EPR and ENDOR characterization of intermediates in the cryoreduced oxy-nitric oxide synthase heme domain with bound L-arginine or N(G)-hydroxyarginine

Reconstitution of the endothelial nitric oxide synthase heme domain (NOS) with the catalytically noncompetent 4-aminotetrahydrobiopterin has allowed us to prepare at -40 degrees C the oxyferrous-NOS-substrate complexes of both L-arginine (Arg) and N(G)-hydroxyarginine (NOHA). We have radiolytically cryoreduced these complexes at 77 K and used EPR and ENDOR spectroscopies to characterize the initial products of reduction, as well as intermediates that arise during stepwise annealing to higher temperatures. Peroxo-ferri-NOS is the primary product of 77 K cryoreduction when either Arg or NOHA is the substrate. Proton ENDOR spectra of this state suggest that the peroxo group is H-bonded to a [guanidinium-water] network that forms because the binding of O2 to the ferroheme of NOS recruits H2O. At no stage of reaction/annealing does one observe an EPR signal from a hydroperoxo-ferri state with either substrate. Instead, peroxo-ferri-NOS-substrate complexes convert to a product-state intermediate at the extremely low temperature of 165-170 K. EPR and proton ENDOR spectra of the intermediate formed with Arg as substrate support the suggestion that the reaction involves the formation and attack of Compound I. Within the time/temperature resolution of the present experiments, samples with Arg and NOHA as substrate behave the same in the initial steps of cryoreduction/annealing, despite the different acid/base characteristics of the two substrates. This leads us to discuss the possibility that ambient-temperature catalytic conversion of both substrates is initiated by reduction of the oxy-ferroheme to the hydroperoxo-ferriheme through a coupled proton-electron transfer from a heme-pocket reductant, and that Arg may provide the stoichiometrically second proton of catalysis.     

The journey from NADPH-cytochrome P450 oxidoreductase to nitric oxide synthases

This mini-review will reflect the perspective of its author on two fields of research, which have merged as the result of the insights of investigators whose work has influenced both areas immeasurably. It cannot be overlooked, however, that the research activities of many during a period of over five decades have produced the chemical and biological bases for the exciting discoveries now encompassing the cytochromes P450 and their redox partners, and the three isoforms of nitric oxide synthase as they function in their respective biological milieux. Following the remarkable discovery that, indeed, molecular oxygen can be adducted to organic molecules by enzymatic systems and that such processes require a supply of reducing equivalents, it is the purpose of this review to provide a chart, with some of its detours, of the road that followed in the pursuit of interesting biological phenomena involving these two major oxygenation systems. It is not intended to be a balanced review and apologies must be offered in advance to those whose contributions may be overlooked or simply were not directly germane to the development of the author's journey.     

A possible role of erythrocytes in storing and distributing arachidonic acid in rat

Young rats weighing 120g and having a packed red cell volume of 37.4% were maintained on a fat free diet. In eight weeks their body weights and packed red cell volume increased to 425g and 45.4%, respectively. However, the proportion of arachidonic acid (20:4) in the total fatty acids was unaltered in erythrocytes but was lowered in other tissues. In adipose tissue, which contained only trace levels of 20:4, about one third of the fatty acid was linoleic acid (18:2). Feeding fat free diet caused a depletion of most of 18:2 in the adipose tissue. Thus, during growth, when 18:2 is excluded from the diet, erythropoiesis is not inhibited. Furthermore, 20:4 produced from stored 18:2 may be used for the production of erythrocytes which retain the tetraenoic acid effectively.     

Functions of Fun30 Chromatin Remodeler in Regulating Cellular Resistance to Genotoxic Stress

The Saccharomyces cerevisiae Fun30 chromatin remodeler has recently been shown to facilitate long-range resection of DNA double strand break (DSB) ends, which proceeds homologous recombination (HR). This is believed to underlie the role of Fun30 in promoting cellular resistance to DSB inducing agent camptothecin. We show here that Fun30 also contributes to cellular resistance to genotoxins methyl methanesulfonate (MMS) and hydroxyurea (HU) that can stall the progression of DNA replication. We present evidence implicating DNA end resection in Fun30-dependent MMS-resistance. On the other hand, we show that Fun30 deletion suppresses the MMS- and HU-sensitivity of cells lacking the Rad5/Mms2/Ubc13-dependent error-free DNA damage tolerance mechanism. This suppression is not the result of a reduction in DNA end resection, and is dependent on the key HR component Rad51. We further show that Fun30 negatively regulates the recovery of rad5Δ mutant from MMS induced G2/M arrest. Therefore, Fun30 has two functions in DNA damage repair: one is the promotion of cellular resistance to genotoxic stress by aiding in DNA end resection, and the other is the negative regulation of a Rad51-dependent, DNA end resection-independent mechanism for countering replicative stress. The latter becomes manifest when Rad5 dependent DNA damage tolerance is impaired. In addition, we find that the putative ubiquitin-binding CUE domain of Fun30 serves to restrict the ability of Fun30 to hinder MMS- and HU-tolerance in the absence of Rad5.     

Did geckos ride the Palawan raft to the Philippines?

Aim We examine the genetic diversity within the lizard genus Gekko in the Philippine islands to understand the role of geography and geological history in shaping species diversity in this group. We test multiple biogeographical hypotheses of species relationships, including the recently proposed Palawan Ark Hypothesis. Location Southeast Asia and the Philippines. Methods Samples of all island endemic and widespread Philippine Gekko species were collected and sequenced for one mitochondrial gene (NADH dehydrogenase subunit 2) and one nuclear gene (phosducin). We used maximum likelihood and Bayesian phylogenetic methods to derive the phylogeny. Divergence time analyses were used to estimate the time tree of Philippine Gekko in order to test biogeographical predictions of species relationships. The phylogenetic trees from the posterior distribution of the Bayesian analyses were used for testing biogeographical hypotheses. Haplotype networks were created for the widespread species Gekko mindorensis to explore genetic variation within recently divergent clades. Results Both maximum likelihood and Bayesian phylogenetic analyses indicated that Philippine Gekko species are a diverse clade with a long history in the archipelago. Ancestral range reconstruction and divergence time analyses suggest a Palawan microcontinental origin for this clade, coinciding with Palawan’s separation from Asia beginning 30 Ma, with subsequent diversification in the oceanic Philippine islands. The widespread species G. mindorensis and G. monarchus diversified in the late Miocene/early Pliocene and are potentially complexes of numerous undescribed species. Main conclusions The view of the Philippine islands as a ‘fringing archipelago’ does not explain the pattern of species diversity in the genus Gekko. Philippine Gekko species have diversified within the archipelago over millions of years of isolation, forming a large diverse group of endemic species. Furthermore, the Philippine radiation of gekkonid lizards demonstrates biogeographical patterns most consistent with stochastic colonization followed by in situ diversification. Our results reveal the need to consider deeper time geological processes and their potential role in the evolution of some Philippine terrestrial organisms.     

Acute toxicity effects of ibuprofen on behaviour and haematological parameters of African catfish Clarias gariepinus (Burchell, 1822)

Indiscriminate discharge of pharmaceutical waste into the aquatic ecosystem may pose serious health challenges to aquatic biota. The effect of acute exposure to ibuprofen was evaluated using changes in behaviour and haematological parameters under static bio-assay method in Clarias gariepinus. Test specimens were exposed to acute concentrations of ibuprofen (0.28, 0.33, 0.38, 0.43 and 0.48 mg l^?1) for 24, 48, 72 and 96 h durations respectively. Behavioural and phenotypic changes were observed in surviving fish. There were significant (p < 0.05) concentration and duration-dependent increases in erythrocyte (RBC), haemoglobin (Hb), pack cell volume (PCV) and leukocytes (WBC) in treated fish compared to the control. Insignificant decreases (p > 0.05) in mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were observed in treated fish compared to the control. Ibuprofen elicited dose and duration- dependent decrease in neutrophil counts with the decreases being significant (p < 0.05) in the higher doses of 0.43 and 0.48 mg l^?1. Ibuprofen did not elicit any significant changes in monocytes, basophils and eosinophils. Changes observed in this study showed that ibuprofen negatively affected the health of the fish and we recommend that discharge of ibuprofen into the aquatic environment should be monitored and controlled.