302,629 research outputs found

    Effects of chilling on the expression of ethylene biosynthetic genes in Passe-Crassane pear (Pyrus communis L.) fruits

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    Passe-Crassane pears require a 3-month chilling treatment at 0 C to be able to produce ethylene and ripen autonomously after subsequent rewarming. The chilling treatment strongly stimulated ACC oxidase activity, and to a lesser extent ACC synthase activity. At the same time, the levels of mRNAs hybridizing to ACC synthase and ACC oxidase probes increased dramatically. Fruit stored at 18 C immediately after harvest did not exhibit any of these changes, while fruit that had been previously chilled exhibited a burst of ethylene production associated with high activity of ACC oxidase and ACC synthase upon rewarming. ACC oxidase mRNA strongly accumulated in rewarmed fruits, while ACC synthase mRNA level decreased. The chilling-induced accumulation of ACC synthase and ACC oxidase transcripts was strongly reduced when ethylene action was blocked during chilling with 1-methylcyclopropene (1-MCP). Upon rewarming ACC synthase and ACC oxidase transcripts rapidly disappeared in 1-MCP-treated fruits. A five-week treatment of non-chilled fruits with the ethylene analog propylene led to increased expression of ACC oxidase and to ripening. However, ethylene synthesis, ACC synthase activity and ACC synthasemRNAs remained at very lowlevel. Our data indicate thatACC synthase gene expression is regulated by ethylene only during, or after chilling treatment, while ACC oxidase gene expression can be induced separately by either chilling or ethylene

    Enzyme chemical engineering and its application to biosensors : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University, Turitea, New Zealand

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    Enzyme chemical engineering is a fast growing area in biotechnology. It has been used to change the stability, solubility, activity and other properties of enzymes for more control over and wider application of enzymes. In this thesis, this technology is applied to another new and fast growing area of research: biosensors. Over the last decade, biosensors are gaining increasing awareness as a highly attractive analytical tool. One of the current challenges in this area is to identify a universal and scaleable way to produce sensitive, stable, instantaneous, and easy to prepare biosensors for mass production. In this study, enzyme chemical engineering is adopted as a new approach and glucose oxidase is served as a model to build a biosensor system in attempting to address the above challenge. In the study, glucose oxidase was used as the catalyst to chemically amplify the redox reaction of glucose. Haemin was employed as the bifunctional promoter to act as a "bridge" to connect glucose oxidase (GOD) and electrode. Haemin, similar to ferrocene also acts as a mediator to transfer electrons between the active center of the enzyme and the electrode. In the construction of a haemin-glucose oxidase biosensor, haemin was covalently bound with glucose oxidase. The haemin-glucose oxidase conjugate was then chemisorbed on to the platinum electrode to modify the electrode surface and form an "enzymatic redox center-bridge-electrode" system. The modification of the glucose oxidase with haemin comprised of two steps: converting the haemin carboxyl group to the reactive enol ester and then covalently bonding to an amino group of glucose oxidase. For chemisorption, the electrode was soaked in a solution of the haemin-glucose oxidase conjugate in phosphate buffer solution (pH 7.0) at 4°C for 16 hours. The same experiment was carried out by using unmodified glucose oxidase as a blank. The following facts proved that the covalently bound haemin-glucose oxidase system was formed successfully: 1) The large molecule fractions eluted from the Sephadex G-10 gel column had the enzyme activity and other characteristics of glucose oxidase. 2) The same fractions retained about 2/3 to 3/4 of the specific activity of original glucose oxidase. 3) The absorbance spectra of these fractions showed the peaks corresponding to both haemin and glucose oxidase. The following evidence suggests that the haemin-glucose oxidase conjugate was successfully chemisorbed on to the electrode surface: 1) The cyclic voltammogram of the electrode chemisorbed with conjugate was completely different from that adsorbed with glucose oxidase alone. 2) The cyclic voltammogram of the conjugate chemisorbed electrode in the solution with glucose was quite distinct from that without glucose. Thus a different species from either glucose oxidase or haemin was chemiabsorbed on to the electrode. Furthermore, the conjugate chemisorbed electrode showed linearity between current response and glucose concentration at a range from 0mM to 10mM. The ratio of the current response to glucose concentration was about 1.6µA/mM. However, the platinum electrode adsorbed by GOD alone had a poor response to glucose. The response time of the system of platinum electrode-haemin-glucose oxidase was very rapid at less than one minute, and the response fell initially but then remained stable over a period of 14 days. Thus the experimental data proved that the system of platinum electrode-haemin-glucose oxidase met the requirements for a glucose sensor in the factors of the sensitivity, linear response range, lifetime, ease of preparation, convenience of operation, non-toxicity and low cost. In other words, it demonstrated the characteristics of a glucose biosensor. Finally, using the preparation of this glucose biosensor as a model, the electrochemical mechanism of the biosensor system was proposed. The model was also used to suggest a systematic approach for constructing amperometric biosensors. The extension of this approach and the potential applications of this type of biosensor are also discussed

    Role of urate, xanthine oxidase and the effects of allopurinol in vascular oxidative stress

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    Oxidative stress plays an important role in the progression of vascular endothelial dysfunction. The two major systems generating vascular oxidative stress are the NADPH oxidase and the xanthine oxidase pathways. Allopurinol, a xanthine oxidase inhibitor, has been in clinical use for over 40 years in the treatment of chronic gout. Allopurinol has also been shown to improve endothelial dysfunction, reduce oxidative stress burden and improve myocardial efficiency by reducing oxygen consumption in smaller mechanistic studies involving various cohorts at risk of cardiovascular events. This article aims to explain the role of xanthine oxidase in vascular oxidative stress and to explore the mechanisms by which allopurinol is thought to improve vascular and myocardial indices

    Peroxisomes in intestinal and gallbladder epithelial cells of the stickleback, Gasterosteus aculeatus L. (Teleostei)

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    The occurrence of microbodies in the epithelial cells of the intestine and gallbladder of the stickleback, Gasterosteus aculeatus L., is described. In the intestine the organelles are predominantly located in the apical and perinuclear zone of the cells and may contain small crystalline cores. In gallbladder epithelial cells the microbodies are distributed randomly. The latter organdies are characterized by the presence of large crystalloids. Cytochemical and biochemical experiments show that catalase and D-amino acid oxidase are main matrix components of the microbodies in both the intestinal and gallbladder epithelia. These organelles therefore are considered peroxisomes. In addition, in intestinal mucosa but not in gallbladder epithelium a low activity of palmitoyl CoA oxidase was detected biochemically. Urate oxidase and L-α hydroxy acid oxidase activities could not be demonstrated.

    Vitamins Reverse Endothelial Dysfunction Through Regulation of eNOS and NAD(P)H Oxidase Activities

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    Antioxidant vitamins C and E have protective properties in genetic hypertension associated with enhanced oxidative stress. This study investigated whether vitamins C and/or E modulate vascular function by regulating enzymatic activities of endothelial nitric oxide synthase (eNOS) and NAD(P)H oxidase using thoracic aortas of 20- to 22-week-old male spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar-Kyoto rats (WKY). SHR aortas had impaired relaxant responses to acetylcholine but not to sodium nitroprusside, despite an 2-fold increase in eNOS activity and NO release. The levels of superoxide anion (O2 ), a potent NO scavenger, and NAD(P)H oxidase activity were also 2-fold higher in SHR aortas. Mechanical but not pharmacological inactivation of endothelium (by rubbing and 100 mol/L L-NAME, respectively) significantly abrogated O2 in both strains. Treatments of SHR aortas with NAD(P)H oxidase inhibitors, namely diphenyleneiodinium and apocynin, significantly diminished O2 production. The incubation of SHR aortas with different concentrations of vitamin C (10 to 100 mol/L) and specifically with high concentrations of vitamin E (100 mol/L) improved endothelial function, reduced superoxide production as well as NAD(P)H oxidase activity, and increased eNOS activity and NO generation in SHR aortas to the levels observed in vitamin C- and E-treated WKY aortas. Our results reveal endothelial NAD(P)H oxidase as the major source of vascular O2 in SHR and also show that vitamins C and E are critical in normalizing genetic endothelial dysfunction through regulation of eNOS and NAD(P)H oxidase activities

    Precursors of Cytochrome Oxidase in Cytochrome-Oxidase-Deficient Cells of Neurospora crassa

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    Three different cell types of Neurospora crassa deficient in cytochrome oxidase were studied: the nuclear mutant cni-1, the cytoplasmic mutant mi-1 and copper-depleted wild-type cells. * 1. The enzyme-deficient cells have retained a functioning mitochondrial protein synthesis. It accounted for 12–16% of the total protein synthesis of the cell. However, the analysis of mitochondrial translation products by gel electrophoresis revealed that different amounts of individual membrane proteins were synthesized. Especially mutant cni-1 produced large amounts of a small molecular weight translation product, which is barely detectable in wild-type. * 2. Mitochondrial preparations of cytochrome-oxidase-deficient cells were examined for precursors of cytochrome oxidase. The presence of polypeptide components of cytochrome oxidase in the mitochondria was established with specific antibodies. On the other hand, no significant amounts of heme a could be extracted. * 3. Radioactively labelled components of cytochrome oxidase were isolated by immunoprecipitation and analysed by gel electrophoresis. All three cell types contained the enzyme components 4–7, which are translated on cytoplasmic ribosomes. The mitochondrially synthesized components 1–3 were present in mi-1 mutant and in copper-depleted wild-type cells. In contrast, components 2 and 3 were not detectable in the nuclear mutant cni-1. Both relative and absolute amounts of these polypeptides in the enzyme-deficient cells were quite different from those in wild-type cells. * 4. The components of cytochrome oxidase found in the enzyme-deficient cells were tightly associated with the mitochondrial membranes. * 5. Processes, which affect and may control the production of enzyme precursors or their assembly to a functional cytochrome oxidase are discussed

    Synthesis and study of polyhydroxylated phenol derivatives with potential cosmetic and phytoiatric applications

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    Tyrosinase (polyphenol oxidase, E.C. 1.14.18.1) and laccase (phenol oxidase, E.C. 1.10.3.2) are multifunctional copper-containing enzymes, that are keys in melanin biosynthesis, melanisation in animals and browning in plants. Our study is aimed to prepare new monomer and dimer phenol derivatives as potential inhibitors of melanin production starting from natural hydroxylated aromatic units

    The nature of CuA in cytochrome c oxidase

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    The isolation and purification of yeast cytochrome c oxidase is described. Characterization of the purified protein indicates that it is spectroscopically identical with cytochrome c oxidase isolated from beef heart. Preparations of isotopically substituted yeast cytochrome c oxidase are obtained incorporating [1,3-15N2]histidine or [beta,beta- 2H2]cysteine. Electron paramagnetic resonance and electron nuclear double resonance spectra of the isotopically substituted proteins identify unambiguously at least 1 cysteine and 1 histidine as ligands to CuA and suggest that substantial spin density is delocalized onto a cysteine sulfur in the oxidized protein to render the site Cu(I)-S

    The L-amino acid oxidase of Neurospora

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    In 1944 one of us described a D-amino acid oxidase in extracts of Neurospora crassa (1). Except for slight activity against L-glutamate, no oxidation of L-amino acids was observed. Recently a means for inducing the formation of a soluble L-amino acid oxidase by the mold was reported by Bender, Krebs, and Horowitz (2). This is accomplished by reducing the biotin content of the basal medium (3) from the 5 γ per liter usually employed to 0.25 γ per liter. When cultured in such a medium, Neurospora produces an active L-amino acid oxidase which can be demonstrated both in extracts and in the medium. Reduction of the biotin level produces no effect on the D-amino acid oxidase, which is still found in extracts but not in the culture medium. The activity of the L-oxidase toward thirty-eight amino acids has been investigated by Bender and Krebs (4). The initial purpose of the present investigation was to explore certain possibilities for a genetic study of the enzyme. Of special interest was the fact, cited by Bender et al. (a), that the oxidase could not be detected in all of the wild type strains tested. The question arose as to whether this is due to the genetic inability of certain strains to form the enzyme. In connection with the investigation of this problem a general survey of the properties of the enzyme was carried out, together with a preliminary study of the mechanism of the biotin effect and of the adaptive formation of the enzyme. The results of these studies are reported below. Simultaneously with our investigation and independently of it, Dr. K. Burton (5), working in Professor Krebs’ laboratory, has carried out a study of the Neurospora L-amino acid oxidase. Where our respective studies overlap mutual confirmation was obtained in most essential points. We wish to thank Dr. Burton for permission to read his manuscript before publication
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