Biosynthesis of flavan 3-ols by leucoanthocyanidin 4-reductases and anthocyanidin reductases in leaves of grape (Vitis vinifera L.), apple (Malus x domestica Borkh.) and other crops.

Catechin and epicatechin biosyntheses were studied of grape (Vitis vinifera L.), apple (Malus x domestica Borkh.) and other crop leaves, since these monomers and the derived proanthocyanidins are important disease resistance factors. Grape and apple leucoanthocyanidin 4-reductase (LAR; EC 1.17.1.3) enzymes were characterized on basis of plant and recombinant enzymes. In case of grape, two LAR cDNAs were cloned by assembling available EST sequences. Grape and apple leaf anthocyanidin reductase (ANR; EC 1.3.1.77) cDNAs were also obtained and the respective plant and recombinant enzymes were characterized. Despite general low substrate specificity, within the respective flavonoid biosyntheses of grape and apple leaves, both enzyme types deliver differently hydroxylated catechins and epicatechins, due to substrate availability in vivo. Furthermore, for LAR enzymes conversion of 3-deoxyleucocyanidin was shown. Beside relevance for plant protection, this restricts the number of possible reaction mechanisms of LAR. ANR enzyme activity was demonstrated for a number of other crop plants and its correlation with (-)-epicatechin and obvious competition with UDP-glycosyl:flavonoid-3-O-glycosyltransferases was considered.     

Cytokines and lipopolysaccharides induce inducible nitric oxide synthase but not enzyme activity in adult rat cardiomyocytes

There is evidence that nitric oxide (NO) formation in adult cardiomyocytes stimulated with lipopolysaccharide (LPS) is not commensurate with iNOS levels. Tetrahydrobiopterin (BH(4)) is a key factor in the stabilization and NO production by iNOS homodimer. Thus we hypothesized that BH(4) is a limiting factor for NO production in adult cardiomyocytes in response to LPS and cytokines (TNF-alpha, IL-1, IFN-gamma alone, or mixed). It was verified that LPS and cytokines induced iNOS expression which did not translate into increased nitrite or [(14)C]citrulline production. This response coincided with defective BH(4) synthesis and low GTP cyclohydrolase activity. Furthermore, supplementation with BH(4) and ascorbate failed to increase iNOS activity. This effect was related to preferential accumulation of BH(2) rather than BH(4) in these cells. Uncoupled iNOS activity in stimulated cells was examined using mitochondrial aconitase activity as an endogenous marker of superoxide anion radical (O(2)(-)) formation, and found not to be significantly inhibited. 2-Hydroxyethidium also was not significantly increased. We conclude that adult cardiomyocytes are an unlikely source of NO and O(2)(-) in inflammatory conditions. This finding adds a new and unexpected layer of complexity to our understanding of the responses of the adult heart to inflammation.     

Effects of brassinosteroid, auxin, and cytokinin on ethylene production in Arabidopsis thaliana plants

Inflorescence stalks produced the highest amount of ethylene in response to IAA as compared with other plant parts tested. Leaf age had an effect on IAA-induced ethylene with the youngest leaves showing the greatest stimulation. The highest amount of IAA-induced ethylene was produced in the root or inflorescence tip with regions below this producing less. Inflorescence stalks treated with IAA, 2,4-D, or NAA over a range of concentrations exhibited an increase in ethylene production starting at 1 microM with increasingly greater responses up to 100 microM, followed by a plateau at 500 microM and a significant decline at 1000 microM. Both 2,4-D and NAA elicited a greater response than IAA at all concentrations tested in inflorescence stalks. Inflorescence leaves treated with IAA, 2,4-D, or NAA exhibited the same trend as inflorescence stalks. However, they produced significantly less ethylene. Inflorescence stalks and leaves treated with 100 microM IAA exhibited a dramatic increase in ethylene production 2 h following treatment initiation. Inflorescence stalks showed a further increase 4 h following treatment initiation and no further increase at 6 h. However, there was a slight decline between 6 h and 24 h. Inflorescence leaves exhibited similar rates of IAA-induced ethylene between 2 h and 24 h. Light and high temperature caused a decrease in IAA-induced ethylene in both inflorescence stalks and leaves. Three auxin-insensitive mutants were evaluated for their inflorescence's responsiveness to IAA. aux2 did not produce ethylene in response to 100 microM IAA, while axr1-3 and axr1-12 showed reduced levels of IAA-induced ethylene as compared with Columbia wild type. Inflorescences treated with brassinolide alone had no effect on ethylene production. However, when brassinolide was used in combination with IAA there was a dramatic increase in ethylene production above the induction promoted by IAA alone.     

Relationship of steroidal structure to ethylene production by etiolated mung bean segments

Several brassinosteroid (BR) analogues, cholesterol and aldosterone were evaluated for their effectiveness alone and in combination with indole-3-acetic acid (IAA) in stimulating ethylene production by etiolated mung bean ( Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. Changing the conformation of the two hydroxyl groups on C-22 and C-23 positions from α to β did not greatly reduce the efficiency of these compounds to stimulate ethylene production alone or in combination with IAA. There was little difference in activity observed when the conformation of the methyl group in the C-24 position was changed from α to β. However, when hydroxyls were deleted from the side chain in the C-22 and C-23 positions, the compound was rendered inactive alone or in combination with IAA. The compound was also inactivated by removing the 7-oxa function on the B-ring and by substituting an ethyl group for the methyl group in the C-24 position. Both aldosterone and cholesterol were ineffective in promoting ethylene production. This study shows that very stringent structural features are required for a steroid to have BR-like activity and to act synergistically with auxin in the promotion of ethylene synthesis.     

AAV-Mediated Gene Therapy for Choroideremia: Preclinical Studies in Personalized Models

Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1^st or 2^nd decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.     

The Role of Tetrahydrobiopterin in Superoxide Generation from eNOS: Enzymology and Physiological Implications

Tetrahydrobiopterin (BH₄) is a ubiquitous pteridine metabolite that serves as a NOS cofactor. Recently, we showed that BH₄ efficiently inhibits superoxide generation from the heme group at the oxygenase domain of eNOS. This role indicates that BH₄ acts as a redox switch in the catalytic mechanism of the enzyme, which may have important consequences in the physiology of the endothelium. Here the mechanism by which BH₄ inhibits superoxide release from eNOS and the "uncoupling" effects of oxidized BH₄ metabolites are presented. The implications of the disparate actions of fully reduced and oxidized BH 4 metabolites in the control of eNOS biochemistry are discussed in the light of clinical data indicating that BH 4 levels are important in the regulation of superoxide levels and of endothelial reactivity.