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

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

Effects of industrial heat treatments on the kinetics of inactivation of antimicrobial bovine milk xanthine oxidase.

Milk is a source of antimicrobial systems such as xanthine oxidoreductase, which has been proposed to modulate the oral and gut microbiota of infants. Heat treatments are applied to milk to ensure its microbial safety, however, the effects of heat on this antimicrobial enzyme are not known. The effects of batch pasteurization (BP), high-temperature short time (HTST), and ultra high temperature (UHT) on kinetics of inactivation of xanthine oxidase and its antimicrobial properties were determined. Xanthine oxidase activity was preserved by HTST (100%). Partial (8%) and nearly complete (95%) enzyme inactivation were observed for BP and UHT milks, respectively. K m values of 100 μM and V max values of 6.85, 5.12, 6.31, and 0.40 μmol/min/mg were determined for xanthine oxidase in raw, BP, HTST, and UHT milks, respectively. These results demonstrate that xanthine oxidase maintains apparent affinity and activity for its substrate when milk is treated by BP and HTST and yet the enzyme is inactivated with UHT. To investigate heat treatment-induced alterations in the biological activity of xanthine oxidase, heat treated milks were compared to raw milk for their ability to inhibit the growth of S. aureus. Raw, BP, and HTST milk xanthine oxidase efficiently inhibited S. aureus growth. However, these antibacterial properties were lost when milk was subjected to UHT. These results demonstrate that HTST and BP preserves bovine milk xanthine oxidase activity compared with UHT and that, the judicious selection of thermal treatments could be exploited to preserve the antimicrobial properties of bovine milk

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

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

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

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.

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

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

The L-amino acid oxidase of Neurospora

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

β-Amyloid peptides induce mitochondrial dysfunction and oxidative stress in astrocytes and death of neurons through activation of NADPH oxidase

β-Amyloid (βA) peptide is strongly implicated in the neurodegeneration underlying Alzheimer's disease, but the mechanisms of neurotoxicity remain controversial. This study establishes a central role for oxidative stress by the activation of NADPH oxidase in astrocytes as the cause of βA-induced neuronal death. βA causes a loss of mitochondrial potential in astrocytes but not in neurons. The mitochondrial response consists of Ca2+-dependent transient depolarizations superimposed on a slow collapse of potential. The slow response is both prevented by antioxidants and, remarkably, reversed by provision of glutamate and other mitochondrial substrates to complexes I and II. These findings suggest that the depolarization reflects oxidative damage to metabolic pathways upstream of mitochondrial respiration. Inhibition of NADPH oxidase by diphenylene iodonium or 4-hydroxy-3-methoxy-acetophenone blocks βA-induced reactive oxygen species generation, prevents the mitochondrial depolarization, prevents βA-induced glutathione depletion in both neurons and astrocytes, and protects neurons from cell death, placing the astrocyte NADPH oxidase as a primary target of βA-induced neurodegeneration

Substrate specificity of amine oxidase

The tyramine oxidase activity of liver extracts found by Hare (1), the aliphatic amine oxidase activity of brain, kidney, and liver extracts observed by Pugh and Quastel (2), and the adrenalin oxidase activity of similar extracts noted by Blaschko, Richter, and Schlossman (3) were brought under a common enzyme view-point by the latter authors. They were able to show (4) that extracts of brain, instestine, kindey, and liver from a number of mammals or representatives of the birds, reptiles, amphibians, and fishes all acted to absorb oxygen in the presence of several amine substrates. Hare (1) had shown that tyramine and phenethylamine form ammonia in the course of such oxidations, and Richter (5) showed that an ethylamino and a dimethylamino compound, as well as a number of methylamino and amino compounds, all yield the corresponding alkyl-amines or ammonia in the enzymic oxidation. The conslusion that the demonstrated variey of such enzymic activity can be acribed to the presence of a single type pf amine oxidase was dependent in large part on observations that the relative activities of a preparation from one source on a series of substrates bear some relation to the relative activities exhibited by a preparation from another source. Further evidence depended on the action of certain amines as inihibitors and apparent competition between substrates when two oxidizable substrates are present in the system. The degree to which relative activities of different enzyme preparations were constant in a series of substrates was not good in the data reported, and the fact that Hare (1) had not been able to note activity of the liver preparations she used upon adrenalin as the substrate appeared to require special explanations

Alternative final steps in berberine biosynthesis in Coptis japonica cell cultures

In Coptis japonica cell cultures an alternative pathway has been discovered which leads from (S)-tetrahydrocolumbamine via (S)-canadine to berberine. The two enzymes involved have been partially purified. (S)-Tetrahydrocolumbamine is stereospecifically transformed into (S)-canadine under formation of the methylenedioxy bridge in ring A. This new enzyme was named (S)-canadine synthase. (S)-Canadine in turn is stereospecifically dehydrogenated to berberine by an oxidase, (S)-canadine oxidase (COX), which was partially purified (25-fold). This enzyme has many physical properties in common with the already known (S)-tetrahydroprotoberberine oxidase from Berberis but grossly differs from the latter enzyme in its cofactor requirement (Fe) and its substrate specificity. Neither (S)-norreticuline nor (S)-scoulerine serves as substrate for the Coptis enzyme, while both substrates are readily oxidized by the Berberis enzyme. The four terminal enzymes catalyzing the pathway from (S)-reticuline to berberine are housed in Berberis as well as in Coptis in smooth vesicles with a density of =1.14 g/ml. These vesicles have been enriched and characterized by electron microscopy

Cytochrome oxidase subunit VI of Trypanosoma brucei is imported without a cleaved presequence and is developmentally regulated at both RNA and protein levels

Mitochondrial respiration in the African trypanosome undergoes dramatic developmental stage regulation. This requires co-ordinated control of components encoded by both the nuclear genome and the kinetoplast, the unusual mitochondrial genome of these parasites. As a model for understanding the co-ordination of these genomes, we have examined the regulation and mitochondrial import of a nuclear-encoded component of the cytochrome oxidase complex, cytochrome oxidase subunit VI (COXVI). By generating transgenic trypanosomes expressing intact or mutant forms of this protein, we demonstrate that COXVI is not imported using a conventional cleaved presequence and show that sequences at the N-terminus of the protein are necessary for correct mitochondrial sorting. Analyses of endogenous and transgenic COXVI mRNA and protein expression in parasites undergoing developmental stage differentiation demonstrates a temporal order of control involving regulation in the abundance of, first, mRNA and then protein. This represents the first dissection of the regulation and import of a nuclear-encoded protein into the cytochrome oxidase complex in these organisms, which were among the earliest eukaryotes to possess a mitochondrion