consequence may account for the chemopreventive propensity of quercetin.

Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase.

PubMed

Hayashi, Junji; Seto, Tomonari; Akita, Hironaga; Watanabe, Masahiro; Hoshino, Tamotsu; Yoneda, Kazunari; Ohshima, Toshihisa; Sakuraba, Haruhiko

2017-06-01

A stable NADP + -dependent d-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso -diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP + and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d-amino acids, but

Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase

PubMed Central

Hayashi, Junji; Seto, Tomonari; Akita, Hironaga; Watanabe, Masahiro; Hoshino, Tamotsu; Yoneda, Kazunari; Ohshima, Toshihisa

2017-01-01

ABSTRACT A stable NADP+-dependent d-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso-diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP+ and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d

Transcriptional response after exposure to domoic acid-producing Pseudo-nitzschia in the digestive gland of the mussel Mytilus galloprovincialis.

PubMed

Pazos, Antonio J; Ventoso, Pablo; Martínez-Escauriaza, Roi; Pérez-Parallé, M Luz; Blanco, Juan; Triviño, Juan C; Sánchez, José L

2017-12-15

Bivalve molluscs are filter feeding species that can accumulate biotoxins in their body tissues during harmful algal blooms. Amnesic Shellfish Poisoning (ASP) is caused by species of the diatom genus Pseudo-nitzschia, which produces the toxin domoic acid. The Mytilus galloprovincialis digestive gland transcriptome was de novo assembled based on the sequencing of 12 cDNA libraries, six obtained from control mussels and six from mussels naturally exposed to domoic acid-producing diatom Pseudo-nitzschia australis. After de novo assembly 94,727 transcripts were obtained, with an average length of 1015 bp and a N50 length of 761 bp. The assembled transcripts were clustered (homology > 90%) into 69,294 unigenes. Differential gene expression analysis was performed (DESeq2 algorithm) in the digestive gland following exposure to the toxic algae. A total of 1158 differentially expressed unigenes (absolute fold change > 1.5 and p-value < 0.05) were detected: 686 up-regulated and 472 down-regulated. Several membrane transporters belonging to the family of the SLC (solute carriers) were over-expressed in exposed mussels. Functional enrichment was performed using Pfam annotations obtained from the genes differentially expressed, 37 Pfam families were found to be significantly (FDR adjusted p-value < 0.1) enriched. Some of these families (sulfotransferases, aldo/keto reductases, carboxylesterases, C1q domain and fibrinogen C-terminal globular domain) could be putatively involved in detoxification processes, in the response against of the oxidative stress and in immunological processes. Protein network analysis with STRING algorithm found alteration of the Notch signaling pathway under the action of domoic acid-producing Pseudo-nitzschia. In conclusion, this study provides a high quality reference transcriptome of M. galloprovincialis digestive gland and identifies potential genes involved in the response to domoic acid. Copyright © 2017 Elsevier Ltd. All rights reserved.

Methyl Salicylate: A Reactive Chemical Warfare Agent Surrogate to Detect Reaction with Hypochlorite (POSTPRINT)

DTIC Science & Technology

2011-05-01

DMeS11,12 (tR = 14.83 min). The 3,5- DMeS crystallized spontaneously11 as colorless needles, melting point 143 146 C; 1HNMRdata: δ 7.785, 1Hd, J = 2.6 Hz, H...dichloro-phenol. Figure 2 shows two possible mecha- nisms, both promoted by the phenolic oxygen center— tautomerization to a β- keto acid and β

[Acute encephalopathy due to late-onset maple syrup urine disease in a school boy].

PubMed

Qu, Su-Qing; Yang, Li-Cai; Luan, Zuo; Du, Kan; Yang, Hui

2012-03-01

Maple syrup urine disease is a common amino acids metabolic disease. In most patients, onset occurs in the neonatal period and infancy. In this study, the case of a school boy with acute encephalopathy due to late-onset maple syrup urine disease is summarized. The boy (8.5 years) was admitted because of acute encephalopathy after suffering from infection for two days at the age of eight and a half years. Metabolic acidosis, hyperuricemia and decreased protein level in cerebrospinal fluid were found by general laboratory tests. Magnetic resonance imaging of the brain revealed signal intensity abnormalities in the bilateral cerebellum dentate nucleus, brainstem, thalamus, putamen, caudate nucleus and cortex of the cerebral hemispheres. On T1WI and T2WI scanning, hyperintensive signal was found. Blood leucine and valine were significantly elevated. Urinary 2-hydroxy isovaleric acid, 3-hydroxybutyric acid, 2-keto isovaleric acid, and 2-keto acid also increased. Both the blood amino acid and urine organic acid profiles led to the diagnosis of maple syrup urine disease. In the acute period, the patient was treated with a large dose of vitamin B1, glucose, L-carnitine and a protein-restrict diet. The patient's condition improved significantly after five days of treatment, and he recovered completely two days later. Afterwards, treatment with vitamin B1, L-carnitine and a protein-restrict diet (1 g/kg/day) was continued. One and a half months later, blood amino acids and urine organic acids returned to normal. Magnetic resonance imaging of the brain also indicated a great improvement. It was concluded that inborn metabolic disease should be considered in the patients with an onset similar to acute encephalopathy. Early diagnosis and proper treatment can prevent brain damage and improve prognosis.

Redesigning Aldolase Stereoselectivity by Homologous Grafting.

PubMed

Bisterfeld, Carolin; Classen, Thomas; Küberl, Irene; Henßen, Birgit; Metz, Alexander; Gohlke, Holger; Pietruszka, Jörg

2016-01-01

The 2-deoxy-d-ribose-5-phosphate aldolase (DERA) offers access to highly desirable building blocks for organic synthesis by catalyzing a stereoselective C-C bond formation between acetaldehyde and certain electrophilic aldehydes. DERA´s potential is particularly highlighted by the ability to catalyze sequential, highly enantioselective aldol reactions. However, its synthetic use is limited by the absence of an enantiocomplementary enzyme. Here, we introduce the concept of homologous grafting to identify stereoselectivity-determining amino acid positions in DERA. We identified such positions by structural analysis of the homologous aldolases 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG) and the enantiocomplementary enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase (KDPGal). Mutation of these positions led to a slightly inversed enantiopreference of both aldolases to the same extent. By transferring these sequence motifs onto DERA we achieved the intended change in enantioselectivity.

LC/MSMS STUDY OF BENZO[A]PYRENE-7,8-QUINONE ADDUCTION TO GLOBIN TRYPTIC PEPTIDES AND N-ACETYLAMINO ACIDS

EPA Science Inventory

Benzo[a]pyrene-7,8-quinone (BPQ) is regarded as a reactive genotoxic compound enzymatically formed from a xenobiotic precursor benzo[a]pyrene-7,8-diol by aldo-keto-reductase family of enzymes. Because BPQ, a Michael electrophile, was previously shown to react with oligonucleotide...

Synthesis, structure and study of azo-hydrazone tautomeric equilibrium of 1,3-dimethyl-5-(arylazo)-6-amino-uracil derivatives.

PubMed

Debnath, Diptanu; Roy, Subhadip; Li, Bing-Han; Lin, Chia-Her; Misra, Tarun Kumar

2015-04-05

Azo dyes, 1,3-dimethyl-5-(arylazo)-6-aminouracil (aryl=-C6H5 (1), -p-CH3C6H4 (2), -p-ClC6H4 (3), -p-NO2C6H4 (4)) were prepared and characterized by UV-vis, FT-IR, 1H NMR, 13C NMR spectroscopic techniques and single crystal X-ray crystallographic analysis. In the light of spectroscopic analysis it evidences that of the tautomeric forms, the azo-enamine-keto (A) form is the predominant form in the solid state whereas in different solvents it is the hydrazone-imine-keto (B) form. The study also reveals that the hydrazone-imine-keto (B) form exists in an equilibrium mixture with its anionic form in various organic solvents. The solvatochromic and photophysical properties of the dyes in various solvents with different hydrogen bonding parameter were investigated. The dyes exhibit positive solvatochromic property on moving from polar protic to polar aprotic solvents. They are fluorescent active molecules and exhibit high intense fluorescent peak in some solvents like DMSO and DMF. It has been demonstrated that the anionic form of the hydrazone-imine form is responsible for the high intense fluorescent peak. In addition, the acid-base equilibrium in between neutral and anionic form of hydrazone-imine form in buffer solution of varying pH was investigated and evaluated the pKa values of the dyes by making the use of UV-vis spectroscopic methods. The determined acid dissociation constant (pKa) values increase according to the sequence of 2>1>3>4. Copyright © 2014 Elsevier B.V. All rights reserved.

Ethylene induced plant stress tolerance by Enterobacter sp. SA187 is mediated by 2‐keto‐4‐methylthiobutyric acid production

PubMed Central

Xie, Yakun; Rolli, Eleonora; Guerard, Florence; Colcombet, Jean; Benhamed, Moussa; Depaepe, Thomas

2018-01-01

Several plant species require microbial associations for survival under different biotic and abiotic stresses. In this study, we show that Enterobacter sp. SA187, a desert plant endophytic bacterium, enhances yield of the crop plant alfalfa under field conditions as well as growth of the model plant Arabidopsis thaliana in vitro, revealing a high potential of SA187 as a biological solution for improving crop production. Studying the SA187 interaction with Arabidopsis, we uncovered a number of mechanisms related to the beneficial association of SA187 with plants. SA187 colonizes both the surface and inner tissues of Arabidopsis roots and shoots. SA187 induces salt stress tolerance by production of bacterial 2-keto-4-methylthiobutyric acid (KMBA), known to be converted into ethylene. By transcriptomic, genetic and pharmacological analyses, we show that the ethylene signaling pathway, but not plant ethylene production, is required for KMBA-induced plant salt stress tolerance. These results reveal a novel molecular communication process during the beneficial microbe-induced plant stress tolerance. PMID:29554117

Molecular description of α-keto-based inhibitors of cruzain with activity against Chagas disease combining 3D-QSAR studies and molecular dynamics.

PubMed

Saraiva, Ádria P B; Miranda, Ricardo M; Valente, Renan P P; Araújo, Jéssica O; Souza, Rutelene N B; Costa, Clauber H S; Oliveira, Amanda R S; Almeida, Michell O; Figueiredo, Antonio F; Ferreira, João E V; Alves, Cláudio Nahum; Honorio, Kathia M

2018-04-22

In this work, a group of α-keto-based inhibitors of the cruzain enzyme with anti-chagas activity was selected for a three-dimensional quantitative structure-activity relationship study (3D-QSAR) combined with molecular dynamics (MD). Firstly, statistical models based on Partial Least Square (PLS) regression were developed employing comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) descriptors. Validation parameters (q 2 and r 2 )for the models were, respectively, 0.910 and 0.997 (CoMFA) and 0.913 and 0.992 (CoMSIA). In addition, external validation for the models using a test group revealed r 2 pred  = 0.728 (CoMFA) and 0.971 (CoMSIA). The most relevant aspect in this study was the generation of molecular fields in both favorable and unfavorable regions based on the models developed. These fields are important to interpret modifications necessary to enhance the biological activities of the inhibitors. This analysis was restricted considering the inhibitors in a fixed conformation, not interacting with their target, the cruzain enzyme. Then, MD was employed taking into account important variables such as time and temperature. MD helped describe the behavior of the inhibitors and their properties showed similar results as those generated by QSAR-3D study. © 2018 John Wiley & Sons A/S.

Keto-adaptation enhances exercise performance and body composition responses to training in endurance athletes.

PubMed

McSwiney, Fionn T; Wardrop, Bruce; Hyde, Parker N; Lafountain, Richard A; Volek, Jeff S; Doyle, Lorna

2018-04-01

Low-carbohydrate diets have recently grown in popularity among endurance athletes, yet little is known about the long-term (>4wk) performance implications of consuming a low-carbohydrate high fat ketogenic diet (LCKD) in well-trained athletes. Twenty male endurance-trained athletes (age 33±11y, body mass 80±11kg; BMI 24.7±3.1kg/m 2 ) who habitually consumed a carbohydrate-based diet, self-selected into a high-carbohydrate (HC) group (n=11, %carbohydrate:protein:fat=65:14:20), or a LCKD group (n=9, 6:17:77). Both groups performed the same training intervention (endurance, strength and high intensity interval training (HIIT)). Prior to and following successful completion of 12-weeks of diet and training, participants had their body composition assessed, and completed a 100km time trial (TT), six second (SS) sprint, and a critical power test (CPT). During post-intervention testing the HC group consumed 30-60g/h carbohydrate, whereas the LCKD group consumed water, and electrolytes. The LCKD group experienced a significantly greater decrease in body mass (HC -0.8kg, LCKD -5.9kg; P=0.006, effect size (ES): 0.338) and percentage body fat percentage (HC -0.7%, LCKD -5.2%; P=0.008, ES: 0.346). Fasting serum beta-hydroxybutyrate (βHB) significantly increased from 0.1 at baseline to 0.5mmol/L in the LCKD group (P=0.011, ES: 0.403) in week 12. There was no significant change in performance of the 100km TT between groups (HC -1.13min·s, LCKD -4.07min·s, P=0.057, ES: 0.196). SS sprint peak power increased by 0.8 watts per kilogram bodyweight (w/kg) in the LCKD group, versus a -0.1w/kg reduction in the HC group (P=0.025, ES: 0.263). CPT peak power decreased by -0.7w/kg in the HC group, and increased by 1.4w/kg in the LCKD group (P=0.047, ES: 0.212). Fat oxidation in the LCKD group was significantly greater throughout the 100km TT. Compared to a HC comparison group, a 12-week period of keto-adaptation and exercise training, enhanced body composition, fat oxidation during

Alterations in protein and amino acid metabolism in rats fed a branched-chain amino acid- or leucine-enriched diet during postprandial and postabsorptive states.

PubMed

Holecek, Milan; Siman, Pavel; Vodenicarovova, Melita; Kandar, Roman

2016-01-01

Many people believe in favourable effects of branched-chain amino acids (BCAAs; valine, leucine, and isoleucine), especially leucine, on muscle protein balance and consume BCAAs for many years. We determined the effects of the chronic intake of a BCAA- or leucine-enriched diet on protein and amino acid metabolism in fed and postabsorptive states. Rats were fed a standard diet, a diet with a high content of valine, leucine, and isoleucine (HVLID), or a high content of leucine (HLD) for 2 months. Half of the animals in each group were sacrificed in the fed state on the last day, and the other half were sacrificed after overnight fast. Protein synthesis was assessed using the flooding dose method (L-[3,4,5-(3)H]phenylalanine), proteolysis on the basis of chymotrypsin-like activity (CHTLA) of proteasome and cathepsin B and L activities. Chronic intake of HVLID or HLD enhanced plasma levels of urea, alanine and glutamine. HVLID also increased levels of all three BCAA and branched-chain keto acids (BCKA), HLD increased leucine, ketoisocaproate and alanine aminotransferase and decreased valine, ketovaline, isoleucine, ketoisoleucine, and LDL cholesterol. Tissue weight and protein content were lower in extensor digitorum longus muscles in the HLD group and higher in kidneys in the HVLID and HLD groups. Muscle protein synthesis in postprandial state was higher in the HVLID group, and CHTLA was lower in muscles of the HVLID and HLD groups compared to controls. Overnight starvation enhanced alanine aminotransferase activity in muscles, and decreased protein synthesis in gastrocnemius (in HVLID group) and extensor digitorum longus (in HLD group) muscles more than in controls. Effect of HVLID and HLD on CHTLA in muscles in postabsorptive state was insignificant. The results failed to demonstrate positive effects of the chronic consumption of a BCAA-enriched diet on protein balance in skeletal muscle and indicate rather negative effects from a leucine-enriched diet. The primary

Branched-chain amino acid restriction in Zucker-fatty rats improves muscle insulin sensitivity by enhancing efficiency of fatty acid oxidation and acyl-glycine export.

PubMed

White, Phillip J; Lapworth, Amanda L; An, Jie; Wang, Liping; McGarrah, Robert W; Stevens, Robert D; Ilkayeva, Olga; George, Tabitha; Muehlbauer, Michael J; Bain, James R; Trimmer, Jeff K; Brosnan, M Julia; Rolph, Timothy P; Newgard, Christopher B

2016-07-01

A branched-chain amino acid (BCAA)-related metabolic signature is strongly associated with insulin resistance and predictive of incident diabetes and intervention outcomes. To better understand the role that this metabolite cluster plays in obesity-related metabolic dysfunction, we studied the impact of BCAA restriction in a rodent model of obesity in which BCAA metabolism is perturbed in ways that mirror the human condition. Zucker-lean rats (ZLR) and Zucker-fatty rats (ZFR) were fed either a custom control, low fat (LF) diet, or an isonitrogenous, isocaloric LF diet in which all three BCAA (Leu, Ile, Val) were reduced by 45% (LF-RES). We performed comprehensive metabolic and physiologic profiling to characterize the effects of BCAA restriction on energy balance, insulin sensitivity, and glucose, lipid and amino acid metabolism. LF-fed ZFR had higher levels of circulating BCAA and lower levels of glycine compared to LF-fed ZLR. Feeding ZFR with the LF-RES diet lowered circulating BCAA to levels found in LF-fed ZLR. Activity of the rate limiting enzyme in the BCAA catabolic pathway, branched chain keto acid dehydrogenase (BCKDH), was lower in liver but higher in skeletal muscle of ZFR compared to ZLR and was not responsive to diet in either tissue. BCAA restriction had very little impact on metabolites studied in liver of ZFR where BCAA content was low, and BCKDH activity was suppressed. However, in skeletal muscle of LF-fed ZFR compared to LF-fed ZLR, where BCAA content and BCKDH activity were increased, accumulation of fatty acyl CoAs was completely normalized by dietary BCAA restriction. BCAA restriction also normalized skeletal muscle glycine content and increased urinary acetyl glycine excretion in ZFR. These effects were accompanied by lower RER and improved skeletal muscle insulin sensitivity in LF-RES fed ZFR as measured by hyperinsulinemic-isoglycemic clamp. Our data are consistent with a model wherein elevated circulating BCAA contribute to development of

Vegetarian low-protein diets supplemented with keto analogues: a niche for the few or an option for many?

PubMed

Piccoli, Giorgina B; Ferraresi, Martina; Deagostini, Maria C; Vigotti, Federica Neve; Consiglio, Valentina; Scognamiglio, Stefania; Moro, Irene; Clari, Roberta; Fassio, Federica; Biolcati, Marilisa; Porpiglia, Francesco

2013-09-01

Low-protein diets are often mentioned but seldom used to slow chronic kidney disease (CKD) progression. The aim of the study was to investigate the potential for implementation of a simplified low-protein diet supplemented with alpha-keto analogues (LPD-KA) as part of the routine work-up in CKD patients. In an implementation study (December 2007-November 2011), all patients with CKD Stages IV-V not on dialysis, rapidly progressive Stage III and/or refractory proteinuria, were offered either a simplified LPD-KA, or commercially available low-protein food. LPD-KA consisted of proteins 0.6 g/kg/day, supplementation with Ketosteril 1 pill/10 Kg, 1-3 free-choice meals/week and a simplified schema based on 'allowed' and 'forbidden' foods. 'Success' was defined as at least 6 months on LPD-KA. Progression was defined as reduction in glomerular filtration rate (GFR)[(Chronic Kidney Disease Epidemiology Collaboration) formula CKD-EPI] in patients with at least 6 months of follow-up. Of about 2500 patients referred (8% CKD Stages IV-V), 139 started LPD-KA; median age (70 years) and prevalence of comorbidity (79%) were in line with the dialysis population. Start of dialysis was the main reason for discontinuation (40 cases, unplanned in 7); clinical reasons were recorded in 7, personal preference in 14 and improvement and death in 8 each. The low gross mortality (4% per year) and the progression rate (from -8 to 0 mL/min/year at 6 months) are reassuring concerning safety. None of the baseline conditions, including age, educational level, comorbidity or kidney function, discriminated the patients who followed the diet for at least 6 months. Our data suggest a wider offer of LPD-KA to patients with severe and progressive CKD. The promising results in terms of mortality and progression need confirmation with different study designs.

STUDIES ON MAMMALIAN AND HUMAN PYRUVATE AND ALPHA-KETOGLUTARATE DEHYDROGENATION COMPLEXES.

DTIC Science & Technology

Enzyme systems that catalyze a coenzyme A- and nicotinamide adenine dinucleotide-linked oxidative decarboxylation of pyruvate and alpha - ketoglutarate ...The pig heart pyruvate dehydrogenase complex was strongly inhibited by EDTA at low concentration, but the pig heart alpha - ketoglutarate ...On the oxidative decarboxylation of alpha -keto acids in pig heart complexes, Ca(2+) was strongly stimulatory to the same or more extent than Mg(2

Serum Markers of Neurodegeneration in Maple Syrup Urine Disease.

PubMed

Scaini, Giselli; Tonon, Tássia; de Souza, Carolina F Moura; Schuk, Patricia F; Ferreira, Gustavo C; Neto, Joao Seda; Amorin, Tatiana; Schwartz, Ida Vanessa D; Streck, Emilio L

2017-09-01

Maple syrup urine disease (MSUD) is an inherited disorder caused by deficient activity of the branched-chain α-keto acid dehydrogenase complex involved in the degradation pathway of branched-chain amino acids (BCAAs) and their respective α-keto-acids. Patients affected by MSUD present severe neurological symptoms and brain abnormalities, whose pathophysiology is poorly known. However, preclinical studies have suggested alterations in markers involved with neurodegeneration. Because there are no studies in the literature that report the neurodegenerative markers in MSUD patients, the present study evaluated neurodegenerative markers (brain-derived neurotrophic factor (BDNF), cathepsin D, neural cell adhesion molecule (NCAM), plasminogen activator inhibitor-1 total (PAI-1 (total)), platelet-derived growth factor AA (PDGF-AA), PDGF-AB/BB) in plasma from 10 MSUD patients during dietary treatment. Our results showed a significant decrease in BDNF and PDGF-AA levels in MSUD patients. On the other hand, NCAM and cathepsin D levels were significantly greater in MSUD patients compared to the control group, while no significant changes were observed in the levels of PAI-1 (total) and PDGF-AB/BB between the control and MSUD groups. Our data show that MSUD patients present alterations in proteins involved in the neurodegenerative process. Thus, the present findings corroborate previous studies that demonstrated that neurotrophic factors and lysosomal proteases may contribute, along with other mechanisms, to the intellectual deficit and neurodegeneration observed in MSUD.

Molecular modelling for the design of chimaeric biomimetic dye-ligands and their interaction with bovine heart mitochondrial malate dehydrogenase.

PubMed Central

Labrou, N E; Eliopoulos, E; Clonis, Y D

1996-01-01

Molecular modelling and kinetic inhibition studies, as well as KD determinations by both difference-spectra and enzyme-inactivation studies, were employed to assess the ability of purpose-designed chimaeric biomimetic dyes (BM dyes) to act as affinity ligands for bovine heart L-malate dehydrogenase (MDH). Each BM dye was composed of two enzyme-recognition moieties. The terminal biomimetic moiety bore a carboxyl or a keto acid structure linked to the triazine ring, thus mimicking the substrate of MDH. The chromophore anthraquinone moiety remained unchanged and the same as that of the parent dye Vilmafix Blue A-R (VBAR), recognizing the nucleotide-binding site of MDH. The monochlorotriazine BM dyes did not inactivate MDH but competitively inhibited inactivation by the parent dichlorotriazine dye VBAR. Dye binding to MDH was accompanied by a characteristic spectral change in the range 500-850 nm. This phenomenon was reversed after titration with increasing amounts of NADH. When compared with VBAR, Cibacron Blue 3GA and two control non-biomimetic anthraquinone dyes, all BM dyes exhibited lower KD values and therefore higher affinity for MDH. The enzyme bound preferably to BM ligands substituted with a biomimetic aromatic moiety bearing an alpha-keto acid group and an amide linkage, rather than a monocarboxyl group. Thus the biomimetic dye bearing p-aminobenzyloxanilic acid as its terminal biomimetic moiety (BM5) exhibited the highest affinity (KD 1.3 microM, which corresponded to a 219-fold decrease over the KD of a control dye). BM5 displayed competitive inhibition with respect to both NADH (Ki 2.7 microM) and oxaloacetate (Ki 9.6 microM). A combination of molecular modelling and experimental studies has led to certain conclusions. The positioning of the dye in the enzyme is primarily achieved by the recognition and positioning of the nucleotide-pseudomimetic anthraquinone moiety. The hydrophobic groups of the dye provide the driving force for positioning of the

78 FR 21818 - Schedules of Controlled Substances: Placement of Methylone Into Schedule I

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-12

...- methylenedioxymethamphetamine (MDMA), cathinone and other related substances. The addition of a beta-keto ([beta]-ketone) substituent to the phenethylamine core structure produces a group of substances that have [beta]-keto-phenethylamine as the core structure. Methylone has a [beta]-keto-phenethylamine core structure. Methylone has...

A novel aldo-keto reductase from Jatropha curcas L. (JcAKR) plays a crucial role in the detoxification of methylglyoxal, a potent electrophile.

PubMed

Mudalkar, Shalini; Sreeharsha, Rachapudi Venkata; Reddy, Attipalli Ramachandra

2016-05-20

Abiotic stress leads to the generation of reactive oxygen species (ROS) which further results in the production of reactive carbonyls (RCs) including methylglyoxal (MG). MG, an α, β-dicarbonyl aldehyde, is highly toxic to plants and the mechanism behind its detoxification is not well understood. Aldo-keto reductases (AKRs) play a role in detoxification of reactive aldehydes and ketones. In the present study, we cloned and characterised a putative AKR from Jatropha curcas (JcAKR). Phylogenetically, it forms a small clade with AKRs of Glycine max and Rauwolfia serpentina. JcAKR was heterologously expressed in Escherichia coli BL-21(DE3) cells and the identity of the purified protein was confirmed through MALDI-TOF analysis. The recombinant protein had high enzyme activity and catalytic efficiency in assays containing MG as the substrate. Protein modelling and docking studies revealed MG was efficiently bound to JcAKR. Under progressive drought and salinity stress, the enzyme and transcript levels of JcAKR were higher in leaves compared to roots. Further, the bacterial and yeast cells expressing JcAKR showed more tolerance towards PEG (5%), NaCl (200mM) and MG (5mM) treatments compared to controls. In conclusion, our results project JcAKR as a possible and potential target in crop improvement for abiotic stress tolerance. Copyright © 2016 Elsevier GmbH. All rights reserved.

Interaction Between Domperidone and Ketoconazole: Toward Prediction of Consequent QTc Prolongation Using Purely In Vitro Information

PubMed Central

Mishra, H; Polak, S; Jamei, M; Rostami-Hodjegan, A

2014-01-01

We aimed to investigate the application of combined mechanistic pharmacokinetic (PK) and pharmacodynamic (PD) modeling and simulation in predicting the domperidone (DOM) triggered pseudo-electrocardiogram modification in the presence of a CYP3A inhibitor, ketoconazole (KETO), using in vitro–in vivo extrapolation. In vitro metabolic and inhibitory data were incorporated into physiologically based pharmacokinetic (PBPK) models within Simcyp to simulate time course of plasma DOM and KETO concentrations when administered alone or in combination with KETO (DOM+KETO). Simulated DOM concentrations in plasma were used to predict changes in gender-specific QTcF (Fridericia correction) intervals within the Cardiac Safety Simulator platform taking into consideration DOM, KETO, and DOM+KETO triggered inhibition of multiple ionic currents in population. Combination of in vitro–in vivo extrapolation, PBPK, and systems pharmacology of electric currents in the heart was able to predict the direction and magnitude of PK and PD changes under coadministration of the two drugs although some disparities were detected. PMID:25116274

High Leucine Diets Stimulate Cerebral Branched-Chain Amino Acid Degradation and Modify Serotonin and Ketone Body Concentrations in a Pig Model

PubMed Central

Wessels, Anna G.; Kluge, Holger; Hirche, Frank; Kiowski, Andreas; Schutkowski, Alexandra; Corrent, Etienne; Bartelt, Jörg; König, Bettina; Stangl, Gabriele I.

2016-01-01

In addition to its role as an essential protein component, leucine (Leu) displays several other metabolic functions such as activation of protein synthesis. This property makes it an interesting amino acid for the therapy of human muscle atrophy and for livestock production. However, Leu can stimulate its own degradation via the branched-chain keto acid dehydrogenase complex (BCKDH). To examine the response of several tissues to excessive Leu, pigs were fed diets containing two- (L2) and four-fold (L4) higher Leu contents than the recommended amount (control). We found that the L4 diet led to a pronounced increase in BCKDH activity in the brain (2.5-fold, P < 0.05), liver (1.8-fold, P < 0.05) and cardiac muscle (1.7-fold, P < 0.05), whereas we found no changes in enzyme activity in the pancreas, skeletal muscle, adipose tissue and intestinal mucosa. The L2 diet had only weak effects on BCKDH activity. Both high Leu diets reduced the concentrations of free valine and isoleucine in nearly all tissues. In the brain, high Leu diets modified the amount of tryptophan available: for serotonin synthesis. Compared to the controls, pigs treated with the high Leu diets consumed less food, showed increased plasma concentrations of 3-hydroxybutyrate and reduced levels of circulating serotonin. In conclusion, excessive Leu can stimulate BCKDH activity in several tissues, including the brain. Changes in cerebral tryptophan, along with the changes in amino acid-derived metabolites in the plasma may limit the use of high Leu diets to treat muscle atrophy or to increase muscle growth. PMID:26930301

Characterization of hamster NAD+-dependent 3(17)β-hydroxysteroid dehydrogenase belonging to the aldo-keto reductase 1C subfamily.

PubMed

Endo, Satoshi; Noda, Misato; Ikari, Akira; Tatematsu, Kenjiro; El-Kabbani, Ossama; Hara, Akira; Kitade, Yukio; Matsunaga, Toshiyuki

2015-11-01

The cDNAs for morphine 6-dehydrogenase (AKR1C34) and its homologous aldo-keto reductase (AKR1C35) were cloned from golden hamster liver, and their enzymatic properties and tissue distribution were compared. AKR1C34 and AKR1C35 similarly oxidized various xenobiotic alicyclic alcohols using NAD(+), but differed in their substrate specificity for hydroxysteroids and inhibitor sensitivity. While AKR1C34 showed 3α/17β/20α-hydroxysteroid dehydrogenase activities, AKR1C35 efficiently oxidized various 3β- and 17β-hydroxysteroids, including biologically active 3β-hydroxy-5α/β-dihydro-C19/C21-steroids, dehydroepiandrosterone and 17β-estradiol. AKR1C35 also differed from AKR1C34 in its high sensitivity to flavonoids, which inhibited competitively with respect to 17β-estradiol (Ki 0.11-0.69 μM). The mRNA for AKR1C35 was expressed liver-specific in male hamsters and ubiquitously in female hamsters, whereas the expression of the mRNA for AKR1C34 displayed opposite sexual dimorphism. Because AKR1C35 is the first 317Β-HYDROXYSTEROID DEHYDROGENASE IN THE AKR SUPERFAMILY: , we also investigated the molecular determinants for the 3β-hydroxysteroid dehydrogenase activity by replacement of Val54 and Cys310 in AKR1C35 with the corresponding residues in AKR1C34, Ala and Phe, respectively. The mutation of Val54Ala, but not Cys310Phe, significantly impaired this activity, suggesting that Val54 plays a critical role in recognition of the steroidal substrate. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Fluoxetine elevates allopregnanolone in female rat brain but inhibits a steroid microsomal dehydrogenase rather than activating an aldo-keto reductase

PubMed Central

Fry, J P; Li, K Y; Devall, A J; Cockcroft, S; Honour, J W; Lovick, T A

2014-01-01

Background and Purpose Fluoxetine, a selective serotonin reuptake inhibitor, elevates brain concentrations of the neuroactive progesterone metabolite allopregnanolone, an effect suggested to underlie its use in the treatment of premenstrual dysphoria. One report showed fluoxetine to activate the aldo-keto reductase (AKR) component of 3α-hydroxysteroid dehydrogenase (3α-HSD), which catalyses production of allopregnanolone from 5α-dihydroprogesterone. However, this action was not observed by others. The present study sought to clarify the site of action for fluoxetine in elevating brain allopregnanolone. Experimental Approach Adult male rats and female rats in dioestrus were treated with fluoxetine and their brains assayed for allopregnanolone and its precursors, progesterone and 5α-dihydroprogesterone. Subcellular fractions of rat brain were also used to investigate the actions of fluoxetine on 3α-HSD activity in both the reductive direction, producing allopregnanolone from 5α-dihydroprogesterone, and the reverse oxidative direction. Fluoxetine was also tested on these recombinant enzyme activities expressed in HEK cells. Key Results Short-term treatment with fluoxetine increased brain allopregnanolone concentrations in female, but not male, rats. Enzyme assays on native rat brain fractions and on activities expressed in HEK cells showed fluoxetine did not affect the AKR producing allopregnanolone from 5α-dihydroprogesterone but did inhibit the microsomal dehydrogenase oxidizing allopregnanolone to 5α-dihydroprogesterone. Conclusions and Implications Fluoxetine elevated allopregnanolone in female rat brain by inhibiting its oxidation to 5α-dihydroprogesterone by a microsomal dehydrogenase. This is a novel site of action for fluoxetine, with implications for the development of new agents and/or dosing regimens to raise brain allopregnanolone. PMID:25161074

Latent class evaluation of a milk test, a urine test, and the fat-to-protein percentage ratio in milk to diagnose ketosis in dairy cows.

PubMed

Krogh, M A; Toft, N; Enevoldsen, C

2011-05-01

In this study, 3 commonly used tests to diagnose ketosis were evaluated with a latent class model to avoid the assumption of an available perfect test. The 3 tests were the KetoLac BHB (Sanwa Kagaku Kenkyusho Co. Ltd., Nagoya, Japan) test strip that tests milk for β-hydroxybutyrate, the KetoStix (Bayer Diagnostics Europe Ltd., Dublin, Ireland) test strip that tests urine for acetoacetate, and the fat-to-protein percentage ratio (FPR) in milk. A total of 8,902 cows were included in the analysis. The cows were considered to be a random sample from the population of Danish dairy cattle under intensive management, thus representing a natural spectrum of ketosis as a disease. All cows had a recorded FPR between 7 and 21 d postpartum. The KetoLac BHB recordings were available from 2,257 cows and 6,645 cows had a KetoStix recording. The recordings were analyzed with a modified Hui-Walter model, in a Bayesian framework. The specificity of the KetoLac BHB test and the KetoStix test were both high [0.99 (0.97-0.99)], whereas the specificity of FPR was somewhat lower [0.79 (0.77-0.81)]. The best sensitivity was for the KetoStix test [0.78 (0.55-0.98)], followed by the FPR [0.63 (0.58-0.71)] and KetoLac BHB test [0.58 (0.35-0.93)]. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

French, Jarrod B.; Ealick, Steven E.

Many plants, fungi, and bacteria catabolize allantoin as a mechanism for nitrogen assimilation. Recent reports have shown that in plants and some bacteria the product of hydrolysis of allantoin by allantoinase is the unstable intermediate ureidoglycine. While this molecule can spontaneously decay, genetic analysis of some bacterial genomes indicates that an aminotransferase may be present in the pathway. Here we present evidence that Klebsiella pneumoniae HpxJ is an aminotransferase that preferentially converts ureidoglycine and an {alpha}-keto acid into oxalurate and the corresponding amino acid. We determined the crystal structure of HpxJ, allowing us to present an explanation for substrate specificity.

Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose▿

PubMed Central

Higashide, Wendy; Li, Yongchao; Yang, Yunfeng; Liao, James C.

2011-01-01

Producing biofuels directly from cellulose, known as consolidated bioprocessing, is believed to reduce costs substantially compared to a process in which cellulose degradation and fermentation to fuel are accomplished in separate steps. Here we present a metabolic engineering example for the development of a Clostridium cellulolyticum strain for isobutanol synthesis directly from cellulose. This strategy exploits the host's natural cellulolytic activity and the amino acid biosynthesis pathway and diverts its 2-keto acid intermediates toward alcohol synthesis. Specifically, we have demonstrated the first production of isobutanol to approximately 660 mg/liter from crystalline cellulose by using this microorganism. PMID:21378054

Molecular Mechanism by Which Retinoids Prevent Breast Cancer Development

DTIC Science & Technology

2007-06-01

10.11 AFFX-r2-Ec- bioB -M_at E. coli /GEN= bioB /DB_XREF=gb:J04423.1 /NOTE=SIF corresponding to nucleotides 2393-2682 of gb:J04423.1 /DEF=E.coli...7,8-diamino-pelargonic acid (bioA), biotin synthetase ( bioB ), 7-keto-8-amino-pelargonic acid synthetase (bioF), bioC protein, and dethiobiot 9.76...9.42 AFFX- BioB - M_at E. coli /GEN= bioB /DB_XREF=gb:J04423.1 /NOTE=SIF corresponding to nucleotides 2482-2739 of gb:J04423.1 /DEF=E.coli 7,8

Gluconacetobacter medellinensis sp. nov., cellulose- and non-cellulose-producing acetic acid bacteria isolated from vinegar.

PubMed

Castro, Cristina; Cleenwerck, Ilse; Trcek, Janja; Zuluaga, Robin; De Vos, Paul; Caro, Gloria; Aguirre, Ricardo; Putaux, Jean-Luc; Gañán, Piedad

2013-03-01

The phylogenetic position of a cellulose-producing acetic acid bacterium, strain ID13488, isolated from commercially available Colombian homemade fruit vinegar, was investigated. Analyses using nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rRNA gene internal transcribed spacer (ITS) sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated the micro-organism to the genus Gluconacetobacter, and more precisely to the Gluconacetobacter xylinus group. Moreover, the data suggested that the micro-organism belongs to a novel species in this genus, together with LMG 1693(T), a non-cellulose-producing strain isolated from vinegar by Kondo and previously classified as a strain of Gluconacetobacter xylinus. DNA-DNA hybridizations confirmed this finding, revealing a DNA-DNA relatedness value of 81 % between strains ID13488 and LMG 1693(T), and values <70 % between strain LMG 1693(T) and the type strains of the closest phylogenetic neighbours. Additionally, the classification of strains ID13488 and LMG 1693(T) into a single novel species was supported by amplified fragment length polymorphism (AFLP) and (GTG)5-PCR DNA fingerprinting data, as well as by phenotypic data. Strains ID13488 and LMG 1693(T) could be differentiated from closely related species of the genus Gluconacetobacter by their ability to produce 2- and 5-keto-d-gluconic acid from d-glucose, their ability to produce acid from sucrose, but not from 1-propanol, and their ability to grow on 3 % ethanol in the absence of acetic acid and on ethanol, d-ribose, d-xylose, sucrose, sorbitol, d-mannitol and d-gluconate as carbon sources. The DNA G+C content of strains ID13488 and LMG 1693(T) was 58.0 and 60.7 mol%, respectively. The major ubiquinone of LMG 1693(T) was Q-10. Taken together these data indicate that strains ID13488 and LMG 1693(T) represent a novel species of the genus Gluconacetobacter for which the name Gluconacetobacter

α-Ketoglutaramate: An overlooked metabolite of glutamine and a biomarker for hepatic encephalopathy and inborn errors of the urea cycle

PubMed Central

Cooper, Arthur J. L.; Kuhara, Tomiko

2013-01-01

Glutamine metabolism is generally regarded as proceeding via glutaminase-catalyzed hydrolysis to glutamate and ammonia, followed by conversion of glutamate to α-ketoglutarate catalyzed by glutamate dehydrogenase or by a glutamate-linked aminotransferase (transaminase). However, another pathway exists for the conversion of glutamine to α-ketoglutarate that is often overlooked, but is widely distributed in nature. This pathway, referred to as the glutaminase II pathway, consists of a glutamine transaminase coupled to ω-amidase. Transamination of glutamine results in formation of the corresponding α-keto acid, namely, α-ketoglutaramate (KGM). KGM is hydrolyzed by ω-amidase to α-ketoglutarate and ammonia. The net glutaminase II reaction is: L-Glutamine + α-keto acid + H2O → α-ketoglutarate + L-amino acid + ammonia. In this mini-review the biochemical importance of the glutaminase II pathway is summarized, with emphasis on the key component KGM. Forty years ago it was noted that the concentration of KGM is increased in the cerebrospinal fluid (CSF) of patients with hepatic encephalopathy (HE) and that the level of KGM in the CSF correlates well with the degree of encephalopathy. In more recent work, we have shown that KGM is markedly elevated in the urine of patients with inborn errors of the urea cycle. It is suggested that KGM may be a useful biomarker for many hyperammonemic diseases including hepatic encephalopathy, inborn errors of the urea cycle, citrin deficiency and lysinuric protein intolerance. PMID:24234505

Validation of highly sensitive simultaneous targeted and untargeted analysis of keto-steroids by Girard P derivatization and stable isotope dilution-liquid chromatography-high resolution mass spectrometry.

PubMed

Frey, Alexander J; Wang, Qingqing; Busch, Christine; Feldman, Daniel; Bottalico, Lisa; Mesaros, Clementina A; Blair, Ian A; Vachani, Anil; Snyder, Nathaniel W

2016-12-01

A multiplexed quantitative method for the analysis of three major unconjugated steroids in human serum by stable isotope dilution liquid chromatography-high resolution mass spectrometry (LC-HRMS) was developed and validated on a Q Exactive Plus hybrid quadrupole/Orbitrap mass spectrometer. This quantification utilized isotope dilution and Girard P derivatization on the keto-groups of testosterone (T), androstenedione (AD) and dehydroepiandrosterone (DHEA) to improve ionization efficiency using electrospray ionization. Major isomeric compounds to T and DHEA; the inactive epimer of testosterone (epiT), and the metabolite of AD, 5α-androstanedione (5α-AD) were completely resolved on a biphenyl column within an 18min method. Inter- and intra-day method validation using LC-HRMS with qualifying product ions was performed and acceptable analytical performance was achieved. The method was further validated by comparing steroid levels from 100μL of serum from young vs older subjects. Since this approach provides high-dimensional HRMS data, untargeted analysis by age group was performed. DHEA and T were detected among the top analytes most significantly different across the two groups after untargeted LC-HRMS analysis, as well as a number of other still unknown metabolites, indicating the potential for combined targeted/untargeted analysis in steroid analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes

PubMed Central

Vivar, Juan C.; Knight, Megan S.; Pointer, Mildred A.; Gwathmey, Judith K.; Ghosh, Sujoy

2014-01-01

The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of

A New Model to Study the Role of Arachidonic Acid in Colon Cancer Pathophysiology.

PubMed

Fan, Yang-Yi; Callaway, Evelyn; M Monk, Jennifer; S Goldsby, Jennifer; Yang, Peiying; Vincent, Logan; S Chapkin, Robert

2016-09-01

A significant increase in cyclooxygenase 2 (COX2) gene expression has been shown to promote cylcooxygenase-dependent colon cancer development. Controversy associated with the role of COX2 inhibitors indicates that additional work is needed to elucidate the effects of arachidonic acid (AA)-derived (cyclooxygenase and lipoxygenase) eicosanoids in cancer initiation, progression, and metastasis. We have recently developed a novel Fads1 knockout mouse model that allows for the investigation of AA-dependent eicosanoid deficiency without the complication of essential fatty acid deficiency. Interestingly, the survival rate of Fads1-null mice is severely compromised after 2 months on a semi-purified AA-free diet, which precludes long-term chemoprevention studies. Therefore, in this study, dietary AA levels were titrated to determine the minimal level required for survival, while maintaining a distinct AA-deficient phenotype. Null mice supplemented with AA (0.1%, 0.4%, 0.6%, 2.0%, w/w) in the diet exhibited a dose-dependent increase (P < 0.05) in AA, PGE2, 6-keto PGF1α, TXB2, and EdU-positive proliferative cells in the colon. In subsequent experiments, null mice supplemented with 0.6% AA diet were injected with a colon-specific carcinogen (azoxymethane) in order to assess cancer susceptibility. Null mice exhibited significantly (P < 0.05) reduced levels/multiplicity of aberrant crypt foci (ACF) as compared with wild-type sibling littermate control mice. These data indicate that (i) basal/minimal dietary AA supplementation (0.6%) expands the utility of the Fads1-null mouse model for long-term cancer prevention studies and (ii) that AA content in the colonic epithelium modulates colon cancer risk. Cancer Prev Res; 9(9); 750-7. ©2016 AACR. ©2016 American Association for Cancer Research.

Selectivity in progesterone and androgen receptor binding of progestagens used in oral contraceptives

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kloosterboer, H.J.; Vonk-Noordegraaf, C.A.; Turpijn, E.W.

1988-09-01

The relative binding affinities (RBAs) of four progestational compounds (norethisterone, levonorgestrel, 3-keto-desogestrel and gestodene) for the human progesterone and androgen receptors were measured in MCF-7 cytosol and intact MCF-7 cells. For the binding to the progesterone receptor, both Org 2058 and Org 3236 (or 3-keto-desogestrel) were used as labelled ligands. The following ranking (low to high) for the RBA of the nuclear (intact cells) progesterone receptor irrespective of the ligand used is found: norethisterone much less than levonorgestrel less than 3-keto-destogestrel less than gestodene. The difference between the various progestagens is significant with the exception of that between 3-keto-desogestrel andmore » gestodene, when Org 2058 is used as ligand. For the cytosolic progesterone receptor, the same order is found with the exception that similar RBAs are found for gestodene and 3-keto-desogestrel. The four progestagens clearly differ with respect to binding to the androgen receptor using dihydrotestosterone as labelled ligand in intact cells; the ranking (low to high) is: norethisterone less than 3 keto-desogestrel less than levonorgestrel and gestodene. The difference between 3-keto-desogestrel and levonorgestrel or gestodene is significant. The selectivity indices (ratio of the mean RBA for the progesterone receptor to that of androgen receptor) in intact cells are significantly higher for 3-keto-desogestrel and gestodene than for levonorgestrel and norethisterone. From these results we conclude that the introduction of the 18-methyl in norethisterone (levonorgestel) increases both the binding to the progesterone and androgen receptors.« less

Identification of aldo-keto reductase (AKR7A1) and glutathione S-transferase pi (GSTP1) as novel renal damage biomarkers following exposure to mercury.

PubMed

Shin, Y-J; Kim, K-A; Kim, E-S; Kim, J-H; Kim, H-S; Ha, M; Bae, O-N

2017-01-01

The kidney is one of the main targets for toxicity induced by xenobiotics. Sensitive detection of early impairment is critical to assess chemical-associated renal toxicity. The aim of this study was to identify potential nephrotoxic biomarkers in rat kidney tissues after exposure to mercury (Hg), a representative nephrotoxicant, and to evaluate these new biomarkers employing in vivo and in vitro systems. Mercuric chloride was administered orally to Sprague-Dawley rats for 2 weeks. Proteomic analysis revealed that aldo-keto reductase (AKR7A1) and glutathione S-transferase pi (GSTP1) were significantly elevated in kidney after Hg exposure. While the levels of conventional nephrotoxic clinical markers including blood urea nitrogen and serum creatinine were not elevated, the mRNA and protein levels of AKR7A1 and GSTP1 were increased upon Hg exposure in a dose-dependent manner. The increases in AKR7A1 and GSTP1 were also observed in rat kidneys after an extended exposure for 6 weeks to low-dose Hg. In in vitro rat kidney proximal tubular cells, changes in AKR7A1 and GSTP1 levels correlated well with the extent of cytotoxicity induced by Hg, cadmium, or cisplatin. AKR7A1 and GSTP1 were identified as new candidates for Hg-induced nephrotoxicity, suggesting that these biomarkers have potential for evaluating or predicting nephrotoxicity.

Platelet aggregation, eicosanoid production and thrombogenic ratio in individuals at high cardiovascular risk consuming meat enriched in walnut paste. A crossover, placebo-controlled study.

PubMed

Canales, Amaia; Bastida, Sara; Librelottto, Josana; Nus, Meritxell; Sánchez-Muniz, Francisco J; Benedi, Juana

2009-07-01

Walnut consumption produces beneficial cardiovascular effects. The aim of the present study is to compare the effects of meat enriched in walnut paste (WM) and low-fat meat (LM) consumptions on platelet aggregation, plasma thromboxane A2 (TXA2, measured as TXB2), prostacyclin I2 (PGI2, as 6-keto-PGF1alpha) and the thrombogenic ratio (TXB2/6-keto-PGF1alpha) in volunteers at high CVD risk. Twenty-two adults were placed on a random, non-blinded crossover study involving two test periods (five portions WM/week for 5 week; five portions LM/week for 5 week) separated by a 4- to 6-week washout period. The participants were asked to complete a diet record throughout the study. Platelet aggregation, plasma TXB2, 6-keto-PGF1alpha production and the TXB2/6-keto-PGF1alpha ratio were determined at baseline and at weeks 3 and 5 for the two dietary periods. The WM diet contains a lower SFA content, a higher concentration of PUFA and a more favourable n-6/n-3 ratio than the LM diet. Significant time x treatment interactions were observed for TXB2 (P = 0.048) and the TXB2/6-keto-PGF1alpha ratio (P = 0.028). The WM diet significantly increased the level of 6-keto-PGF1alpha (P = 0.037) and decreased the TXB2/6-keto-PGF1alpha ratio (P = 0.048). At week 5, significant differences (P < 0.05) between treatments were found for maximum aggregation rate, TXB2 values and the TXB2/6-keto-PGF1alpha ratio. The effects on TXB2 and the TXB2/6-keto-PGF1alpha ratio were time-course dependent (P = 0.019 and 0.011, respectively). The WM and LM diets reduced TXB2 levels most (P = 0.050) in obese individuals, while the TXB2/6-keto-PGF1alpha ratio decreased most (P = 0.066) in volunteers whose serum cholesterol levels were > or = 2200 mg/l. The WM diet should be considered a functional meat because it improves the thrombogenic status mainly in individuals with high-cholesterol levels or high BMI.

The position of a key tyrosine in dTDP-4-Keto-6-deoxy-D-glucose-5-epimerase (EvaD) alters the substrate profile for this RmlC-like enzyme.

PubMed

Merkel, Alexandra B; Major, Louise L; Errey, James C; Burkart, Michael D; Field, Robert A; Walsh, Christopher T; Naismith, James H

2004-07-30

Vancomycin, the last line of defense antibiotic, depends upon the attachment of the carbohydrate vancosamine to an aglycone skeleton for antibacterial activity. Vancomycin is a naturally occurring secondary metabolite that can be produced by bacterial fermentation. To combat emerging resistance, it has been proposed to genetically engineer bacteria to produce analogues of vancomycin. This requires a detailed understanding of the biochemical steps in the synthesis of vancomycin. Here we report the 1.4 A structure and biochemical characterization of EvaD, an RmlC-like protein that is required for the C-5' epimerization during synthesis of dTDP-epivancosamine. EvaD, although clearly belonging to the RmlC class of enzymes, displays very low activity in the archetypal RmlC reaction (double epimerization of dTDP-6-deoxy-4-keto-D-glucose at C-3' and C-5'). The high resolution structure of EvaD compared with the structures of authentic RmlC enzymes indicates that a subtle change in the enzyme active site repositions a key catalytic Tyr residue. A mutant designed to re-establish the normal position of the Tyr increases the RmlC-like activity of EvaD.

Keto-enol tautomerism of (E)-2-[(3,4-dimethylphenylimino)methyl]-4-nitrophenol: Synthesis, X-ray, FT-IR, UV-Vis, NMR and quantum chemical characterizations

NASA Astrophysics Data System (ADS)

Özek Yıldırım, Arzu; Yıldırım, M. Hakkı; Albayrak Kaştaş, Çiǧdem

2017-01-01

(E)-2-((3,4-dimethylphenylimino)methyl)-4-nitrophenol, which is a new Schiff base compound, was synthesized and characterized by experimental and computational methods. Molecular geometry, harmonic oscillator model of aromaticity (HOMA) indices, intra- and inter-molecular interactions in the crystal structure were determined by using single crystal X-ray diffraction technique. The optimized structures, which are obtained by Gaussian and Slater type orbitals, were compared to experimental structures to determine how much correlation is found between the experimental and the calculated properties. Intramolecular and hyperconjugative interactions of bonds have been found by Natural Bond Orbital analysis. The experimental infrared spectrum of the compound has been analyzed in detail by the calculated infrared spectra and Potential Energy Distribution analysis. To find out about the correlation between the solvent polarity and the enol-keto equilibrium, experimental UV-Visible spectra of the compound were obtained in benzene, CHCl3, EtOH and DMSO solvents. In these solvents, the UV-Vis spectra and relaxed potential energy surface scan (PES) calculations have been performed to get more insight into the equilibrium dynamics. Solvent effects in UV-Vis and PES calculations have been taken into account by using Polarizable Continuum Modelling method. 1H and 13C NMR spectra of the compound (in DMSO) were analyzed. The computational study of nonlinear optical properties shows that the compound can be used for the development of nonlinear optical materials.

Flavonoids, flavonoid metabolites, and phenolic acids inhibit oxidative stress in the neuronal cell line HT-22 monitored by ECIS and MTT assay: a comparative study.

PubMed

Kling, Beata; Bücherl, Daniel; Palatzky, Peter; Matysik, Frank-Michael; Decker, Michael; Wegener, Joachim; Heilmann, Jörg

2014-03-28

A real-time and label-free in vitro assay based on electric cell-substrate impedance sensing (ECIS) was established, validated, and compared to an end-point MTT assay within an experimental trial addressing the cytoprotective effects of 19 different flavonoids, flavonoid metabolites, and phenolic acids and their methyl esters on the HT-22 neuronal cell line, after induction of oxidative stress with tert-butyl hydroperoxide. Among the flavonoids under study, only those with a catechol unit and an additional 4-keto group provided cytoprotection. The presence of a 2,3-double bond was not a structural prerequisite for a neuroprotective effect. In the case of the phenolics, catechol substitution was the only structural requirement for activity. The flavonoids and other phenolics with a ferulic acid substitution or a single hydroxy group showed no activity. Electrochemical characterization of all compounds via square-wave voltammetry provided a rather specific correlation between cytoprotective activity and redox potential for the active flavonoids, but not for the active phenolics with a low molecular weight. Moreover this study was used to compare label-free ECIS recordings with results of the established MTT assay. Whereas the former provides time-resolved and thus entirely unbiased information on changes of cell morphology that are unequivocally associated with cell death, the latter requires predefined exposure times and a strict causality between metabolic activity and cell death. However, MTT assays are based on standard lab equipment and provide a more economic way to higher throughput.

Intermittent maple syrup urine disease: two case reports.

PubMed

Axler, Olof; Holmquist, Peter

2014-02-01

The presenting symptoms and clinical course of 2 cases of intermittent maple syrup urine disease (MSUD) are described. Intermittent MSUD is a potentially life-threatening metabolic disorder caused by a deficiency of branched-chain α-keto acid dehydrogenase, the enzyme complex that decarboxylates the 3 branched-chain amino acids. In contrast to classic MSUD, children with the intermittent form show normal development with normal intelligence and, when asymptomatic, normal levels of branched-chain amino acids. Symptoms usually appear between 5 months and 2 years of age, when a trivial infection such as otitis media or viral gastroenteritis triggers catabolism of muscle protein. Intermittent MSUD should be suspected in cases of common infections with a clinically atypical course, especially in children displaying ataxia or marked drowsiness.

Operando X-ray absorption and EPR evidence for a single electron redox process in copper catalysis

DOE PAGES

Lu, Qingquan; Zhang, Jian; Peng, Pan; ...

2015-05-26

An unprecedented single electron redox process in copper catalysis is confirmed using operando X-ray absorption and EPR spectroscopies. The oxidation state of the copper species in the interaction between Cu(II) and a sulfinic acid at room temperature, and the accurate characterization of the formed Cu(I) are clearly shown using operando X-ray absorption and EPR evidence. Further investigation of anion effects on Cu(II) discloses that bromine ions can dramatically increase the rate of the redox process. Moreover, it is proven that the sulfinic acids are converted into sulfonyl radicals, which can be trapped by 2-arylacrylic acids and various valuable β-keto sulfonesmore » are synthesized with good to excellent yields under mild conditions.« less

A historical sketch of the discovery and development of HIV-1 integrase inhibitors.

PubMed

Savarino, Andrea

2006-12-01

The long process of HIV-1 integrase inhibitor discovery and development can be attributed to both the complexity of HIV-1 integration and poor 'integration' of these researches into mainstream investigations on antiretroviral therapy in the mid-1990s. Of note, some fungal extracts investigated during this period contain the beta-hydroxyketo group, later recognised to be a key structural requirement for keto-enol acids (also referred to as diketo acids) and other integrase inhibitors. This review reconstructs (in the general context of the history of AIDS research) the principal steps that led to the integrase inhibitors currently in clinical trials, and discusses possible future directions.

Biosynthetic Origin of Hygromycin A

PubMed Central

Habib, El-Sayed E.; Scarsdale, J. Neel; Reynolds, Kevin A.

2003-01-01

Hygromycin A, an antibiotic produced by Streptomyces hygroscopicus, is an inhibitor of bacterial ribosomal peptidyl transferase. The antibiotic binds to the ribosome in a distinct but overlapping manner with other antibiotics and offers a different template for generation of new agents effective against multidrug-resistant pathogens. Reported herein are the results from a series of stable-isotope-incorporation studies demonstrating the biosynthetic origins of the three distinct structural moieties which comprise hygromycin A. Incorporation of [1-13C]mannose and intact incorporation of d-[1,2-13C2]glucose into the 6-deoxy-5-keto-d-arabino-hexofuranose moiety are consistent with a pathway in which mannose is converted to an activated l-fucose, via a 4-keto-6-deoxy-d-mannose intermediate, with a subsequent unusual mutation of the pyranose to the corresponding furanose. The aminocyclitol moiety was labeled by d-[1,2-13C2]glucose in a manner consistent with formation of myo-inositol and a subsequent unprecedented oxidation and transamination of the C-2 hydroxyl group to generate neo-inosamine-2. Incorporation of [carboxy-13C]-4-hydroxybenzoic acid and intact incorporation of [2,3-13C2]propionate are consistent with a polyketide synthase-type decarboxylation condensation to generate the 3,4-dihydroxy-α-methylcinnamic acid moiety of hygromycin A. No labeling of hygromycin A was observed when [3-13C]tyrosine, [3-13C]phenylalanine, or [carboxy-13C]benzoic acid was used, suggesting that the 4-hydroxybenzoic acid is derived directly from chorismic acid. Consistent with this hypothesis was the observation that hygromycin A titers could be reduced by addition of N-(phosphonomethyl)-glycine (an inhibitor of chorismic acid biosynthesis) and restored by coaddition of 4-hydroxybenzoic acid. The convergent biosynthetic pathway established for hygromycin A offers significant versatility for applying the techniques of combinatorial and directed biosynthesis to production of new

Pseudomonas kribbensis sp. nov., isolated from garden soils in Daejeon, Korea.

PubMed

Chang, Dong-Ho; Rhee, Moon-Soo; Kim, Ji-Sun; Lee, Yookyung; Park, Mi Young; Kim, Haseong; Lee, Seung-Goo; Kim, Byoung-Chan

2016-11-01

Two bacterial strains, 46-1 and 46-2 T , were isolated from garden soil. These strains were observed to be aerobic, Gram-stain negative, rod-shaped, non-spore-forming, motile and catalase and oxidase positive. Phylogenetic analysis based on 16S rRNA gene sequences showed that the two strains shared 100 % sequence similarity with each other and belong to the genus Pseudomonas in the class Gammaproteobacteria. The concatenated 16S rRNA, gyrB, rpoB and rpoD gene sequences further confirmed that the isolates belong to the Pseudomonas koreensis subgroup (SG), with P. koreensis Ps 9-14 T , Pseudomonas moraviensis 1B4 T and Pseudomonas granadensis F-278,770 T as their close relatives (>96 % pairwise similarity). DNA-DNA hybridization with the closely related type strain P. koreensis SG revealed a low level of relatedness (<50 %). A cladogram constructed using whole-cell matrix-assisted laser desorption/ionization time-of-flight (WC-MALDI-TOF) MS analysis showed the isolates formed a completely separate monophyletic group. The isolates were negative for utilization of glycogen, D-psicose, α-keto butyric acid, α-keto valeric acid, succinamic acid and D, L-α-glycerol phosphate. In contrast, all these reactions were positive in P. koreensis JCM 14769 T and P. moraviensis DSM 16007 T . The fatty acid C 17:0 cyclo was detected as one of the major cellular fatty acids (>15 %) in the isolates but it was a minor component (<4 %) in both reference type strains. In contrast, the fatty acid, C 12:0 was not observed in the isolates but was present in both reference strains. Based on differences such as phylogenetic position, low-level DNA-DNA hybridization, WC-MALDI-TOF MS analysis, fluorescence pigmentation, fatty acid profiles, and substrate utilization, we propose that the isolates 46-1 and 46-2 T represent a novel species of the genus Pseudomonas, for which the name Pseudomonas kribbensis sp. nov. is proposed; the type strain is 46-2 T (=KCTC 32541 T  = DSM 100278 T ).

Opposing roles of the aldo-keto reductases AKR1B1 and AKR1B10 in colorectal cancer.

PubMed

Taskoparan, Betul; Seza, Esin Gulce; Demirkol, Secil; Tuncer, Sinem; Stefek, Milan; Gure, Ali Osmay; Banerjee, Sreeparna

2017-12-01

Aldo-keto reductases (including AKR1B1 and AKR1B10) constitute a family of oxidoreductases that have been implicated in the pathophysiology of diabetes and cancer, including colorectal cancer (CRC). Available data indicate that, despite their similarities in structure and enzymatic functions, their roles in CRC may be divergent. Here, we aimed to determine the expression and functional implications of AKR1B1 and AKR1B10 in CRC. AKR1B1 and AKR1B10 gene expression levels were analyzed using publicly available microarray data and ex vivo CRC-derived cDNA samples. Gene Set Enrichment Analysis (GSEA), The Cancer Genome Atlas (TCGA) RNA-seq data and The Cancer Proteome Atlas (TCPA) proteome data were analyzed to determine the effect of high and low AKR1B1 and AKR1B10 expression levels in CRC patients. Proliferation, cell cycle progression, cellular motility, adhesion and inflammation were determined in CRC-derived cell lines in which these genes were either exogenously overexpressed or silenced. We found that the expression of AKR1B1 was unaltered, whereas that of AKR1B10 was decreased in primary CRCs. GSEA revealed that, while high AKR1B1 expression was associated with increased cell cycle progression, cellular motility and inflammation, high AKR1B10 expression was associated with a weak inflammatory phenotype. Functional studies carried out in CRC-derived cell lines confirmed these data. Microarray data analysis indicated that high expression levels of AKR1B1 and AKR1B10 were significantly associated with shorter and longer disease-free survival rates, respectively. A combined gene expression signature of AKR1B10 (low) and AKR1B1 (high) showed a better prognostic stratification of CRC patients independent of confounding factors. Despite their similarities, the expression levels and functions of AKR1B1 and AKR1B10 are highly divergent in CRC, and they may have prognostic implications.

The Impact and Oxidation Survival of Selected Meteoritic Compounds: Signatures of Asteroid Organic Material on Planetary Surfaces

NASA Technical Reports Server (NTRS)

Cooper, George; Horz, Fred; Oleary, Alanna; Chang, Sherwood

2013-01-01

Polar, non-volatile organic compounds may be present on the surfaces (or near surfaces) of multiple Solar System bodies. If found, by current or future missions, it would be desirable to determine the origin(s) of such compounds, e.g., asteroidal or in situ. To test the possible survival of meteoritic compounds both during impacts with planetary surfaces and under subsequent (possibly) harsh ambient conditions, we subjected known meteoritic compounds to relatively high impact-shock pressures and/or to varying oxidizing/corrosive conditions. Tested compounds include sulfonic and phosphonic acids (S&P), polyaromatic hydrocarbons (PAHs) amino acids, keto acids, dicarboxylic acids, deoxy sugar acids, and hydroxy tricarboxylic acids (Table 1). Meteoritic sulfonic acids were found to be relatively abundant in the Murchison meteorite and to possess unusual S-33 isotope anomalies (non mass-dependent isotope fractionations). Combined with distinctive C-S and C-P bonds, the S&P are potential signatures of asteroidal organic material.

Specific coupling between the 13-keto carbonyl and chlorin skeletal vibrational modes of synthetic 13 1- 18O-(un)labelled metallochlorophyll derivatives

NASA Astrophysics Data System (ADS)

Morishita, Hidetada; Tamiaki, Hitoshi

2009-03-01

Metal complexes of methyl 13 1- 18O-labelled pyropheophorbide- a1-M- 18O (M = Zn, Cu and Ni) were prepared by metallation of the 18O-labelled free base ( 1- 18O) and 18O-labelling of unlabelled nickel complex ( 1-Ni). The FT-IR spectra of 1-Zn and 1-Zn- 18O in CH 2Cl 2 showed that the 13-keto carbonyl stretching vibration mode moved to about a 30-cm -1 lower wavenumber by 18O-labelling of the 13 1-oxo moiety. In 1-Cu- 18O and 1-Ni- 18O, the 13-C dbnd 18O stretching modes were close to the highest-energy wavenumber mode of chlorin skeletal C-C/C-N vibrations at around 1650 cm -1 and they were coupled in CH 2Cl 2 to give two split IR bands (Fermi resonance). A similar coupling was observed in the resonance Raman scattering of 1-Ni- 18O in the solid state. The hydrogen-bonded 13-C dbnd 16O vibration mode of 1-Ni similarly coupled with the skeletal C-C/C-N mode in CCl 4 containing 1% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, while such a coupling was not observed in a neat CCl 4 solution of 1-Ni possessing the 13-C dbnd 16O free from any interaction. The skeletal C-C/C-N band selectively coupled with the 13-C dbnd O, not with the 3-C dbnd O, when the difference in their peak maxima was less than 20 cm -1.

Metabolism of pentose sugars in the hyperthermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius.

PubMed

Nunn, Charlotte E M; Johnsen, Ulrike; Schönheit, Peter; Fuhrer, Tobias; Sauer, Uwe; Hough, David W; Danson, Michael J

2010-10-29

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, D-xylose and L-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of D-xylose and L-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.

Phenylbutyrate therapy for maple syrup urine disease

PubMed Central

Brunetti-Pierri, Nicola; Lanpher, Brendan; Erez, Ayelet; Ananieva, Elitsa A.; Islam, Mohammad; Marini, Juan C.; Sun, Qin; Yu, Chunli; Hegde, Madhuri; Li, Jun; Wynn, R. Max; Chuang, David T.; Hutson, Susan; Lee, Brendan

2011-01-01

Therapy with sodium phenylacetate/benzoate or sodium phenylbutyrate in urea cycle disorder patients has been associated with a selective reduction in branched-chain amino acids (BCAA) in spite of adequate dietary protein intake. Based on this clinical observation, we investigated the potential of phenylbutyrate treatment to lower BCAA and their corresponding α-keto acids (BCKA) in patients with classic and variant late-onset forms of maple syrup urine disease (MSUD). We also performed in vitro and in vivo experiments to elucidate the mechanism for this effect. We found that BCAA and BCKA are both significantly reduced following phenylbutyrate therapy in control subjects and in patients with late-onset, intermediate MSUD. In vitro treatment with phenylbutyrate of control fibroblasts and lymphoblasts resulted in an increase in the residual enzyme activity, while treatment of MSUD cells resulted in the variable response which did not simply predict the biochemical response in the patients. In vivo phenylbutyrate increases the proportion of active hepatic enzyme and unphosphorylated form over the inactive phosphorylated form of the E1α subunit of the branched-chain α-keto acid dehydrogenase complex (BCKDC). Using recombinant enzymes, we show that phenylbutyrate prevents phosphorylation of E1α by inhibition of the BCKDC kinase to activate BCKDC overall activity, providing a molecular explanation for the effect of phenylbutyrate in a subset of MSUD patients. Phenylbutyrate treatment may be a valuable treatment for reducing the plasma levels of neurotoxic BCAA and their corresponding BCKA in a subset of MSUD patients and studies of its long-term efficacy are indicated. PMID:21098507

Phenylbutyrate therapy for maple syrup urine disease.

PubMed

Brunetti-Pierri, Nicola; Lanpher, Brendan; Erez, Ayelet; Ananieva, Elitsa A; Islam, Mohammad; Marini, Juan C; Sun, Qin; Yu, Chunli; Hegde, Madhuri; Li, Jun; Wynn, R Max; Chuang, David T; Hutson, Susan; Lee, Brendan

2011-02-15

Therapy with sodium phenylacetate/benzoate or sodium phenylbutyrate in urea cycle disorder patients has been associated with a selective reduction in branched-chain amino acids (BCAA) in spite of adequate dietary protein intake. Based on this clinical observation, we investigated the potential of phenylbutyrate treatment to lower BCAA and their corresponding α-keto acids (BCKA) in patients with classic and variant late-onset forms of maple syrup urine disease (MSUD). We also performed in vitro and in vivo experiments to elucidate the mechanism for this effect. We found that BCAA and BCKA are both significantly reduced following phenylbutyrate therapy in control subjects and in patients with late-onset, intermediate MSUD. In vitro treatment with phenylbutyrate of control fibroblasts and lymphoblasts resulted in an increase in the residual enzyme activity, while treatment of MSUD cells resulted in the variable response which did not simply predict the biochemical response in the patients. In vivo phenylbutyrate increases the proportion of active hepatic enzyme and unphosphorylated form over the inactive phosphorylated form of the E1α subunit of the branched-chain α-keto acid dehydrogenase complex (BCKDC). Using recombinant enzymes, we show that phenylbutyrate prevents phosphorylation of E1α by inhibition of the BCKDC kinase to activate BCKDC overall activity, providing a molecular explanation for the effect of phenylbutyrate in a subset of MSUD patients. Phenylbutyrate treatment may be a valuable treatment for reducing the plasma levels of neurotoxic BCAA and their corresponding BCKA in a subset of MSUD patients and studies of its long-term efficacy are indicated.

A complex effect of arsenite on the formation of alpha-ketoglutarate in rat liver mitochondria.

PubMed

Lenartowicz, E

1990-12-01

This investigation presents disturbances of the mitochondrial metabolism by arsenite, a hydrophilic dithiol reagent known as an inhibitor of mitochondrial alpha-keto acid dehydrogenases. Arsenite at concentrations of 0.1-1.0 mM was shown to induce a considerable oxidation of intramitochondrial NADPH, NADH, and glutathione without decreasing the mitochondrial membrane potential. The oxidation of NAD(P)H required the presence of phosphate and was sensitive to ruthenium red, but occurred without the addition of calcium salts. Mitochondrial reactions producing alpha-ketoglutarate from glutamate and isocitrate were modulated by arsenite through various mechanisms: (i) both glutamate transaminations, with oxaloacetate and with pyruvate, were inhibited by accumulating alpha-ketoglutarate; however, at low concentrations of alpha-ketoglutarate the aspartate aminotransferase reaction was stimulated due to the increase of NAD+ content; (ii) the oxidation of isocitrate was stimulated at its low concentration only, due to the oxidation of NADPH and NADH; this oxidation was prevented by concentrations of citrate or isocitrate greater than 1 mM; (iii) the conversion of isocitrate to citrate was suppressed, presumably as a result of the decrease of Mg2+ concentration in mitochondria. Thus the depletion of mitochondrial vicinal thiol groups in hydrophilic domains disturbs the mitochondrial metabolism not only by the inhibition of alpha-keto acid dehydrogenases but also by the oxidation of NAD(P)H and, possibly, by the change in the ion concentrations.

Metabolomic Analysis of Key Central Carbon Metabolism Carboxylic Acids as Their 3-Nitrophenylhydrazones by UPLC/ESI-MS

PubMed Central

Han, Jun; Gagnon, Susannah; Eckle, Tobias; Borchers, Christoph H.

2014-01-01

Multiple hydroxy-, keto-, di-, and tri-carboxylic acids are among the cellular metabolites of central carbon metabolism (CCM). Sensitive and reliable analysis of these carboxylates is important for many biological and cell engineering studies. In this work, we examined 3-nitrophenylhydrazine as a derivatizing reagent and optimized the reaction conditions for the measurement of ten CCM related carboxylic compounds, including glycolate, lactate, malate, fumarate, succinate, citrate, isocitrate, pyruvate, oxaloacetate, and α-ketoglutarate as their 3-nitrophenylhydrazones using LC/MS with electrospray ionization. With the derivatization protocol which we have developed, and using negative-ion multiple reaction monitoring on a triple-quadrupole instrument, all of the carboxylates showed good linearity within a dynamic range of ca. 200 to more than 2000. The on-column limits of detection and quantitation were from high femtomoles to low picomoles. The analytical accuracies for eight of the ten analytes were determined to be between 89.5 to 114.8% (CV≤7.4%, n=6). Using a quadrupole time-of-flight instrument, the isotopic distribution patterns of these carboxylates, extracted from a 13C-labeled mouse heart, were successfully determined by UPLC/MS with full-mass detection, indicating the possible utility of this analytical method for metabolic flux analysis. In summary, this work demonstrates an efficient chemical derivatization LC/MS method for metabolomic analysis of these key CCM intermediates in a biological matrix. PMID:23580203

Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD + and NADP +

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leitgeb, Stefan; Petschacher, Barbara; Wilson, David K.

2005-01-11

Aldo-keto reductases of family 2 employ single site replacement Lys → Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD + and NADP + were determined at a resolution of 2.4 and 2.3 Å, respectively. Due to steric conflicts in the NADP +-bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2'-phosphate and 3'-hydroxy groups. Because anchoring contactsmore » of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P) + in the wild-type remains partly disordered in the NADP +-bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type.« less

Biofuel and chemical production by recombinant microorganisms via fermentation of proteinaceous biomass

DOEpatents

Liao, James C.; Cho, Kwang Myung; Yan, Yajun; Huo, Yixin

2016-03-15

Provided herein are metabolically modified microorganisms characterized by having an increased keto-acid flux when compared with the wild-type organism and comprising at least one polynucleotide encoding an enzyme that when expressed results in the production of a greater quantity of a chemical product when compared with the wild-type organism. The recombinant microorganisms are useful for producing a large number of chemical compositions from various nitrogen containing biomass compositions and other carbon sources. More specifically, provided herein are methods of producing alcohols, acetaldehyde, acetate, isobutyraldehyde, isobutyric acid, n-butyraldehyde, n-butyric acid, 2-methyl-1-butyraldehyde, 2-methyl-1-butyric acid, 3-methyl-1-butyraldehyde, 3-methyl-1-butyric acid, ammonia, ammonium, amino acids, 2,3-butanediol, 1,4-butanediol, 2-methyl-1,4-butanediol, 2-methyl-1,4-butanediamine, isobutene, itaconate, acetoin, acetone, isobutene, 1,5-diaminopentane, L-lactic acid, D-lactic acid, shikimic acid, mevalonate, polyhydroxybutyrate (PHB), isoprenoids, fatty acids, homoalanine, 4-aminobutyric acid (GABA), succinic acid, malic acid, citric acid, adipic acid, p-hydroxy-cinnamic acid, tetrahydrofuran, 3-methyl-tetrahydrofuran, gamma-butyrolactone, pyrrolidinone, n-methylpyrrolidone, aspartic acid, lysine, cadeverine, 2-ketoadipic acid, and/or S-adenosyl-methionine (SAM) from a suitable nitrogen rich biomass.

Annual Report on Electronics Research at The University of Texas at Austin.

DTIC Science & Technology

1982-05-15

Professor, Physics, 471-5747 L. Frommhold, Professor, Physics, 471-5100 J. Keto , Associate Professor, Physics, 471-4151 H.J. Kimble, Assistant Professor...Scattering Cross Section of Argon Diatom," Canad. J. Physics, 59, 1418 (1981). *Michael H. Proffitt, J.W. Keto and Lothar Frommhold, "Col- lision Induced...Elec- tron Diffraction Study of the Structure of Anthraquinone and Anthracene," J. Mol. Struct. 77, 127-138 (1981). J.W. Keto , T.D. Raymond and Chien-Yu

Prostaglandin reductase-3 negatively modulates adipogenesis through regulation of PPARγ activity[S

PubMed Central

Yu, Yu-Hsiang; Chang, Yi-Cheng; Su, Tseng-Hsiung; Nong, Jiun-Yi; Li, Chao-Chin; Chuang, Lee-Ming

2013-01-01

Adipocyte differentiation is a multistep program under regulation by several factors. Peroxisome proliferator-activated receptor γ (PPARγ) serves as a master regulator of adipogenesis. However, the endogenous ligand for PPARγ remained elusive until 15-keto-PGE2 was identified recently as an endogenous PPARγ ligand. In this study, we demonstrate that zinc-containing alcohol dehydrogenase 2 (ZADH2; here termed prostaglandin reductase-3, PTGR-3) is a new member of prostaglandin reductase family that converts 15-keto-PGE2 to 13,14-dihydro-15-keto-PGE2. Adipogenesis is accelerated when endogenous PTGR-3 is silenced in 3T3-L1 preadipocytes, whereas forced expression of PTGR-3 significantly decreases adipogenesis. PTGR-3 expression decreased during adipocyte differentiation, accompanied by an increased level of 15-keto-PGE2. 15-keto-PGE2 exerts a potent proadipogenic effect by enhancing PPARγ activity, whereas overexpression of PTGR-3 in 3T3-L1 preadipocytes markedly suppressed the proadipogenic effect of 15-keto-PGE2 by repressing PPARγ activity. Taken together, these findings demonstrate for the first time that PTGR-3 is a novel 15-oxoprostaglandin-Δ13-reductase and plays a critical role in modulation of normal adipocyte differentiation via regulation of PPARγ activity. Thus, modulation of PTGR-3 might provide a novel avenue for treating obesity and related metabolic disorders. PMID:23821743

Conformational analysis, tautomerization, IR, Raman, and NMR studies of benzyl acetoacetate

NASA Astrophysics Data System (ADS)

Tayyari, Sayyed Faramarz; Naghavi, Farnaz; Pojhan, Sahar; McClurg, Ryan W.; Sammelson, Robert E.

2011-02-01

A complete conformational analysis of the keto and enol forms of benzyl acetoacetate (BAA), a β-dicarbonyl compound, was carried out by ab initio calculations, at the density functional theory (DFT) level. By inspection of all possible conformers and tautomers, 22 stable cis-enol, 28 stable trans-enol, and five keto conformers were obtained. Among all stable cis-enol forms only six of them are engaged in intramolecular hydrogen bond. The hydrogen bond strength of the most stable conformer of BAA is compared with that of acetylacetone (AA) and dimethyl oxaloacetate (DMOA). Harmonic vibrational frequencies of the most stable enol and keto forms and their deuterated analogues were also calculated and compared with the experimental data. According to the theoretical calculations, the hydrogen bond strength of the most stable enol conformer of BAA is 56.7 kJ/mol (calculated at the B3LYP/6-311++G ∗∗ level), about 10 kJ/mol less than that of AA. This weakening of hydrogen bond is consistent with the spectroscopic results. NMR studies indicate that BAA exists mainly as a keto tautomer in all considered solutions. The Gibbs energies for keto/enol tautomerization were calculated at the B3LYP level, with several basis sets, in both gas phase and CH 3CN solution (using PCM model), for the most stable enol and keto conformers.

Hic-5’s Regulatory Role in TGFB Signaling in Prostate Stroma

DTIC Science & Technology

2012-06-01

the androgen metabolites 3α-Adiol and 3β-Adiol, and their importance is underscored by high expression levels of the aldo keto reductase (AKR1C...known as aldo -keto reductases (AKR1C) [33]. DU145 cells express AKR1C enzymes and are capable of catalyzing redox reactions at the C17 position of...584-95. 37. Bauman, D.R., et al., Development of nonsteroidal anti-inflammatory drug analogs and steroid carboxylates selective for human aldo -keto

Functional characteristics of spirilloxanthin and keto-bearing Analogues in light-harvesting LH2 complexes from Rhodobacter sphaeroides with a genetically modified carotenoid synthesis pathway.

PubMed

Niedzwiedzki, Dariusz M; Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Bocian, David F; Holten, Dewey; Hunter, C Neil

2015-01-01

Light-harvesting 2 (LH2) complexes from a genetically modified strain of the purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. Carotenoid synthesis in the Rba. sphaeroides strain was engineered to redirect carotenoid production away from spheroidene into the spirilloxanthin synthesis pathway. The strain assembles LH2 antennas with substantial amounts of spirilloxanthin (total double-bond conjugation length N=13) if grown anaerobically and of keto-bearing long-chain analogs [2-ketoanhydrorhodovibrin (N=13), 2-ketospirilloxanthin (N=14) and 2,2'-diketospirilloxanthin (N=15)] if grown semi-aerobically (with ratios that depend on growth conditions). We present the photophysical, electronic, and vibrational properties of these carotenoids, both isolated in organic media and assembled within LH2 complexes. Measurements of excited-state energy transfer to the array of excitonically coupled bacteriochlorophyll a molecules (B850) show that the mean lifetime of the first singlet excited state (S1) of the long-chain (N≥13) carotenoids does not change appreciably between organic media and the protein environment. In each case, the S1 state appears to lie lower in energy than that of B850. The energy-transfer yield is ~0.4 in LH2 (from the strain grown aerobically or semi-aerobically), which is less than half that achieved for LH2 that contains short-chain (N≤11) analogues. Collectively, the results suggest that the S1 excited state of the long-chain (N≥13) carotenoids participates little if at all in carotenoid-to-BChl a energy transfer, which occurs predominantly via the carotenoid S2 excited state in these antennas. Copyright © 2015 Elsevier B.V. All rights reserved.

Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation.

PubMed Central

Condemine, G; Hugouvieux-Cotte-Pattat, N; Robert-Baudouy, J

1986-01-01

Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis. A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source. Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon. Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression. In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism. Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation. 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage. These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis. PMID:3949717

Anthracycline resistance mediated by reductive metabolism in cancer cells: The role of aldo-keto reductase 1C3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hofman, Jakub; Malcekova, Beata; Skarka, Adam

2014-08-01

Pharmacokinetic drug resistance is a serious obstacle that emerges during cancer chemotherapy. In this study, we investigated the possible role of aldo-keto reductase 1C3 (AKR1C3) in the resistance of cancer cells to anthracyclines. First, the reducing activity of AKR1C3 toward anthracyclines was tested using incubations with a purified recombinant enzyme. Furthermore, the intracellular reduction of daunorubicin and idarubicin was examined by employing the transfection of A549, HeLa, MCF7 and HCT 116 cancer cells with an AKR1C3 encoding vector. To investigate the participation of AKR1C3 in anthracycline resistance, we conducted MTT cytotoxicity assays with these cells, and observed that AKR1C3 significantlymore » contributes to the resistance of cancer cells to daunorubicin and idarubicin, whereas this resistance was reversible by the simultaneous administration of 2′-hydroxyflavanone, a specific AKR1C3 inhibitor. In the final part of our work, we tracked the changes in AKR1C3 expression after anthracycline exposure. Interestingly, a reciprocal correlation between the extent of induction and endogenous levels of AKR1C3 was recorded in particular cell lines. Therefore, we suggest that the induction of AKR1C3 following exposure to daunorubicin and idarubicin, which seems to be dependent on endogenous AKR1C3 expression, eventually might potentiate an intrinsic resistance given by the normal expression of AKR1C3. In conclusion, our data suggest a substantial impact of AKR1C3 on the metabolism of daunorubicin and idarubicin, which affects their pharmacokinetic and pharmacodynamic behavior. In addition, we demonstrate that the reduction of daunorubicin and idarubicin, which is catalyzed by AKR1C3, contributes to the resistance of cancer cells to anthracycline treatment. - Highlights: • Metabolism of anthracyclines by AKR1C3 was studied at enzyme and cellular levels. • Anthracycline resistance mediated by AKR1C3 was demonstrated in cancer cells. • Induction of

DNA Carrier Testing and Newborn Screening for Maple Syrup Urine Disease in Old Order Mennonite Communities

PubMed Central

Carleton, Stephanie M.; Peck, Dawn S.; Grasela, Julie; Dietiker, Kristin L.

2010-01-01

Maple syrup urine disease (MSUD) is an inherited metabolic disorder caused by mutations in the branched chain α-keto acid dehydrogenase complex. Worldwide incidence of MSUD is 1:225,000 live births. However, within Old Order Mennonite communities, the incidence is 1:150 live births and results from a common tyrosine to asparagine substitution (Y438N) in the E1α subunit of branched chain α-keto acid dehydrogenase. We developed a new DNA diagnostic assay utilizing TaqMan® technology and compared its efficacy, sensitivity, and duration with an existing polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay. Carrier testing was performed by both TaqMan technology and PCR-RFLP on DNA isolated from buccal swabs of 160 individuals as well as from buccal swabs and blood spots of nine at-risk newborns; assay time, sensitivity, and reliability were also evaluated. The TaqMan assay, like the PCR-RFLP assay, accurately determined Y438N E1α allele status. However, the TaqMan assay appeared (1) more sensitive than the PCR-RFLP assay, requiring 10-fold less DNA (10 ng) to reliably determine genotype status and (2) faster, reducing the assay time required for diagnosis from ∼12 to 5 h. TaqMan technology allowed more rapid DNA diagnoses of MSUD in the neonate, thereby reducing the likelihood of neurological impairment while enhancing health and prognosis for affected infants. PMID:20136525

Food Overconsumption in Healthy Adults Triggers Early and Sustained Increases in Serum Branched-Chain Amino Acids and Changes in Cysteine Linked to Fat Gain.

PubMed

Elshorbagy, Amany K; Samocha-Bonet, Dorit; Jernerén, Fredrik; Turner, Cheryl; Refsum, Helga; Heilbronn, Leonie K

2018-06-13

Plasma concentrations of branched-chain amino acids (BCAAs) and the sulfur-containing amino acid cysteine are associated with obesity and insulin resistance. BCAAs predict future diabetes. We investigated amino acid changes during food overconsumption. Forty healthy men and women with a body mass index (mean ± SEM) of 25.6 ± 0.6 were overfed by 1250 kcal/d for 28 d, increasing consumption of all macronutrients. Insulin sensitivity and body composition were assessed at baseline (day 0) and day 28. Fasting serum amino acids were measured at days 0, 3, and 28. Linear mixed-effects models evaluated the effect of time in the total group and separately in those with low and high body fat gain (below compared with at or above median fat gain, 1.95 kg). At days 0 and 28, insulin-induced suppression of serum amino acids during a hyperinsulinemic-euglycemic clamp test and, in a subset (n = 20), adipose tissue mRNA expression of selected amino acid metabolizing enzymes were assessed. Weight increased by 2.8 kg. High fat gainers gained 2.6 kg fat mass compared with 1.1 kg in low fat gainers. Valine and isoleucine increased at day 3 (+17% and +22%, respectively; P ≤ 0.002) and remained elevated at day 28, despite a decline in valine (P = 0.019) from day 3 values. Methionine, cystathionine, and taurine were unaffected. Serum total cysteine (tCys) transiently increased at day 3 (+11%; P = 0.022) only in high fat gainers (P-interaction = 0.043), in whom the cysteine catabolic enzyme cysteine dioxygenase (CDO1) was induced (+26%; P = 0.025) in adipose tissue (P-interaction = 0.045). Overconsumption did not alter adipose tissue mRNA expression of the BCAA-metabolizing enzymes branched-chain keto acid dehydrogenase E1α polypeptide (BCKDHA) or branched-chain amino transferase 1 (BCAT1). In the total population at day 0, insulin infusion decreased all serum amino acids (-11% to -47%; P < 0.01), except for homocysteine and tCys, which were unchanged, and

Chiral allene-containing phosphines in asymmetric catalysis

PubMed Central

Cai, Feng; Pu, Xiaotao; Qi, Xiangbing; Lynch, Vincent; Radha, Akella; Ready, Joseph M.

2011-01-01

Traditionally, ligands used in asymmetric catalysis have contained either stereogenic atoms or hindered single bonds (atropisomerism), or both. Here we demonstrate that allenes, chiral 1,2-dienes, appended with basic functionality can serve as ligands for transition metals. We describe an allene-containing bisphosphine that, when coordinated to Rh(I), promotes the asymmetric addition of aryl boronic acids to α-keto esters with high enantioselectivity. Solution and solid-state structural analysis reveals that one olefin of the allene can coordinate to transition metals generating bi- and tri-dentate ligands. PMID:21972824

Femtosecond dynamics in ionic structures of a heart medicine

NASA Astrophysics Data System (ADS)

Gil, M.; Douhal, A.

2006-12-01

Femtosecond studies of ionic structures of milrinone - a medicine used to help the heart to recuperate its life - in acidic and alkaline water solutions show that the intramolecular charge transfer in the cation and in the anion happen in 550 fs and ˜1.2 ps, respectively. These times are longer than 100 fs, observed in the keto (inotropic) form. The transients also show a 2-3 ps component, assigned to cooling and twisting motion in the produced states. The result might be used for a better understanding of other functional molecules.

[A new eremophilane derivative from Senecio dianthus].

PubMed

Han, He-Dong; Hu, Hai-Qing; Li, Yan; Wang, Xiao-Ling

2013-10-01

A new eremophilane derivative, 4,5,11-trimethyl-9( 10), 7 ( 11) -eremophiladien-8-keto-12-carboxylic acid-beta-D-glucopyranoside( which named dianthuside A) 1 and four known compounds, 5,7,4'-trihydroxy-flavonone-3-0-beta-D-glucoside (2), quercetin-3-0-beta-D-glucoside(3) ,hyperin(4) and rutin(5) have been isolated from the aerial part of Senecio dianthus. Their structures were elucidated by physicochemical properties and spectroscopic data analysis. Compounds 2, 4 and 5 were isolated from this plant for the first time.

Dihydronaphthalenone Carboxylates - Spectral Characteristics and Structure

NASA Astrophysics Data System (ADS)

Bakalova, Sn.; Georgieva, A.; Nikolov, P.; Stanoeva, E.

1997-05-01

The absorption and luminescence characteristics of a group of newly synthesized methyl esters of 2-alkyl (p-substituted-aryl) -aminomethylene-3,4-dihydro-1(2 H)-naphthalenone-4-carboxylic acids have been investigated. The studied compounds may exist in three tautomeric forms. On the basis of comparison of their electronic spectra to those of similar substances, the observed substituent effect on the position of the UV-VIS absorption bands, the IR spectra and the results of PPP-SCF-CI quantum-chemical calculations it is concluded that the keto tautomer predominates in solution.

γ-Sultam-cored N,N-ligands in the ruthenium(ii)-catalyzed asymmetric transfer hydrogenation of aryl ketones.

PubMed

Rast, Slavko; Modec, Barbara; Stephan, Michel; Mohar, Barbara

2016-02-14

The synthesis of new enantiopure syn- and anti-3-(α-aminobenzyl)-benzo-γ-sultam ligands 6 and their application in the ruthenium(ii)-catalyzed asymmetric transfer hydrogenation (ATH) of ketones using formic acid/triethylamine is described. In particular, benzo-fused cyclic ketones afforded excellent enantioselectivities in reasonable time employing a low loading of the syn ligand-containing catalyst. A never-before-seen dynamic kinetic resolution (DKR) during reduction of a γ-keto carboxylic ester (S7) derivative of 1-indanone is realized leading as well to excellent induction.

X-ray crystallographic study of 3-Oxo-2-{[4-(thiazol-2-ylsulfamoyl)-phenyl]-hydrazono}-butyric acid ethyl ester and its application in the solvent assisted naked eye sensing of Hg(II)

NASA Astrophysics Data System (ADS)

Upadhyay, K. K.; Upadhyay, Shalini; Kumar, Kamlesh; Prasad, Rajendra

2009-06-01

The 3-Oxo-2-{[4-(thiazol-2-ylsulfamoyl)-phenyl]-hydrazono}-butyric acid ethyl ester (OSPBE) was studied through single crystal structure analysis revealing some interesting supramolecular architectural patterns. The N(3)-N(4) bond length of OSPBE was found to be 1.36 Å matching well with reported N-N bond length in the literature and hence clearly proved that it is the keto form of OSPBE which is stable. Full structural optimization of OSPBE using density functional theory (DFT) at the HCTH407/6-31G ∗∗ level also proved that the keto form of OSPBE is stable. The UV-Vis absorption peaks for OSPBE predicted by the time dependent DFT at B3LYP/6-311G ∗∗ level matched quite well with the experimentally observed UV-Vis bands for OSPBE. The OSPBE was successfully tested as the naked eye sensor for Hg(II) as its chloride salt at the millimolar level in dimethylsulfoxide. A color change from red orange to olive green was observed on addition of 1.0 equiv. of Hg(II) to the 1.0 × 10 -3 M DMSO solution of the chemosensor. The role of DMSO in the sensing process appears to be the crucial one because the intramolecular charge transfer (ICT) band of OSPBE in DMSO observed at 489 nm did not appear in the UV-Vis spectrum of OSPBE in nujol. The UV-Vis and 1H NMR titrations revealed that formation of six membered 1:1 chelate between OSPBE and Hg(II) along with reversible supramolecular association of DMSO with NH at N-2 position in OSPBE may be responsible for its Hg(II) sensing. No sensing for other d 10 metal ions like Zn(II) and Cd(II) were observed with OSPBE under similar conditions. Besides DMSO, some other polar aprotic solvents like DMF and acetone having X dbnd O (where X = C) also produced similar type of color change on the addition of 1.0 equiv. of Hg(II) to their respective 1.0 × 10 -3 M OSPBE solutions. Nevertheless, polar aprotic solvent like acetonitrile not having X dbnd O or non-polar aprotic solvent like chloroform no color change was observed under

Use of urinary 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) concentrations to diagnose pregnancy and predict parturition in the giant panda (Ailuropoda melanolecua).

PubMed

Roberts, Beth M; Brown, Janine L; Kersey, David C; Snyder, Rebecca J; Durrant, Barbara S; Kouba, Andrew J

2018-01-01

Pregnancy determination is difficult in the giant panda (Ailuropoda melanolecua), representing a challenge for ex situ conservation efforts. Research in other species experiencing pseudopregnancy indicates that urinary/fecal concentrations of 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) can accurately determine pregnancy status. Our objective was to determine if urinary PGFM concentrations are associated with pregnancy status in the giant panda. Urinary PGFM concentrations were measured in female giant pandas (n = 4) throughout gestation (n = 6) and pseudopregnancy (n = 4) using a commercial enzyme immunoassay. Regardless of pregnancy status, PGFM excretion followed a predictable pattern: 1) baseline concentrations for 11-19 weeks following ovulation; 2) a modest, initial peak 14-36 days after the start of the secondary urinary progestagen rise; 3) a subsequent period of relatively low concentrations; and 4) a large, terminal peak at the end of the luteal phase. Pregnant profiles were distinguished by an earlier initial peak (P = 0.024), higher inter-peak concentrations (P < 0.001), and a larger terminal peak (P = 0.003) compared to pseudopregnancy profiles. Parturition occurred 23 to 25 days from the initial PGFM surge and within 24 hours of the start of the terminal increase. These pattern differences indicate that urinary PGFM monitoring can be used to predict pregnancy status and time parturition in the giant panda. Furthermore, this is the only species known to exhibit a significant PGFM increase during pseudopregnancy, suggesting a unique physiological mechanism for regulating the end of the luteal phase in the giant panda.

Use of urinary 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) concentrations to diagnose pregnancy and predict parturition in the giant panda (Ailuropoda melanolecua)

PubMed Central

Brown, Janine L.; Kersey, David C.; Snyder, Rebecca J.; Durrant, Barbara S.; Kouba, Andrew J.

2018-01-01

Pregnancy determination is difficult in the giant panda (Ailuropoda melanolecua), representing a challenge for ex situ conservation efforts. Research in other species experiencing pseudopregnancy indicates that urinary/fecal concentrations of 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) can accurately determine pregnancy status. Our objective was to determine if urinary PGFM concentrations are associated with pregnancy status in the giant panda. Urinary PGFM concentrations were measured in female giant pandas (n = 4) throughout gestation (n = 6) and pseudopregnancy (n = 4) using a commercial enzyme immunoassay. Regardless of pregnancy status, PGFM excretion followed a predictable pattern: 1) baseline concentrations for 11–19 weeks following ovulation; 2) a modest, initial peak 14–36 days after the start of the secondary urinary progestagen rise; 3) a subsequent period of relatively low concentrations; and 4) a large, terminal peak at the end of the luteal phase. Pregnant profiles were distinguished by an earlier initial peak (P = 0.024), higher inter-peak concentrations (P < 0.001), and a larger terminal peak (P = 0.003) compared to pseudopregnancy profiles. Parturition occurred 23 to 25 days from the initial PGFM surge and within 24 hours of the start of the terminal increase. These pattern differences indicate that urinary PGFM monitoring can be used to predict pregnancy status and time parturition in the giant panda. Furthermore, this is the only species known to exhibit a significant PGFM increase during pseudopregnancy, suggesting a unique physiological mechanism for regulating the end of the luteal phase in the giant panda. PMID:29718929

Novel evidence for curcumin and boswellic acid induced chemoprevention through regulation of miR-34a and miR-27a in colorectal cancer

PubMed Central

Toden, Shusuke; Okugawa, Yoshinaga; Buhrmann, Constanze; Nattamai, Durgha; Anguiano, Esperanza; Baldwin, Nicole; Shakibaei, Mehdi; Boland, C. Richard; Goel, Ajay

2015-01-01

Colorectal cancer (CRC) is one of the most common causes of cancer-associated mortality worldwide, but it is truly a preventable disease. Both curcumin and boswellic acids are well-established dietary botanicals with potent anti-tumorigenic properties which have been shown to modulate multiple oncogenic pathways. Recent data suggest that the chemopreventive effects of these botanicals may in part be mediated through regulation of key cancer-related microRNAs (miRNAs) and their downstream gene targets. Here, we investigated the anti-tumorigenic effects of curcumin and 3 acetyl-11-keto-β-boswellic acid (AKBA) on modulation of specific cancer-related miRNAs in CRC cells and validated their protective effects in vivo using a xenograft mouse model. Both curcumin and AKBA inhibited cellular proliferation, induced apoptosis and cell cycle arrest in CRC cell lines, and these effects were significantly enhanced with combined treatment. Gene-expression arrays revealed that curcumin and AKBA regulated distinct cancer signaling pathways including key cell-cycle regulatory genes. Combined bioinformatics and in-silico analysis identified apoptosis, proliferation and cell-cycle regulatory signaling pathways as key modulators of curcumin and AKBA-induced anti-cancer effects. We discovered that curcumin and AKBA induced upregulation of tumor-suppressive miR-34a and downregulation of miR-27a in CRC cells. Furthermore, we demonstrated in a mouse xenograft model that both curcumin and AKBA treatments suppressed tumor growth, which corresponded with alterations in the expression of miR-34a and miR-27a, consistent with our in vitro findings. Herein we provide novel mechanistic evidence for the chemopreventive effects of curcumin and AKBA through regulation of specific miRNAs in colorectal cancer. PMID:25712055

Hybrid pigments resulting from several guest dyes onto γ-alumina host: A spectroscopic analysis

NASA Astrophysics Data System (ADS)

Pérez, Erik; Ibarra, Ilich A.; Guzmán, Ariel; Lima, Enrique

2017-02-01

The synthesis of hybrid pigments was made from combination of γ-Al2O3 and some organic chromophores such as carminic acid, alizarin, purpurin, curcumin, fluorescein and betacyanins. The γ-Al2O3 was obtained through sol-gel synthesis with 2-propanol and aluminium tri-sec-butoxide (ATB). This article presents some spectroscopic evidences related to the formation of aluminium complexes between coordinative unsaturated sites (CUS) of aluminium and some organic groups (carboxylic acid, quaternary ammonium and β-keto enol) present in the chromophores structure. The physicochemical properties upcoming from a spectroscopic analysis point out that these materials can be applied in the design of new materials with potential uses in artworks and in the field of cultural heritage.

Cyclic azole-homologated peptides from Marine sponges.

PubMed

Molinski, Tadeusz F

2017-12-19

This review discusses the chemistry of cyclic azole-homologated peptides (AHPs) from the marine sponges, Theonella swinhoei, other Theonella species, Calyx spp. and Plakina jamaicensis. The origin, distribution of AHPs and molecular structure elucidations of AHPs are described followed by their biosynthesis, bioactivity, and synthetic efforts towards their total synthesis. Reports of partial and total synthesis of AHPs extend beyond peptide coupling reactions and include creative construction of the non-proteinogenic amino acid components, mainly the homologated heteroaromatic and α-keto-β-amino acids. A useful conclusion is drawn regarding AHPs: despite their rarity, exotic structures and the potent protease inhibitory properties of some members, their synthesis is under-developed and beckons solutions for outstanding problems towards their efficient assembly.

Molecular modeling and simulation of FabG, an enzyme involved in the fatty acid pathway of Streptococcus pyogenes.

PubMed

Shafreen, Rajamohmed Beema; Pandian, Shunmugiah Karutha

2013-09-01

Streptococcus pyogenes (SP) is the major cause of pharyngitis accompanied by strep throat infections in humans. 3-keto acyl reductase (FabG), an important enzyme involved in the elongation cycle of the fatty acid pathway of S. pyogenes, is essential for synthesis of the cell-membrane, virulence factors and quorum sensing-related mechanisms. Targeting SPFabG may provide an important aid for the development of drugs against S. pyogenes. However, the absence of a crystal structure for FabG of S. pyogenes limits the development of structure-based drug designs. Hence, in the present study, a homology model of FabG was generated using the X-ray crystallographic structure of Aquifex aeolicus (PDB ID: 2PNF). The modeled structure was refined using energy minimization. Furthermore, active sites were predicted, and a large dataset of compounds was screened against SPFabG. The ligands were docked using the LigandFit module that is available from Discovery Studio version 2.5. From this list, 13 best hit ligands were chosen based on the docking score and binding energy. All of the 13 ligands were screened for Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties. From this, the two best descriptors, along with one descriptor that lay outside the ADMET plot, were selected for molecular dynamic (MD) simulation. In vitro testing of the ligands using biological assays further substantiated the efficacy of the ligands that were screened based on the in silico methods. Copyright © 2013 Elsevier Inc. All rights reserved.

Identification of cytotoxic mediators and their putative role in the signaling pathways during docosahexaenoic acid (DHA)-induced apoptosis of cancer cells.

PubMed

Das, Moitreyi; Das, Sumantra

2016-12-01

Docosahexaenoic acid (DHA), an important w-3 fatty acid exhibits differential behavior in cancer cells of neural origin when compared to that in normal healthy astrocytes. Treatment of C6 glioma and SH-SY5Y cell lines and primary astrocytes, representing the neoplastic cells and normal healthy cells respectively, with 100 µM DHA for 24 h showed significant loss of cell viability in the both the cancer cells as determined by MTT assay, whereas the primary astrocytes cultures were unaffected. Such loss of cell viability was due to apoptosis as confirmed by TUNEL staining and caspase-3 activation in cancer cells. Proteomic approach, employing 2-dimensional gel electrophoresis (2DE), difference gel electrophoresis (DIGE), and MALDI-TOF-TOF analysis identified six proteins which unlike in the astrocytes, were differently altered in the cancer cells upon exposure to DHA, suggesting their putative contribution in causing apoptosis in these cells. Of these, annexin A2, calumenin, pyruvate kinase M2 isoform, 14-3-3ζ were downregulated while aldo keto reductase-1B8 (AKR1B8) and glutathione-S-transferase P1 subunit (GSTP1) showed upregulation by DHA in the cancer cells. siRNA-mediated knockdown of AKR1B8 and GSTP1 inhibit DHA-induced apoptosis confirming their role in apoptotic process. Furthermore, western blot analysis identified upregulation of PPARα and the MAP kinases, JNK and p38 as well as increased ROS production selectively in the cell lines. Results suggest that DHA selectively induces apoptosis in the neural cell lines by regulating the expression of the above proteins to activate multiple apoptotic pathways which in association with excess ROS and activated MAPKs promote cell death.

Oxygen optical gas sensing by reversible fluorescence quenching in photo-oxidized poly(9,9-dioctylfluorene) thin films.

PubMed