A microbiological assay for host-specific fungal polyketide toxins

Genetic analysis of biosynthetic pathways for fungal secondary metabolites depends on availability of efficient and dependable assays for the end products. Some fungal plant pathogens produce secondary metabolites called host-specific toxins. Until recently, all bioassays for these toxins required use of whole plants or plant parts (Yoder 1981 In: Toxins in Plant Disease, Durbin ed., pp. 45-78). Since host-specific toxins, by definition, affect only plants that are susceptible to the toxin-producing fungus, other plants, animals and microorganisms are not sensitive and therefore cannot be used in bioassays

Passive phloem loading and long-distance transport in a synthetic tree-on-a-chip

Vascular plants rely on differences of osmotic pressure to export sugars from regions of synthesis (mature leaves) to sugar sinks (roots, fruits). In this process, known as M\"unch pressure flow, the loading of sugars from photosynthetic cells to the export conduit (the phloem) is crucial, as it sets the pressure head necessary to power long-distance transport. Whereas most herbaceous plants use active mechanisms to increase phloem concentration above that of the photosynthetic cells, in most tree species, for which transport distances are largest, loading seems to occur via passive symplastic diffusion from the mesophyll to the phloem. Here, we use a synthetic microfluidic model of a passive loader to explore the nonlinear dynamics that arise during export and determine the ability of passive loading to drive long-distance transport. We first demonstrate that in our device, phloem concentration is set by the balance between the resistances to diffusive loading from the source and convective export through the phloem. Convection-limited export corresponds to classical models of M\"unch transport, where phloem concentration is close to that of the source; in contrast, diffusion-limited export leads to small phloem concentrations and weak scaling of flow rates with the hydraulic resistance. We then show that the effective regime of convection-limited export is predominant in plants with large transport resistances and low xylem pressures. Moreover, hydrostatic pressures developed in our synthetic passive loader can reach botanically relevant values as high as 10 bars. We conclude that passive loading is sufficient to drive long-distance transport in large plants, and that trees are well suited to take full advantage of passive phloem loading strategies

Saisonnalité du transport de carbone organique dissous dans le ruisseau de l'Hermine, un bassin versant de tête de réseau du Bouclier Canadien

Nous avons étudié la variabilité saisonnière de la relation entre les fluctuations des concentrations en carbone organique dissous (COD) dans le ruisseau de l'Hermine (Québec, Canada) et les changements du débit (Q). Un total de 93 événements hydrologiques échantillonnés de 1994 à 2003 et regroupés sur une base saisonnière (hiver-printemps, été, automne) a été analysé. Le modèle de régression linéaire est utilisé afin de déterminer, pour chaque événement, la pente de la relation entre la concentration en COD dans le ruisseau et le débit. Ces pentes sont regroupées par saison et selon un seuil arbitraire de un qui permet de contraster les conditions hydrologiques et climatiques initiales des événements répertoriés. Les résultats du test de Kruskal-Wallis, visant la comparaison entre les événements de pentes supérieures et inférieures à un, montrent clairement la saisonnalité de la relation entre le COD et le débit. La saisonnalité de la relation COD/Q est ensuite mise en relation avec des variables climatiques et hydrologiques susceptibles de conditionner le transport du COD dans le bassin de l'Hermine. Les résultats montrent que les changements saisonniers des conditions climatiques et hydrologiques dans le bassin versant ont un impact significatif sur la relation entre le COD et le débit. Ainsi, le volume de précipitation tombé durant l'événement, la température moyenne de l'air et la température du sol régissent significativement (p =0,041; 0,001 et 0,009 respectivement) le transport du COD pour la période hiver-printemps. Les basses températures du sol et l'apport élevé en eau via les précipitations et la fonte favorisent le lessivage intense du COD soluble déjà limité par les basses températures. Au cours de l'été, l'état initial d'humidité du bassin est le principal facteur contrôlant l'évolution des concentrations de COD lors d'une crue; les fortes relations avec le pourcentage d'humidité des sols et le débit total 24 h avant l'événement le prouvent (p =0,039 et 0,0003 respectivement). Les changements les plus prononcés du COD surviennent, au cours de l'été, suite à une période prolongée de sécheresse. À l'automne, le transport du COD est influencé par le volume de précipitation tombé durant l'événement (p =0,031) et la température du sol (p =0,042). La modélisation de la relation COD/Q par les variables hydro-climatiques montre que 40% de la relation COD/Q s'explique par la température du sol durant la période d'hiver-printemps. Durant l'été, les conditions initiales d'humidité du bassin, traduites par le débit 24 h avant l'événement, expliquent à 51% la relation COD/Q. À l'automne, la relation COD/Q est gouvernée à 50% à la fois par le volume de précipitation tombé durant l'événement et la température du sol. L'analyse de ces données établit clairement la saisonnalité de la relation COD/Q et que des variables climatiques et hydrologiques permettent de quantifier ces fluctuations saisonnières.The terrestrial organic carbon (C) pool, estimated to 1.5 x 1015 kg C for the first meter of soil (Amundson, 2001), represents a major terrestrial elemental stock for which the recycling rate and the response to perturbations are still unknown. Under the present changing climatic conditions, C fluxes in terrestrial ecosystems could be significantly disturbed during the next decades. Indeed, the multi-annual changes in temperature and precipitation are likely to have a major impact on the net primary production and on organic matter decomposition in soils. This situation influences the production of the dissolved organic carbon (DOC) in soils, its transport to surface waters and hence, water quality. In this context, a better knowledge of the climatic and hydrologic factors influencing seasonal variations in DOC export is crucial to improve our understanding of the potential transformation of carbon stocks and fluxes in terrestrial ecosystems.The objectives of the present study were 1) to evaluate the seasonality in the relationship between dissolved organic carbon (DOC) concentrations in the stream and streamflow (Q) and 2) to quantify the impact of seasonal changes in climatic and hydrological conditions in the watershed on the DOC/Q relationship.The Hermine catchment is located about 80 km north of Montréal, Québec, Canada. An intermittent first-order stream drains the 5.1 ha catchment. Soils are Orthic and Gleyed Humo-Ferric and Ferro-Humic Podzols. The stream water was sampled daily, from 1994 to 2003, with an automatic sampler. The stream discharge was calculated from the water level above a 90º V-notch weir using a Global level sensor bubbler. Soil organic C content was analysed by the modified Walkley-Black method. Because of the high cost of DOC analysis for numerous samples, the DOC content was estimated by the relationship obtained between eight stream water samples analysed with a Shimadzu TOC analyser (Shimadzu, Kyoto, Japan) and the corresponding absorbance measured at 254 nm. From the initial year of the project, 1994, the regression used was Y=-0.05 + 32.60 X with an r2 value of 0.58 and a precision of 0.05 mg·L-1.The relationship between the DOC concentration and Q at the Hermine was positive and significant (p < 0.01) when all data were considered (n=1960). Because of the weakness of this relation (r2 =0.12), the stream samples, from 1994 to 2003, were seasonally split into 93 distinct hydrological events: 33 for winter-spring, 34 for summer and 26 for fall. A linear regression model was used to determine, for each event, the slope of the relationship between the DOC concentrations in the stream and Q. To contrast the antecedent conditions of the Hermine watershed, the events from a given season were divided into two groups. The Kruskal-Wallis test was then used to establish the link between the slope of the DOC/Q relationships on the one hand, and the environmental watershed conditions on the other hand: the climatic variables (volume of precipitation during event, mean air and soil temperatures) and the hydrological variables (stream discharge 24 h before the event, soil moisture, and ground water level).The DOC concentrations in the stream varied on an annual, a seasonal and an event basis. For the period 1994 to 2003, the annual mean concentrations, calculated from daily samples, varied from 2.0 to 2.5 mg DOC·L-1. On a seasonal basis, mean daily DOC was higher during the summer and the fall (2.9 and 2.8 mg DOC·L-1 respectively), and lower in the winter-spring (2.1 mg DOC·L-1). The relation between DOC concentrations and Q fluctuated as a function of the seasonal evolution of climatic and hydrological conditions in the Hermine catchment. For winter-spring events, 79% of the events had a DOC/Q slope lower than one. This period was characterised by high streamflow levels and high total DOC fluxes even though the daily mean DOC concentrations were low (2.1 mg DOC·L-1). The volume of precipitation during the event (p =0.041), the mean air temperature (p =0.001) and the soil temperature (p =0.009) were significantly related to the difference between events with slopes lower and higher than one. Indeed the slope of the relation increases when soil temperatures are elevated. When the temperatures are higher, DOC export increases and subsurface flow in soil horizon is enriched in DOC. Under colder temperature, the DOC production is limited and the soluble organic substances stored in soils are leached out the catchment with the high volume of precipitation and with the water coming from the snowmelt. For the summer period, there were 20 events with slopes greater than one against 14 with slopes lower than one. The soil humidity (p =0.039) and the total streamflow 24 h before the event (p=0.0003), were the two variables that significantly distinguished both slope groups. Rapid changes in DOC concentration occur during hydrological events following a long drought period. Under dry conditions, the subsurface flow in soil horizons rich in organic matter, the re-hydration of bed sediments and the hydrophobic behaviour of soil particles can all contribute to the export of very high DOC concentrations, even during small events. The relationships between DOC and Q, for the fall season, were significantly influenced by the volume of precipitation during the event (p =0.031) and the mean soil temperature (p =0.042). The events with the lower slopes showed the highest volume of precipitation during event and the lowest soil temperature. For these events occurring under wet conditions, the water originates essentially from the B and C horizons, and DOC fluctuations are then limited because of the low concentrations of the DOC in these horizons (anionic sorption of soluble organic substances by iron oxides).Best-fit from multiple regressions indicated that 40% of the link between DOC and Q was explained by the soil temperature during the winter-spring period (p =0.0001). For summer, the streamflow 24 h before events accounted for 51% of the variation in DOC/Q relationships (p =0.00001). For the fall period, the volume of precipitation during event and the soil temperature both contributed equally to the DOC/Q relationships (p =0.001). From these results, obtained from a multi-year project, it is clear that the relation between DOC and Q is a function of the variability in the climatic and hydrological watershed conditions. In a context of global warming, it is possible that warmer air temperatures have an effect on soil temperature. Thus, during winter-spring and fall periods, the duration and the intensity of the DOC production in soils will increase and the export of DOC from the watershed to other surface water system could become more important under equivalent or higher streamflow. Higher air temperature also means higher evapotranspiration by the forest during the summer period, and consequently dryer watershed conditions. A low streamflow and a low soil humidity level could be expected and then, brief rain events will sporadically flush the soluble organic carbon accumulated in the soil. The DOC export would be insignificant for that period, but the DOC would reach the highest annual level. The new knowledge on the DOC/Q relationships, at the hydrological event scale, will be added to the accumulated data on the possible effects of global warming on the carbon cycle in forested ecosystems

Zinc modulates GABAB binding in rat brain

The effects of ZnCl2 on [3H]GABA binding to GABAA and GABAB binding sites were investigated using receptor autoradiography. At concentrations exceeding 100 [mu]M, zinc non-competitively inhibited GABAB binding in a dose dependent fashion. GABAA binding was not inhibited significantly by zinc eliminating the possibility of a non-specific effect of zinc. Increased calcium concentrations up to 10 MM enhanced total GABAB binding but did not prevent zinc induced inhibition of GABAB binding, indicating a separate site of action for these cations at the GABAB binding site. In some regions, zinc modulates GABAB binding in a biphasic manner as concentrations of 10-100 [mu]M zinc significantly enhanced GABAB binding in the hippocampus and the molecular layer of the cerebellum but not in the thalamus. These results provide further evidence for a neuromodulatory role for zinc in the central nervous system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29723/1/0000057.pd

GABAB binding sites in early adult and aging rat brain

The effect of aging on GABAB binding was investigated in rat brain. Receptor autoradiography was used to investigate both GABAB and GABAA binding at 2 months, 3 months, 13 months, and 23 months. GABAB binding decreases significantly between 2 months and 23 months of age, as does GABAA binding, with was investigated in rat brain. Receptor autoradiography was used to investigate both GABAB and GABAA binding at 2 months, 3 months, 13 months, and 23 months. GABAB binding decreases significantly between 2 months and 23 months of age, as does GABAA binding, with greatest decrease between 2 and 3 months. The decrease in GABAB binding appears to be due to a decrease in binding site affinity rather than a decrease in receptor density. The noncompetitive GABAB antagonist zinc, the competitive GABAB antagonist CGP 35348, and the guanyl nucleotide analogue GTP-[gamma]-S all inhibit GABAB binding identically in 2 month and 23 month brain. These data indicate subtle age-related changes in the GABAB binding in early adult life but little change with senescence.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31228/1/0000130.pd

Changes in Photosynthate Unloading from Perfused Seed Coats of Phaseolus vulgaris L Induced by Osmoticum and Ethylenediaminetetraacetate (EDTA)

ABSTRACT Photosynthate unloading in Phaseolus vulgaris L. seed coats was studied by treating perfused seed coats with differing concentrations of an osmoticum and ethylenediaminetetraacetate (EDTA). Large changes in osmoticum concentration typically produced rapid changes in efflux of unlabelled sugar and steady-state-labelled l4 C-photosynthate. Osmoticum-induced changes in photosynthate efflux were caused by phloem import stimulation at low cell turgor and net efflux stimulation by high cell turgor. Even though rapid changes in sugar and tracer efflux were often induced by osmoticum treatments, the specific activity of sugar released from seed coats was not greatly affected by these treatments and was similar to the specific activity of sugar remaining in the seed coat after perfusion. Thus, tracer was transported from the phloem throughout the seed coat sugar pool before it was released to the apoplast. This result is most consistent with symplastic phloem unloading throughout perfused seed coats, because apoplastic transport between cells within the seed coat was blocked by perfusion. Photosynthate efflux was stimulated by simultaneous treatment of seed coats with EDTA and different concentrations of an osmoticum; loss of photosynthate from seed coats did not appear to be tissue-specific

Development of exchange lists for M editerranean and H ealthy E ating D iets: implementation in an intervention trial

Background There has been little research published on the adaptation of diabetic exchange list diet approaches for the design of intervention diets in health research despite their clinical utility. The exchange list approach can provide clear and precise guidance on multiple dietary changes simultaneously. The present study aimed to develop exchange list diets for M editerranean and H ealthy E ating, and to evaluate adherence, dietary intakes and markers of health risks with each counselling approach in 120 subjects at increased risk for developing colon cancer. Methods A randomised clinical trial was implemented in the USA involving telephone counselling. The M editerranean diet had 10 dietary goals targeting increases in mono‐unsaturated fats, n ‐3 fats, whole grains and the amount and variety of fruits and vegetables. The Healthy Eating diet had five dietary goals that were based on the US H ealthy P eople 2010 recommendations. Results Dietary compliance was similar in both diet arms, with 82–88% of goals being met at 6 months, although subjects took more time to achieve the M editerranean goals than the H ealthy E ating goals. The relatively modest fruit and vegetable goals in the Healthy Eating arm were exceeded, resulting in fruit and vegetable intakes of approximately eight servings per day in each arm after 6 months. A significant ( P  < 0.05) weight loss and a decrease in serum C ‐reactive protein concentrations were observed in the overweight/obese subgroup of subjects in the M editerranean arm in the absence of weight loss goals. Conclusions Counselling for the M editerranean diet may be useful for both improving diet quality and for achieving a modest weight loss in overweight or obese individuals.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108685/1/jhn12158.pd

P450 3A activity and cyclosporine dosing in kidney and heart transplant recipients

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109917/1/cptclpt1994135.pd

Quantum mechanics/molecular mechanics modeling of drug metabolism:Mexiletine N-hydroxylation by cytochrome P450 1A2

The mechanism of cytochrome P450­(CYP)-catalyzed hydroxylation of primary amines is currently unclear and is relevant to drug metabolism; previous small model calculations have suggested two possible mechanisms: direct N-oxidation and H-abstraction/rebound. We have modeled the N-hydroxylation of (<i>R</i>)-mexiletine in CYP1A2 with hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing a more detailed and realistic model. Multiple reaction barriers have been calculated at the QM­(B3LYP-D)/MM­(CHARMM27) level for the direct N-oxidation and H-abstraction/rebound mechanisms. Our calculated barriers indicate that the direct N-oxidation mechanism is preferred and proceeds via the doublet spin state of Compound I. Molecular dynamics simulations indicate that the presence of an ordered water molecule in the active site assists in the binding of mexiletine in the active site, but this is not a prerequisite for reaction via either mechanism. Several active site residues play a role in the binding of mexiletine in the active site, including Thr124 and Phe226. This work reveals key details of the N-hydroxylation of mexiletine and further demonstrates that mechanistic studies using QM/MM methods are useful for understanding drug metabolism