Substrate inhibition of xanthine oxidase and its influence on superoxide radical production.

The influence of substrate inhibition on xanthine oxidase-intramolecular electron transport was studied by steady-state kinetic analysis. Experiments with hypoxanthine and xanthine up to 900 microM indicated an inhibition pattern which fitted an equation of the general form nu 0 = nu max . [S]/(Km + a[S] + b[S]2/Ki). Univalent electron flux to oxygen was favored at substrate concentrations above 50 microM. This augmentation of univalent flux percentage that appeared at a high substrate concentration was greater for hypoxanthine that xanthine and at pH 8.3 than at 9.5. Our results support a mechanism of inhibition in which a substrate-reduced enzyme, non-productive Michaelis complex was formed. It is possible that this non-productive complex favored the univalent pathway of enzyme reoxidation (superoxide production) by increasing the midpoint redox potential of the molybdenum active site.     

Inhibition of xanthine oxidase by uric acid and its influence on superoxide radical production.

The inhibition of xanthine oxidase by its reaction product, uric acid, was studied by steady state kinetic analysis. Uric acid behaved as an uncompetitive inhibitor of xanthine oxidase with respect to the reducing substrate, xanthine. Under 50 microM xanthine and 210 microM oxygen, the apparent K(i) for uric acid was 70 microM. Uric acid-mediated xanthine oxidase inhibition also caused an increase in the percentage of univalent reoxidation of the enzyme (superoxide radical production). Steady-state rate equations derived by the King-Altman method support the formation of an abortive-inhibitory enzyme-uric acid complex (dead-end product inhibition). Alternatively, inhibition could also depend on the reversibility of the classical ping-pong mechanism present in xanthine oxidase-catalyzed reactions.     

Autoinactivation of xanthine oxidase: the role of superoxide radical and hydrogen peroxide.

Xanthine oxidase suffers autoinactivation in the course of catalyzing the oxidation of acetaldehyde. When no special efforts were made to maintain a high pO2 in these reaction mixtures catalase protected the xanthine oxidase, but superoxide dismutase did not. However, when oxygen depletion was slowed or prevented by working at lower concentrations of xanthine oxidase, at lower temperatures or by vigorous agitation under an atmosphere of 100% oxygen, superoxide dismutase or catalase protected markedly when added separately and protected almost completely when added together. This result correlates with the greater production of O2-, relative to H2O2, by xanthine oxidase, at elevated pO2. Since histidine also provided some protection and the high levels of acetaldehyde used would have precluded any significant effect of OH., we conclude that singlet oxygen, or something with similar reactivity, was generated from O2- plus H2O2 and contributed significantly to the observed autoinactivation.     

Release of iron from ferritin by xanthine oxidase. Role of the superoxide radical.

Mobilization of iron from ferritin by xanthine oxidase was studied under aerobic and anaerobic conditions. Aerobic iron release amounted to approx. 3.7 nmol/ml in 10 min. This amount was decreased by approx. 30% under anaerobic conditions. Aerobic iron mobilization involved two mechanisms. About 70% was released by O2.- generated by xanthine oxidase. The rest was released by O2(.-)-independent mechanisms, which also accounted for the total iron release when O2 was absent. A possible transfer of reducing equivalents directly from xanthine oxidase to ferritin is discussed. The results imply that, in pathological conditions with increased formation of O2.-, iron may be released from ferritin. Furthermore, in hypoxic tissues xanthine oxidase can release iron from ferritin by an O2(.-)-independent process. Free iron is liable to catalyse the formation of the extremely reactive and damaging OH. radical.     

Synthesis and characterization of CAPE derivatives as xanthine oxidase inhibitors with radical scavenging properties

Inhibitors of the enzyme xanthine oxidase (XO) with radical scavenging properties hold promise as novel agents against reperfusion injuries after ischemic events. By suppressing the formation of damaging reactive oxygen species (ROS) by XO or scavenging ROS from other sources, these compounds may prevent a buildup of ROS in the aftermath of a heart attack or stroke. To combine these two properties in a single molecule, we synthesized and characterized the non-purine XO inhibitor caffeic acid phenethylester (CAPE) and 19 derivatives using a convenient microwave-assisted Knoevenagel condensation protocol. Varying systematically the number and positions of the hydroxyl groups at the two phenyl rings, we derived structure-activity relationships based on experimentally determined XO inhibition data. Molecular docking suggested that critical enzyme/inhibitor interactions involved π-π interactions between the phenolic inhibitor ring and Tyr914, hydrogen bonds between inhibitor hydroxyl groups and Glu802, and hydrophobic interactions between the CAPE phenyl ring and non-polar residues located at the entrance of the binding site. To effectively scavenge the stable radical DPPH, two hydroxyl groups in 1,2- or 1,4-position at the phenyl ring were required. Among all compounds tested, E-phenyl 3-(3,4-dihydroxyphenyl)acrylate, a CAPE analog without the ethyl tether, showed the most promising properties.     

A reconsideration of the Rhus glutinosa complex (Anacardiaceae)

R. glutinosa Hochst. ex. A. Rich. is lectotypified. R. abyssinica Oliv. and R. neoglutinosa M. Gilbert are reduced to subspecies of R. glutinosa.     

Effect of normothermic ischemic cardiac arrest and of reperfusion on the free oxygen radical scavenger enzymes and xanthine oxidase (a generator of superoxide anions)

Recent evidence suggests that free oxygen radicals are produced by ischaemic tissues, accounting for at least part of the damage that results. These free oxygen radicals are produced by xanthine oxidase, amongst others, and removed by scavenger enzymes (catalase, superoxide dismutase and glutathione peroxidase) and anti-oxidants. As mitochondria are oxygen-utilising organelles, they are capable of producing free oxygen radicals. Our results indicate that the removal of free oxygen radicals are not diminished during ischaemia, but the activity of the free oxygen radical generator, xanthine oxidase, is increased. This could lead to an increased superoxide anion concentration.     

Reevaluation of the sections of Rhus L. subgenus lobadium (Raf.) T. & G. (Anacardiaceae)

Species ofRhus subgen.Lobadium have in the past been grouped into five sections:Pseudosumac, Stypkonia,Pseudoschmaltzia, Rhoeidium andLobadium. Reexamination of the morphological criteria used to delimit the sections, as well as consideration of data from flavonoid chemistry and wood anatomy, suggest that the affinities of this group of taxa are best reflected by recognizing three sections and three subsections. The namesPseudosumac andPseudoschmaltzia are rejected as beingnomina nuda.     

Phylogenetic analysis based on structural and combined analyses of Rhus s.s. (Anacardiaceae)

Structural data were combined with trnL‐F and internal transcribed spacer sequences from other studies and with new sequences representing ten additional species to clarify the phylogenetic relationships of Rhus s.s. These data indicate that Rhus s.s and both subgenera, Rhus and Lobadium, are monophyletic. The genus Rhus is supported as monophyletic by the presence of red glandular hairs on the berries and inflorescence axis, cilia on the sepals and glands on the leaf blades. Subgenus Rhus can be identified by the presence of more than seven resin channels in the petiole, weakly percurrent tertiary veins and a type I vascular system in the mid‐vein. Subgenus Lobadium is characterized by the presence of short bracteoles and pedicels. This subgenus is divided into four sections, Lobadium, Rhoeidium, Styphonia and Terebinthifolia. Section Lobadium has trifoliate leaves; section Rhoeidium is monotypic, including only Rhus microphylla; section Styphonia is supported by five synapomorphies, including an incomplete marginal vein, fibres in the petiole, a thick cuticle, two layers of palisade parenchyma and prismatic crystals in the mesophyll; and section Terebinthifolia has gelatinous xylary fibres in the petiole. Hypotheses about the evolutionary changes of these characters are presented based on the cladograms. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 452–468.     

Polyphenols of Rosa L. leaves extracts and their radical scavenging activity

Antioxidant potential of Rosa L. leaves methanolic extracts was evaluated in vitro using a spectrophotometric method based on measuring the radical scavenging effect on 2,2-di-phenyl-1-picrylhydrazyl (DPPH) radicals. The contents of ellagic acid, quercetin and kaempferol in the extracts from leaves of seventeen rose species were determined using SPE-RP-HPLC methods. Additionally, total phenolic content was determined spectrophotometrically according to the Folin-Ciocalteu procedure and calculated as gallic acid equivalents (GAE). Remarkable high antioxidant activity and high total phenolic content (5.7% < GAE < 15.2%), large ellagic acid (EA) content from 9.37 to 19.42 mg/g of dry weight, a quercetin content ranging from 3.68 to 15.81 mg/g of dry weight and kaempferol content from 1.25 to 9.41mg/g of dry weight were found in rose leaves. Significant correlation between EA (r(2) = 0.6131), quercetin (r(2) = 0.5158), total phenolic content (r(2)= 0.8485) and antioxidant activity was observed. Basing on the studies conducted one may assume that the extracts of rose leaves are a rich source of natural antioxidants and could be used to prevent free-radical-induced deleterious effects.     

Comparative study of chicken liver xanthine dehydrogenase and bovine liver xanthine oxidase. dehydrogenase activity of xanthine oxidase (author's transl)]

A method to purify bovine liver xanthine oxidase in described, with which samples of 256-fold specific activity with respect to the initial homogenate are obtained. Bovine liver xanthine oxidase and chicken liver xanthine dehydrogenase with oxygen as electron acceptor exhibit similar profile in pKM and log V versus pH plots. With NAD+ as electron acceptor a different profile in the pKM xanthine plot is obtained for chicken liver xanthine dehydrogenase. However three inflection points at the same pH values appear in all plots. Both enzymes are irreversibly inhibited by pCMB and reversibly by N-ethylmaleimide and by iodoacetamide, with competitive and uncompetitive type inhibitions respectively. These results suggest that NAD+ alters the enzymatic action since its binding to the enzyme antecedes the binding of xanthine to the xanthine oxidase molecule, without undergoing itself any modification. 0.15 M DDT of DTE treatment of bovine liver xanthine oxidase gives to the enzyme a permanent activity with NAD+ without modifying its activity with oxygen. The enzyme thus treated produces parallel straight lines in Lineweaver-Burk plots.     

Introgressive hybridization in two southern California species of Rhus (Anacardiaceae)

Five populations ofRhus integrifolia andR. ovata were studied and, based upon morphological characteristics and pollen viability, a number of putative hybrids were found in those areas where the two species are sympatric. Diagnostic characteristics most useful for distinguishing the species and their hybrids are leaf morphology, sepal pubescence, petiole length, and petal size. Introgression was evident in at least three populations, which suggested that hybridization between the two species is an ancient phenomenon. Evolutionarily, hybridization between the two taxa has probably been of little consequence.     

Phylogenetic and biogeographic diversification of Rhus (Anacardiaceae) in the Northern Hemisphere

Sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA, the chloroplast ndhF gene, and chloroplast trnL-F regions (trnL intron, and trnL [UAA] 3' exon-trnF [GAA] intergenic spacer) were used for phylogenetic analyses of Rhus, a genus disjunctly distributed in Asia, Europe, Hawaii, North America, and Northern Central America. Both ITS and cpDNA data sets support the monophyly of Rhus. The monophyly of subgenus Rhus was suggested by the combined cpDNA and ITS data, and largely supported in the cpDNA data except that Rhus microphylla of subgenus Lobadium was nested within it. The monophyly of subgenus Lobadium was strongly supported in the ITS data, whereas the cpDNA data revealed two main clades within the subgenus, which formed a trichotomy with the clade of subgenus Rhus plus R. microphylla. The ITS and cpDNA trees differ in the positions of Rhus michauxii, R. microphylla, and Rhus rubifolia, and hybridization may have caused this discordance. Fossil evidence indicates that Rhus dates back to the early Eocene. The penalized likelihood method was used to estimate divergence times, with fossils of Rhus subgenus Lobadium, Pistacia and Toxicodendron used for age constraints. Rhus diverged from its closest relative at 49.1+/-2.1 million years ago (Ma), the split of subgenus Lobadium and subgenus Rhus was at 38.1+/-3.0 Ma. Rhus most likely migrated from North America into Asia via the Bering Land Bridge during the Late Eocene (33.8+/-3.1 Ma). Rhus coriaria from southern Europe and western Asia diverged from its relatives in eastern Asia at 24.4+/-3.2 Ma. The Hawaiian Rhus sandwicensis diverged from the Asian Rhus chinensis at 13.5+/-3.0 Ma. Subgenus Lobadium was inferred to be of North American origin. Taxa of subgenus Lobadium then migrated southward to Central America. Furthermore, we herein make the following three nomenclatural combinations: (1) Searsia leptodictya (Diels) T. S. Yi, A. J. Miller and J. Wen, comb. nov., (2) Searsia pyroides (A. Rich.) T. S. Yi, A. J. Miller and J. Wen, comb. nov., and (3) Searsia undulata (Jacq.) T. S. Yi, A. J. Miller and J. Wen, because our analyses support the segregation of Searsia from Rhus.     

Superoxide radicals scavenging and xanthine oxidase inhibitory activity of magnesium lithospermate B from Salvia miltiorrhiza

In this study we investigated the superoxide radicals scavenging effect and xanthine oxidase inhibitory activity by magnesium lithospermate B, which was originally isolated from the roots of Salvia miltiorrhiza (also named Danshen or Dansham), an important herb in Oriental medicine. Superoxide radicals were generated both in beta-NADH/PMS system and xanthine/ xanthine oxidase system. Magnesium lithospermate B significantly inhibited the reduction of NBT induced by superoxide radicals with an IC(50) of 29.8 microg/mL and 4.06 microg/mL respectively in the two systems. Further study suggested that magnesium lithospermate B can directly inhibit xanthine oxidase and exhibits competitive inhibition. Magnesium lithospermate B was also found to have the hypouricemic activity in vivo against potassium oxonate-induced hyperuricaemia in mice. After oral administration of magnesium lithospermate B at doses of 10, 20 and 30 mg/kg, there was a significant decrease in the serum urate level when compared to the hyperuricemia control. In addition, magnesium lithospermate B significantly protected HL-60 cells from superoxide radicals-induced apoptosis in the xanthine/ xanthine oxidase reactions. This study provided evidence that magnesium lithospermate B exhibits direct superoxide radicals scavenging and xanthine oxidase inhibitory activity.     

Superoxide radicals scavenging and xanthine oxidase inhibitory activity of magnesium lithospermate B from Salvia miltiorrhiza

In this study we investigated the superoxide radicals scavenging effect and xanthine oxidase inhibitory activity by magnesium lithospermate B, which was originally isolated from the roots of Salvia miltiorrhiza (also named Danshen or Dansham), an important herb in Oriental medicine. Superoxide radicals were generated both in β-NADH/PMS system and xanthine/ xanthine oxidase system. Magnesium lithospermate B significantly inhibited the reduction of NBT induced by superoxide radicals with an IC₅₀ of 29.8 μg/mL and 4.06 μg/mL respectively in the two systems. Further study suggested that magnesium lithospermate B can directly inhibit xanthine oxidase and exhibits competitive inhibition. Magnesium lithospermate B was also found to have the hypouricemic activity in vivo against potassium oxonate-induced hyperuricaemia in mice. After oral administration of magnesium lithospermate B at doses of 10, 20 and 30 mg/kg, there was a significant decrease in the serum urate level when compared to the hyperuricemia control. In addition, magnesium lithospermate B significantly protected HL-60 cells from superoxide radicals-induced apoptosis in the xanthine/ xanthine oxidase reactions. This study provided evidence that magnesium lithospermate B exhibits direct superoxide radicals scavenging and xanthine oxidase inhibitory activity.     

Genetic variation and evidence of hybridization in the genus Rhus (Anacardiaceae)

Rhus michauxii, a rare plant species endemic to the southeastern United States, was previously known only from central North Carolina and one site in Georgia. An additional site, which is now believed to represent the largest known concentration of R. michauxii, was recently discovered at Ft. Pickett near Blackstone, Virginia. Morphological characteristics in several of the Ft. Pickett Rhus populations appear to be intermediate between R. michauxii and the widespread R. glabra, a closely related congener that co-occurs at Ft. Pickett. Although morphological evidence of hybridization between R. michauxii and R. glabra in North Carolina has been provided previously, genetic marker data are lacking. In the present study we examined levels of allozyme variation at 11 polymorphic loci within and among seven populations of R. michauxii, one population of R. glabra, and four putative hybrid populations at Ft. Pickett. Overall, R. michauxii had typical levels of within-population genetic variation when compared to other species with similar life-history characteristics. In contrast, the proportion of genetic variation among populations (G(ST)) was considerably lower than expected. Finally, R. michauxii and R. glabra appear to have a fixed allelic difference at the Idh2 locus. This enabled us to confirm hybridization in all four of the putative hybrid populations and one of the R. michauxii populations.     

Generation of superoxide anion by brain endothelial cell xanthine oxidase.

Bovine brain endothelial cells (EC) that were isolated and propagated in pure culture had increased (greater than 20-fold) levels of xanthine oxidase and xanthine dehydrogenase activity compared to whole brain homogenate. Brain EC also released superoxide anion (O2-) into the extracellular medium. Treatment of EC with tungsten decreased (P less than 0.05) both XO activity and O2- release. XO appears to be highly concentrated in cerebral vascular endothelium and may be an important source of O2-.     

Rhus neoglutinosa sp. nov. (Anacardiaceae) and Teclea borenensis sp. nov. (Rutaceae) from Ethiopia.

Two new species, Rhus neoglutinosa M. Gilbert (Anacardiaceae) and Teclea borenensis M. Gilbert (Rutaceae) are described in preparation for the forthcoming Flora of Ethiopia.     

Ontogeny of the seed-coat of Rhus lancea L. fil., and pachychalazy in the Anacardiaceae

VON TEICHMAN, I., 1991. Ontogeny of the seed-coat of Rhus lancea L. fil., and pachychalazy in the Anacardiaceae. The bitegmic, anatropous ovule develops into an exalbuminous, partially pachychalazal and endotegmic seed. In the mature seed-coat the extensive chalaza with associated tanniniferous hypostase sensu lato manifests externally as a characteristic brown patch. The walls of the cells of the hypostase are impregnated with callose and lipidic substances, which most probably represent cutin. Ultimately the outer integument and outer parts of the inner integument are more or less squashed. However, the cell walls of the inner epidermis of the inner integument show distinct secondary thickening and lignification. The pachychalazal seed with undifferentiated seed-coat characterizes not only a number of the genera of the tribe Anacardieae, but also occurs in Heeria of the tribe Rhoeae. A number of genera of the tribe Spondiadeae have a partially pachychalazal seed. The seed-coat of the latter shows varying degrees of traces of an exo-, meso- and/or endotestal lignification. The seed of certain genera of the Rhoeae, is partially pachychalazal and endotegmic, or probably only endotegmic.