Quantitative studies on brown algal phenols. I. Estimation of absolute polyphenol content of Ascophyllum nodosum (L.) Le Jol. and Fucus vesiculosus (L.)

Methods for estimating the absolute polyphenol content of the brown algae Ascophyllum nodosum (L.) Le Jol. and Fucus vesiculosus (L.) have been developed and tested. Polyphenols were extracted almost quantitatively from Ascophyllum nodosum using aqueous acetone, whereas this procedure was somewhat less efficient with Fucus vesiculosus. Colorimetric methods based on the Folin-Denis reagent, Brentamine Fast Red 2G Salt, and vanillin-H₂SO₄ were applied to acetone-free extracts for determination of polyphenol content relative to suitable reference compounds. Gravimetric methods based on hide powder and on haemoglobin were employed to derive ‘estimation factors’ (EFs) which allow calculation of the absolute polyphenol content from the relative polyphenol content. The values calculated for absolute polyphenol content are considered to be reasonably accurate, despite imprecisions in the methods and despite often large standard deviations, and re-emphasize the potential physiological and ecological significance of brown algal polyphenols. Although the precise EFs calculated here are not valid for other brown algae, the methods are considered to be generally applicable to other Phaeophyceae.     

Development of a novel automated ion channel recording method using "inside-out" whole-cell membranes

Efforts to develop novel methods for recording from ion channels have been receiving increased attention in recent years. In this study, the authors report a unique "inside-out" whole-cell configuration of patch-clamp recording that has been developed. This method entails adding cells into a standard patch pipette and, with positive pressure, obtaining a gigaseal recording from a cell at the inside tip of the electrode. In this configuration, the cell may be moved through the air, first rupturing part of the cellular membrane and enabling bath access to the intracellular side of the membrane, and then into a series of wells containing differing solutions, enabling robotic control of all the steps in an experiment. The robotic system developed here fully automates the electrophysiological experiments, including gigaseal formation, obtaining whole-cell configuration, data acquisition, and drug application. Proof-of-principle experiments consisting of application of intracellularly acting potassium channel blockers to K+ channel cell lines resulted in a very rapid block, as well as block reversal, of the current. This technique allows compound application directly to the intracellular side of ion channels and enables the dissociation of compound in activities due to cellular barrier limitations. This technique should allow for parallel implementation of recording pipettes and the future development of larger array-based screening methods.     

Season of conception in rural gambia affects DNA methylation at putative human metastable epialleles

Throughout most of the mammalian genome, genetically regulated developmental programming establishes diverse yet predictable epigenetic states across differentiated cells and tissues. At metastable epialleles (MEs), conversely, epigenotype is established stochastically in the early embryo then maintained in differentiated lineages, resulting in dramatic and systemic interindividual variation in epigenetic regulation. In the mouse, maternal nutrition affects this process, with permanent phenotypic consequences for the offspring. MEs have not previously been identified in humans. Here, using an innovative 2-tissue parallel epigenomic screen, we identified putative MEs in the human genome. In autopsy samples, we showed that DNA methylation at these loci is highly correlated across tissues representing all 3 embryonic germ layer lineages. Monozygotic twin pairs exhibited substantial discordance in DNA methylation at these loci, suggesting that their epigenetic state is established stochastically. We then tested for persistent epigenetic effects of periconceptional nutrition in rural Gambians, who experience dramatic seasonal fluctuations in nutritional status. DNA methylation at MEs was elevated in individuals conceived during the nutritionally challenged rainy season, providing the first evidence of a permanent, systemic effect of periconceptional environment on human epigenotype. At MEs, epigenetic regulation in internal organs and tissues varies among individuals and can be deduced from peripheral blood DNA. MEs should therefore facilitate an improved understanding of the role of interindividual epigenetic variation in human disease.     

A Paecilomyces fumosoroseus mutant over-producing chitinase displays enhanced virulence against Bemisia tabaci

A Paecilomyces fumosoroseus strain was mutagenized by u.v. Among 200 colonies, one mutant (M84), showed a large and stable chitin hydrolysis-halo. Glucose consumption and biomass production were similar for M84 and the parental strain. Chitinase was inducible by chitin and repressed by glucose in both strains but, when they were grown on minimal medium plus colloidal chitin as sole carbon source, the parental and M84 strains yielded 198 and 690 mol N-acetylglucosamine, respectively. This results indicate that the mutant strain synthesized a chitinase with a higher activity. Bioassays against Bemisia tabaci nymph, showed that M84 incited a 2-fold higher incidence of disease compared to the parental strain.     

Differing roles of mitochondrial nitric oxide synthase in cardiomyocytes and urothelial cells

The existence of mitochondrial nitric oxide (NO) synthase (mtNOS) has been controversial since it was first reported in 1995. We have addressed this issue by making direct microsensor measurements of NO production in the mitochondria isolated from mouse hearts. Mitochondrial NO production was stimulated by Ca2+ and inhibited by blocking electrogenic Ca2+ uptake or by using NOS antagonists. Cardiac mtNOS was identified as the neuronal isoform by the absence of NO production in the mitochondria of mice lacking the neuronal but not the endothelial or inducible isoforms. In cardiomyocytes from dystrophin-deficient (mdx) mice, elevated intracellular Ca2+, increased mitochondrial NO production, slower oxidative phosphorylation, and decreased ATP production were detected. Inhibition of mtNOS increased contractility in mdx but not in wild-type cardiomyocytes, indicating that mtNOS may protect the cells from overcontracting. mtNOS was also implicated in radiation-induced cell damage. In irradiated rat/mouse urinary bladders, we have evidence that mitochondrially produced NO damages the urothelial "umbrella" cells that line the bladder lumen. This damage disrupts the permeability barrier thereby creating the potential to develop radiation cystitis. RT-PCR and Southern blot analyses indicate that mtNOS is restricted to the umbrella cells, which scanning electron micrographs show are selectively damaged by radiation. Simultaneous microsensor measurements demonstrate that radiation increases NO and peroxynitrite (ONOO-) production in these cells, which can be prevented by transfection with manganese superoxide dismutase (MnSOD) or instillation of NOS antagonists during irradiation or irradiation of bladders devoid of mtNOS. These studies demonstrate that mtNOS is in the cardiomyocytes and urothelial cells, that it is derived from the neuronal isoform, and that it can be either protective or detrimental.     

Definition of the structural elements in plasminogen required for high-affinity binding to apolipoprotein(a): a study utilizing surface plasmon resonance

Lipoprotein(a) [Lp(a)] is suggested to link atherosclerosis and thrombosis owing to the similarity between the apolipoprotein(a) [apo(a)] moiety of Lp(a) and plasminogen. Lp(a) may interfere with tPA-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoaguable state in vivo. The present study employed surface plasmon resonance (SPR) to examine the binding interaction between plasminogen and a physiologically relevant, 17-kringle recombinant apo(a) species [17K r-apo(a)] in real time. Native, intact Glu(1)-plasminogen bound to apo(a) with substantially higher affinity (K(D) approximately 0.3 microM) compared to a series of plasminogen fragments (K1-5, K1-3, K4, K5P, and tail domain) that interacted weakly with apo(a) (K(D) > 50 microM). Treatment of Glu(1)-plasminogen with citraconic anhydride (a lysine modification reagent) completely abolished binding to wild-type 17K r-apo(a), whereas citraconylated 17K r-apo(a) decreased binding to wild-type Glu(1)-plasminogen by approximately 50%; inhibition of binding was also observed using the lysine analogue epsilon-aminocaproic acid. Whereas native Glu(1)-plasminogen exhibited monophasic binding to 17K r-apo(a), truncated Lys(78)-plasminogen exhibited biphasic binding. Altering Glu(1)-plasminogen from its native, closed conformation (in chloride buffer) to an open conformation (in acetate buffer) also yielded biphasic isotherms. These SPR data are consistent with a two-state kinetic model in which a conformational change in the plasminogen-apo(a) complex may occur following the initial binding event. Differential binding kinetics between Glu(1)-/Lys(78)-plasminogen and apo(a) may explain why Lp(a) is a stronger inhibitor of tPA-mediated Glu(1)-plasminogen activation compared to Lys(78)-plasminogen activation.     

Livestock Transfers, Risk Management, and Human Careers in a West African Pastoral System

Pastoralists employ different strategies to minimize the risk of losing their livelihood due to drought, disease, and other disasters. Livestock transfers have been considered critical because they provide not only a safety net during disasters but also contribute to the resilience of pastoral societies by allowing pastoralists to rebuild their herds after disasters. I examine whether and how livestock transfers serve as risk management strategies in a comparative, ethnographic study of three pastoralist communities in the Far North Region of Cameroon. The findings show that livestock transfers contribute to short-term survival of households but not long-term viability of family herds. Here I argue for a more holistic, anthropological approach that considers the social and cultural aspects of strategic decisions that individual pastoralists make when they engage in these transfers.     

Determination of a unique solution to parallel proton transfer reactions using the genetic algorithm

Kinetic analysis of the dynamics as measured in multiequilibria systems is readily attained by curve-fitting methodologies, a treatment that can accurately retrace the shape of the measured signal. Still, these reconstructions are not related to the detailed mechanism of the process. In this study we subjected multiple proton transfer reactions to rigorous kinetic analysis, which consists of solving a set of coupled-nonlinear differential rate equations. The manual analysis of such systems can be biased by the operator; thus the analysis calls for impartial corroboration. What is more, there is no assurance that such a complex system has a unique solution. In this study, we used the Genetic Algorithm to investigate whether the solution of the system will converge into a single global minimum in the multidimensional parameter space. The experimental system consisted of proton transfer between four proton-binding sites with seven independent adjustable parameters. The results of the search indicate that the solution is unique and all adjustable parameters converge into a single minimum in the multidimensional parameter space, thus corroborating the accuracy of the manual analysis.     

Responses of Amazonian ecosystems to climatic and atmospheric carbon dioxide changes since the last glacial maximum

The aims of this paper are to review previously published palaeovegetation and independent palaeoclimatic datasets together with new results we present from dynamic vegetation model simulations and modern pollen rain studies to: (i) determine the responses of Amazonian ecosystems to changes in temperature, precipitation and atmospheric CO2 concentrations that occurred since the last glacial maximum (LGM), ca. 21 000 years ago; and (ii) use this long-term perspective to predict the likely vegetation responses to future climate change. Amazonia remained predominantly forested at the LGM, although the combination of reduced temperatures, precipitation and atmospheric CO2 concentrations resulted in forests structurally and floristically quite different from those of today. Cold-adapted Andean taxa mixed with rainforest taxa in central areas, while dry forest species and lianas probably became important in the more seasonal southern Amazon forests and savannahs expanded at forest-savannah ecotones. Net primary productivity (NPP) and canopy density were significantly lower than today. Evergreen rainforest distribution and NPP increased during the glacial-Holocene transition owing to ameliorating climatic and CO2 conditions. However, reduced precipitation in the Early-Mid-Holocene (ca. 8000-3600 years ago) caused widespread, frequent fires in seasonal southern Amazonia, causing increased abundance of drought-tolerant dry forest taxa and savannahs in ecotonal areas. Rainforests expanded once more in the Late Holocene owing to increased precipitation caused by greater austral summer insolation, although some of this forest expansion (e.g. in parts of the Bolivian Beni) is clearly caused by palaeo Indian landscape modification. The plant communities that existed during the Early-Mid-Holocene may provide insights into the kinds of vegetation response expected from similar increases in temperature and aridity predicted for the twenty-first century. We infer that ecotonal areas near the margins of the Amazon Basin are liable to be most sensitive to future environmental change and should therefore be targeted with conservation strategies that allow 'natural' species movements and plant community re-assortments to occur.