Pycnogenol® and vitamin E inhibit ethanol‐induced apoptosis in rat cerebellar granule cells

Pycnogenol® (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), scavenges free radicals and promotes cellular health. The protective capacity of PYC against ethanol toxicity of neurons has not previously been explored. The present study demonstrates that in postnatal day 9 (P9) rat cerebellar granule cells the antioxidants vitamin E (VE) and PYC (1) dose dependently block cell death following 400, 800, and 1600 mg/dL ethanol exposure (2) inhibit the ethanol‐induced activation of caspase‐3 in the same model system; and (3) reduce neuronal membrane disruption as assayed by phosphatidylserine translocation to the cell surface. These results suggest that both PYC and VE have the potential to act as therapeutic agents, antagonizing the induction of neuronal cell death by ethanol exposure. © 2004 Wiley Periodicals, Inc. J Neurobiol 59: 261–271, 2004     

Comparison of the properties of detergent-solubilized NADPH-cytochrome P-450 reductases from pig liver and kidney immunochemical,kinetic, and reconstitutive properties

NADPH-cytochrome P-450 reductases from pig liver and kidney and rabbit liver microsomes were purified to a specific activity of 50–62 μmol cytochrome c reduced/min/mg. All reductase preparations were separated into one major and one minor fraction on Sephadex G-200 columns. The molecular weights of the major fractions of the reductases were estimated to be 74,000, 75,000, and 75,500 for rabbit liver, pig kidney, and liver reductases, respectively, whereas the molecular weight of the minor fractions of these reductases, 67,000, was the same as that of the steapsin-solubilized pig liver reductase on SDS-polyacrylamide gel electrophoresis. K_m values for NADPH and cytochrome c were: 20 and 29 μm or 14 and 28 μm for the pig kidney or liver reductase, respectively. Immunochemical studies, including Ouchterlony double diffusion experiments and inhibition of benzphetamine N-demethylation activity in microsomes by antibody against pig liver NADPH-cytochrome P-450 reductase, indicated the similarity of the purified liver and kidney reductases. There were no differences in the ability to reconstitute NADPH-mediated benzphetamine N-demethylation and laurate hydroxylation in reconstituted systems between the pig liver and kidney reductases, indicating that the reductase did not determine substrate specificity in these systems.     

Radical mechanism of aminopyrine oxidation by cumene hydroperoxide catalyzed by purified liver microsomal cytochrome P-450

Under identical experimental conditions, purified preparations of rabbit liver microsomal cytochrome P-450 and beef heart metmyoglobin were equally effective at stimulating the oxidation of aminopyrine to a free radical species by cumene hydroperoxide. Mannitol had no effect on radical levels produced with either hemeprotein-hydroperoxide system; however, specific ligands of the two hemeproteins, substrates of cytochrome P-450, and phospholipid affected the two systems quite differently. Only the metmyo-globindependent oxidation of aminopyrine was significantly inhibited by fluoride and cyanide. Metyrapone, a specific ligand of cytochrome P-450, and benzphetamine, which was N-demethylated by cumene hydroperoxide only in the presence of cytochrome P-450, inhibited only the cytochrome P-450-stimulated oxidation of aminopyrine. Moreover, only with the solubilized liver hemeprotein was aminopyrine radical generation markedly stimulated by phospholipid. Similar properties of aminopyrine N-demethylation and radical formation by the cytochrome P-450-cumene hydroperoxide system have strongly implicated the radical as a requisite intermediate in product formation. Micromolar concentrations of metyrapone caused parallel inhibition, by at least 50%, of both radical generation and formaldehyde production. These results support a radical pathway of N-demethylation proposed for other hemeprotein-hydroperoxide systems (B. W. Griffin and P. L. Ting, 1978, Biochemistry, 17, 2206–2211), in which the substrate undergoes two successive one-electron abstractions, followed by hydrolysis of the iminium cation intermediate. Thus, for this class of substrates, the experimental data are consistent with the oxygen atom of the product arising from H₂O and not directly from the hydroperoxide, which has been previously proposed as a general mechanism for cytochrome P-450 peroxidatic activities.     

Survivorship and Spatial Patterns of an Urban Population of Texas Horned Lizards

Habitat fragmentation has negative consequences on threatened and endangered species by creating isolated populations. The Texas horned lizard (Phrynosoma cornutum) is experiencing population declines and localized extirpations throughout its range and has been classified as a species of greatest conservation need in Oklahoma, USA. Younger age classes have been poorly studied but may be vital to the stability of remaining populations. To address gaps in knowledge concerning subadult (hatchling and juvenile) morphometrics, survivorship, and home range sizes, we studied 2 cohorts of subadults, for 2 years each, covering their hatching and juvenile years (2016–2019). We used a combination of radio-telemetry and novel harmonic radar methodology to study a closed population of Texas horned lizards in 15 ha of native grassland at Tinker Air Force Base, Oklahoma. Population abundance for adults and juveniles was estimated as 56.5 ± 5.5 lizards and density as 7.96 lizards/ha. Our lowest estimates of survival indicated an average survival probability for the hatchling life stage of 0.285 (95% CI = 0.15–0.44), which is lower than for adults on the site. Average home range size increased from hatchling to adult life stages. Our results will have an immediate effect on the planning and assessment of ongoing headstart and management programs for Texas horned lizards. © 2021 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society.     

Scaling-up the delivery of dog vaccination campaigns against rabies in Tanzania

An increasing number of countries are committing to meet the global target to eliminate human deaths from dog-mediated rabies by 2030. Mass dog vaccination is central to this strategy. To interrupt rabies transmission from dogs to humans, the World Health Organization recommends that vaccination campaigns should be carried out every year in all dog-owning communities vaccinating 70% of their susceptible dogs. Monitoring and evaluation of dog vaccination campaigns are needed to measure progress towards elimination. In this study, we measured the delivery performance of large-scale vaccination campaigns implemented in 25 districts in south-east Tanzania from 2010 until 2017. We used regression modelling to infer the factors associated with, and potentially influencing the successful delivery of vaccination campaigns. During 2010–2017, five rounds of vaccination campaigns were carried out, vaccinating in total 349,513 dogs in 2,066 administrative vaccination units (rural villages or urban wards). Progressively more dogs were vaccinated over the successive campaigns. The campaigns did not reach all vaccination units each year, with only 16–28% of districts achieving 100% campaign completeness (where all units were vaccinated). During 2013–2017 when vaccination coverage was monitored, approximately 20% of vaccination units achieved the recommended 70% coverage, with average coverage around 50%. Campaigns were also not completed at annual intervals, with the longest interval between campaigns being 27 months. Our analysis revealed that districts with higher budgets generally achieved higher completeness, with a twofold difference in district budget increasing the odds of a vaccination unit being reached by a campaign by slightly more than twofold (OR: 2.29; 95% CI: 1.69–3.09). However, higher budgets did not necessarily result in higher coverage within vaccination units that were reached. We recommend national programs regularly monitor and evaluate the performance of their vaccination campaigns, so as to identify factors hindering their effective delivery and to guide remedial action.     

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

A short-oligonucleotide microarray that allows improved detection of gastrointestinal tract microbial communities

Background  

The human gastrointestinal (GI) tract contains a diverse collection of bacteria, most of which are unculturable by conventional microbiological methods. Increasingly molecular profiling techniques are being employed to examine this complex microbial community. The purpose of this study was to develop a microarray technique based on 16S ribosomal gene sequences for rapidly monitoring the microbial population of the GI tract.     

Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase. Comparisons with NADPH-cytochrome P450 oxidoreductase

Nitric-oxide synthase (NOS) is composed of a C-terminal, flavin-containing reductase domain and an N-terminal, heme-containing oxidase domain. The reductase domain, similar to NADPH-cytochrome P450 reductase, can be further divided into two different flavin-containing domains: (a) the N terminus, FMN-containing portion, and (b) the C terminus FAD- and NADPH-binding portion. The crystal structure of the FAD/NADPH-containing domain of rat neuronal nitric-oxide synthase, complexed with NADP(+), has been determined at 1.9 A resolution. The protein is fully capable of reducing ferricyanide, using NADPH as the electron donor. The overall polypeptide fold of the domain is very similar to that of the corresponding module of NADPH-cytochrome P450 oxidoreductase (CYPOR) and consists of three structural subdomains (from N to C termini): (a) the connecting domain, (b) the FAD-binding domain, and (c) the NADPH-binding domain. A comparison of the structure of the neuronal NOS FAD/NADPH domain and CYPOR reveals the strict conservation of the flavin-binding site, including the tightly bound water molecules, the mode of NADP(+) binding, and the aromatic residue that lies at the re-face of the flavin ring, strongly suggesting that the hydride transfer mechanisms in the two enzymes are very similar. In contrast, the putative FMN domain-binding surface of the NOS protein is less positively charged than that of its CYPOR counterpart, indicating a different nature of interactions between the two flavin domains and a different mode of regulation in electron transfer between the two flavins involving the autoinhibitory element and the C-terminal 33 residues, both of which are absent in CYPOR.     

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.