Involvement of the fatty acid oxidation complex in acetyl-CoA-dependent chain elongation of fatty acids in Escherichia coli

The activity of acetyl-CoA-dependent chain elongation of fatty acids in Escherichia coli was enhanced when the organism was grown on oleic acid as the sole carbon source, but not detected when grown on glucose. Antibodies raised against fatty acid oxidation complex of E. coli inhibited both the reaction catalyzed by crotonase and the chain elongation in a similar manner, showing that the oxidation complex participates in the chain elongation. The activities of condensation and the activities of NADH- and NADPH-dependent 3-ketoacyl reduction in the cell-free extract were precipitated by antibodies to the complex in parallel with those of 3-ketoacyl-CoA thiolase and crotonase. These results together with the presence of NADPH-dependent trans-2-enoyl-CoA reductase in E. coli (Mizugaki, et al. (1982) Chem. Pharm. Bull. 30, 2503-2511) indicate that the acetyl-CoA-dependent chain elongation of fatty acids in E. coli occurs by the reversal of fatty acid oxidation other than the step of enoyl reduction.     

Glutamine Synthetase of the Human Brain: Purification and Characterization

Glutamine synthetase (GS) isolated from human brain formed a single band on sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 44,000. The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of gamma-glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than that in the sheep enzyme. Antibodies were raised in rabbits against the purified enzyme. The double-immunodiffusion technique disclosed cross-reactivities among GSs isolated from human, sheep, and rat brains, but the enzymes were not immunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytes of the liver.     

Studies on the metabolism of unsaturated fatty acids. XVI. Purification and general properties of 2,4-dienoyl-CoA reductase from Candida lipolytica

2,4-Dienoyl-CoA reductase has been purified to homogeneity from Candida lipolytica cultivated in the presence of linoleic acid. The native enzyme had a molecular weight close to 360,000 as estimated by gel filtration on Sepharose CL-4B, whereas the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 33,000. The purified 2,4-dienoyl-CoA reductase from C. lipolytica gave a single precipitin line with antibodies raised against the purified enzyme from C. lipolytica. The general properties of the 2,4-dienyl-CoA reductase from C. lipolytica were examined. The enzyme had optimal pH at 6.5 and was inactivated by heat treatment at 50 degrees C for 10 min. trans-2,trans-4-Octadienoyl-CoA was the most active substrate of the dienoyl-CoA esters examined.     

Purification and characterization of N-ethylmaleimide reducing enzyme from Candida lipolytica

N-Ethylmaleimide (NEM) reducing enzyme was purified to homogeneity from cell-free extracts of Candida lipolytica by chromatography techniques. The molecular weight of the native enzyme was estimated to be about 43,000 by gel filtration using Superose 12 and to be 47,000 by SDS-PAGE. This enzyme can use both NADPH and NADH as an electron donor, and catalyzes the reduction of the carbon-carbon double bond of five membered ring compounds which have two conjugated carbonyl groups on both sides of a double bond.     

Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector

Replication-defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida-free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SRα promoter (SV40 early promoter-fused HTLV-I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for β-galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula-blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2-cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector-treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development. © 1995 wiley-Liss, Inc.     

Extra-weak chemiluminescence of drugs XV. Method for determining stability of Toki-shakuyaku-san extract granules

The purpose of the study was to evaluate the quality of Toki-shakuyaku-san extract granules (TJ-23) using chemiluminescence (CL). A linear relationship was obtained between the log value of the CL of TJ-23 and the reaction temperature. An excellent correlation (r= 0.999) was found between the slope of this curve (ΔA) and the colour intensity due to the browning reaction occurring at the early stage of the Maillard reaction.     

Analysis of the thromboxane/prostacyclin balance in human urine by gas chromatography/selected ion monitoring: abnormalities in diabetics

We microanalyzed 2,3-dinor-6-keto-prostaglandin F_1α (2,3-dinor-6-keto-PGF_1α 1) and 11-dehydrothromboxane B₂ (11-dehydro-TXB₂, 2) in human urine. Samples containing a [²H₄]-analogue as an internal standard were extracted by chromatography using Sep Pak tC18 and silica gel. The compounds were then analysed by means of the lactone ring opening reaction and dimethylisopropylsilylation. The conversion of 1 to 1-methyl ester (ME)-propylamide (PA)-9,12,15-dimethylisopropylsilyl (DMIPS) ether derivative and of 2 to 1-ME-6-methoxime (MO)-9,12,15-tris-DMIPS ether derivative was followed by gas chromatography/selected ion monitoring (GC/SIM). Interfering substances from the urine matrix were eliminated during GC/SIM analysis using a DB-5 column. We were able to detect 1 (222–1031 pg/mg creatinine) and 2 (18–155 pg/mg creatinine) in human urine. Furthermore, the thromboxane/prostacyclin (IX/PGI) ratio in the urine of diabetics was higher than that of healthy volunteers. This method can be used to determine the TX/PGI balance in human urine.     

Separation and concentration of delta 17-6-keto-PGF1 alpha using monoclonal antibody to omega 3-olefin structure of trienoic prostanoids.

A monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate delta 17-6-keto-prostaglandin F1 alpha (PGF1 alpha) in the human sera. cis-3-Hexen-1-ol was conjugated with the human serum albumin (HSA) according to the N-succinimidylester method and hyperimmunized to BALB/c mouse. The monoclonal antibodies were obtained from hybridoma clones established by a fusion between SP2/0-Ag14-k13 mouse myeloma cells and splenocytes of a mouse. A monoclonal antibody, named 4G9-12B, recognized the epitope characteristic for omega 3-olefin structure. The 4G9-12B antibody became more specific for delta 17-6-keto-PGF1 alpha than 6-keto-PGF1 alpha by applying inhibition ELISA using amino-residue coating plates. Using the prepared immunoaffinity columns of this antibody, delta 17-6-keto-PGF1 alpha was clearly detected in 6 pg/ml of the human blood sera by GC/MS analysis. These results suggest that the monoclonal antibody to the partial structure of trienoic prostanoid, omega 3-olefin unit, and that its immunoaffinity columns are useful in separating and concentrating delta 17-6-keto-PGF1 alpha in the human blood or urine.     

Oxidative stress triggered by aluminum in plant roots

Aluminum (Al) is a major growth-limiting factor for plants in acid soils. The primary site of Al accumulation and toxicity is the root meristem, and the inhibition of root elongation is the most sensitive response to Al. Al cannot catalyze redox reactions but triggers lipid peroxidation and reactive oxygen species (ROS) production in roots. Furthermore, Al causes respiration inhibition and ATP depletion. Comparative studies of Al toxicity in roots with that in cultured plant cells suggest that Al causes dysfunction and ROS production in mitochondria, and that ROS production, but not lipid peroxidation, seems to be a determining factor of root-elongation inhibition by Al.