Developing Demonstration Test Catchments as a platform for transdisciplinary land management research in England and Wales.

Whilst a large body of plot and field-scale research exists on the sources, behaviour and mitigation of diffuse water pollution from agriculture, putting this evidence into a practical, context at large spatial scales to inform policy remains challenging. Understanding the behaviour of pollutants (nutrients, sediment, microbes and pesticides) and the effectiveness of mitigation strategies over whole catchments and long timeframes requires new, interdisciplinary approaches to organise and undertake research. This paper provides an introduction to the demonstration test catchments (DTC) programme, which was established in 2009 to gather empirical evidence on the cost-effectiveness of combinations of diffuse pollution mitigation measures at catchment scales. DTC firstly provides a physical platform of instrumented study catchments in which approaches for the mitigation of diffuse agricultural water pollution can be experimentally tested and iteratively improved. Secondly, it has established national and local knowledge exchange networks between researchers and stakeholders through which research has been co-designed. These have provided a vehicle to disseminate emerging findings to inform policy and land management practice. The role of DTC is that of an outdoor laboratory to develop knowledge and approaches that can be applied in less well studied locations. The research platform approach developed through DTC has brought together disparate research groups from different disciplines and institutions through nationally coordinated activities. It offers a model that can be adopted to organise research on other complex, interdisciplinary problems to inform policy and operational decision-making.

Field drains as a route of rapid nutrient export from agricultural land receiving biosolids.

We report research on the environmental risk of incidental nutrient transfers from land to water for biosolids amended soils. We show that subsurface (drainflow) pathways of P transport may result in significant concentrations, up to 10 mg total P l(-1), in the drainage network of an arable catchment when a P source (recent biosolids application) coincides with a significant and active transport pathway (rainfall event). However, the high P concentrations were short-lived, with drainage ditch total P concentrations returning to pre-storm concentrations within a few days of the storm event. In the case of the drainflow concentrations reported here, the results are unusual in that they describe an 'incidental event' for a groundwater catchment where such events might normally be expected to be rare owing to the capacity of the hydrological system to attenuate nutrient fluxes for highly adsorbed elements such as P. Consequently, there is a potential risk of P transfers to shallow groundwater systems. We suggest that the findings are not specific to biosolids-alone, which is a highly regulated industry, but that similar results may be anticipated had livestock waste or mineral fertilizer been applied, although the magnitude of losses may differ. The risk appears to be more one of timing and the availability of a rapid transport pathway than of P source.

First results from the Cryogenic Dark Matter Search in the Soudan Underground Laboratory.

We report the first results from a search for weakly interacting massive particles (WIMPs) in the Cryogenic Dark Matter Search experiment at the Soudan Underground Laboratory. Four Ge and two Si detectors were operated for 52.6 live days, providing 19.4 kg d of Ge net exposure after cuts for recoil energies between 10 and 100 keV. A blind analysis was performed using only calibration data to define the energy threshold and selection criteria for nuclear-recoil candidates. Using the standard dark-matter halo and nuclear-physics WIMP model, these data set the world's lowest exclusion limits on the coherent WIMP-nucleon scalar cross section for all WIMP masses above 15 GeV/c2, ruling out a significant range of neutralino supersymmetric models. The minimum of this limit curve at the 90% C.L. is 4 x 10(-43) cm2 at a WIMP mass of 60 GeV/c2.

The NF2 interactor, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), associates with merlin in the "open" conformation and suppresses cell growth and motility.

The neurofibromatosis 2 tumor suppressor protein, merlin or schwannomin, functions as a negative growth regulator; however, its mechanism of action is not known. In an effort to determine how merlin regulates cell growth, we analyzed a recently identified novel merlin interactor, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS). We demonstrate that regulated overexpression of HRS in rat schwannoma cells results in similar effects as overexpression of merlin, including growth inhibition, decreased motility and abnormalities in cell spreading. Previously, we showed that merlin forms an intramolecular association between the N- and C-termini and exists in "open" and "closed" conformations. Merlin interacts with HRS in the unfolded, or open, conformation. This HRS binding domain maps to merlin residues 453-557. Overexpression of C-terminal merlin has no effect on HRS function, arguing that merlin binding to HRS does not negatively regulate HRS growth suppressor activity. These results suggest the possibility that merlin and HRS may regulate cell growth in schwannoma cells through interacting pathways.

The neurofibromatosis 2 tumor suppressor protein interacts with hepatocyte growth factor-regulated tyrosine kinase substrate.

The neurofibromatosis 2 tumor suppressor protein schwannomin/merlin is commonly mutated in schwannomas and meningiomas. Schwannomin, a member of the 4.1 family of proteins, which are known to link the cytoskeleton to the plasma membrane, has little known function other than its ability to suppress tumor growth. Using yeast two-hybrid interaction cloning, we identified the HGF-regulated tyrosine kinase substrate (HRS) as a schwannomin interactor. We verified the interaction by both immunoprecipitation of endogenous HRS with endogenous schwannomin in vivo as well as by using bacterially purified HRS and schwannomin in vitro. We narrowed the regions of interaction to include schwannomin residues 256-579 and HRS residues from 480 to the end of either of two HRS isoforms. Schwannomin molecules with a L46R, L360P, L535P or Q538P missense mutation demonstrated reduced affinity for HRS binding. As HRS is associated with early endosomes and may mediate receptor translocation to the lysosome, we demonstrated that schwannomin and HRS co-localize at endosomes using the early endosome antigen 1 in STS26T Schwann cells by indirect immunofluorescence. The identification of schwannomin as a HRS interactor implicates schwannomin in HRS-mediated cell signaling.

A meta-analysis of mandibular intercanine width in treatment and postretention.

The meta-analysis technique of literature review was applied to a total of 26 previous studies to assess the longitudinal stability of postretention mandibular intercanine width. Weighted averages and standard deviations for the means of 1,233 subjects were compared for linear changes in intercanine transverse dimensions during treatment (T1), immediately after treatment (T2), and after removal of all retention (T3). Net change was defined as the difference between means at T3 and T1. Dimensional changes were also evaluated on the basis of patient pretreatment Angle classification, extraction, and nonextraction treatment modalities of each group. Paired two-tail t-tests were performed between T3 and T1 means on all groups at the a priori level of significance set at a < or = 0.05. Statistically significant differences were observed for the following groups: all patients; nonextraction; extraction; Class I; Class I extraction; Class II extraction; and, Class I Division 1 nonextraction. The findings of this study indicate that regardless of patient diagnostic and treatment modalities, mandibular intercanine width tends to expand during treatment on the order of one to two millimeters, and to contract postretention to approximately the original dimension. While statistically significant differences could be demonstrated within various groups, the magnitudes of the differences were not considered clinically important.

Kinetic analysis of the catalytic domain of human cdc25B.

The Cdc25 cell cycle regulator is a member of the dual-specificity class of protein-tyrosine phosphatases that hydrolyze phosphotyrosine- and phosphothreonine-containing substrates. To study the mechanism of Cdc25B, we have overexpressed and purified the catalytic domain of human Cdc25B (Xu, X., and Burke, S. P. (1996) J. Biol. Chem. 271, 5118-5124). In the present work, we have analyzed the kinetic properties of the Cdc25B catalytic domain using the artificial substrate 3-O-methylfluorescein phosphate (OMFP). Steady-state kinetic analysis indicated that the kcat/Km for OMFP hydrolysis is almost 3 orders of magnitude greater than that for p-nitrophenyl phosphate hydrolysis. Like other dual-specificity phosphatases, Cdc25 exhibits a two-step catalytic mechanism, characterized by formation and breakdown of a phosphoenzyme intermediate. Pre-steady-state kinetic analysis of OMFP hydrolysis indicated that formation of the phosphoenzyme intermediate is approximately 20 times faster than subsequent phosphoenzyme breakdown. The resulting burst pattern of product formation allowed us to derive rate constants for enzyme phosphorylation (26 s-1) and dephosphorylation (1.5 s-1) as well as the dissociation constant for OMFP (0.3 mM). Calculations suggest that OMFP binds with higher affinity and reacts faster with Cdc25B than does p-nitrophenyl phosphate. OMFP is a highly efficient substrate for the dual-specificity protein-tyrosine phosphatases VHR and rVH6, but not for two protein-tyrosine phosphatases, PTP1 and YOP. The ability to observe distinct phases of the reaction mechanism during OMFP hydrolysis will facilitate future analysis of critical catalytic residues in Cdc25 and other dual-specificity phosphatases.

Roles of active site residues and the NH2-terminal domain in the catalysis and substrate binding of human Cdc25.

Human Cdc25 proteins are dual specific protein phosphatases that play important roles in cell cycle regulation. In this study, the catalytic mechanism and substrate binding specificity of human Cdc25A and -B proteins were investigated by site-directed and deletion mutagenesis methods. Mutations of the cysteine or the arginine residues in the active site motif abolished the Cdc25 phosphatase activity. However, the cysteine mutation in both Cdc25A and -B created enzymes that still retain the ability to bind their substrates. This allowed us to test the ability of Cdc25A and -B to bind various cyclin-Cdk complexes in vitro. While Cdc25A Cys --> Ser could interact with cyclin A-Cdk2, cyclin B-Cdc2, and cyclin E-Cdk2 strongly, Cdc25B mutant was only found to bind to cyclin A-Cdk2 at significant levels. We also identified Arg452 and Ser449 as two crucial residues that could be directly involved in the molecular interactions between Cdc25 and cyclin-Cdk proteins. Deletion mutagenesis data also indicate that the phosphatase catalytic domains of Cdc25A and -B proteins are located within their carboxyl terminus.

Hippocampal slices: glutamate overflow and cellular damage from ischemia are reduced by sodium-channel blockade.

We evaluated concentrations of excitatory amino acids released from slices into the superfusing solution and also evaluated extracellular field potential recordings and histological appearance of slice tissues to evaluate several sodium-channel modulating drugs as potential treatments for ischemia. The selective sodium-channel blocker tetrodotoxin (TTX, 1 microM) reduced glutamate release from deprivation of oxygen and D-glucose, while calcium-channel blockade was ineffective. Thus, during ischemia, we propose that glutamate may be released from the free cytosolic pool ('metabolic' glutamate) rather than by exocytosis. TTX (100-500 nM) and voltage-dependent sodium-channel blockers (phenytoin, 20-100 microM; lidocaine, 2-200 microM) each prevented damage to slices without blocking action potentials. The reduction of cellular depolarization and sodium loading during ischemia may explain the neuroprotective action of several sodium-channel modulating drugs in our in vitro studies and also in animal models.

Inhibition of endogenous glutamate release from hippocampal tissue by Ca2+ channel toxins.

The pharmacology of voltage-dependent Ca2+ channels involved in the release of endogenous neurotransmitter amino acids was measured from small tissue slices of rat hippocampal CA1 region. Application of 50 mM KCl induced large increases in Ca(2+)-dependent overflow of endogenous glutamate, aspartate and gamma-aminobutyric acid (GABA). Pretreatment of tissues with the funnel-web spider toxin omega-Aga-IVA (200 nM) reduced KCl-induced overflow of all three amino acids. Pretreatment with the cone snail toxin omega-CgTx-GVIA (2 microM) caused similar but smaller reductions in amino acid overflow (aspartate overflow was not reduced significantly). These results indicate that P-type Ca2+ channels, and to a lesser extent, N-type Ca2+ channels, are involved in the release of transmitter amino acids under these conditions.

Endogenous extracellular glutamate accumulation in rat neocortical cultures by reversal of the transmembrane sodium gradient.

Glutamate excites receptors located on neurons that cause calcium and sodium influx involved in excitatory synaptic transmission. During ischemia, excess glutamate is present in the extracellular space of brain tissue, leading to abnormal levels of calcium influx and eventually to cell death. In mixed neuronal/glial cell cultures we have found that reduction of extracellular sodium concentration below approximately 10 mM causes marked increases in glutamate and aspartate in medium collected 10 min after changing to low sodium. Various data indicate that the accumulated glutamate comes from reversal of normal cellular glutamate uptake, a process also thought to occur during ischemia.

Reflections on the 101st congress.

Periodically, Washington, D.C. nursing leaders and nurse staffers on Capitol Hill have dinner meetings to exchange ideas and network. Nursing Economic+ hosted the most recent roundtable dinner on October 25, 1989, prior to its Sixth Annual Conference. In this speech, Sheila Burke, MPA, RN, FAAN, a founder of the Washington Roundtable dinner, reflects on the 101st Congress in action.

Regulation of glutamate and aspartate release from the Schaffer collaterals and other projections of CA3 hippocampal pyramidal cells.

Excitatory synaptic transmission in the CNS can be modulated by endogenous substances and metabolic states that alter release of the transmitter, usually glutamate and/or aspartate. To explore this issue, we have studied the release of endogenous glutamate and aspartate from synaptic terminals of the CA3-derived Schaffer collateral, commissural and ipsilateral associational fibers in slices of hippocampal area CA1. These terminals release glutamate and aspartate in about a 5:1 ratio. The release process is modulated by adenosine, by the transmitters themselves and by nerve terminal metabolism. Adenosine inhibits the release of both amino acids by acting upon an A1 receptor. The transmitters, once released, can regulate their further release by acting upon both an NMDA and a non-NMDA (quisqualate/kainate) receptor. Activation of the NMDA receptor enhances the release of both glutamate and aspartate, whereas activation of the non-NMDA receptor depresses the release of aspartate only. Superfusion of CA1 slices with a glucose-deficient medium increases the release of both amino acids and reduces the glutamate/aspartate ratio. These results have implications for the regulation of excitatory synaptic transmission in the CA1 area and for the mechanism of hypoglycemic damage to CA1 pyramidal cells.

Effects of glucose deficiency on glutamate/aspartate release and excitatory synaptic responses in the hippocampal CA1 area in vitro.

The effects of glucose deficiency on (1) the K+-evoked release of glutamate and aspartate and (2) excitatory synaptic transmission were studied in the Schaffer collateral-commissural-ipsilateral associational (SCCIA) projection to area CA1 of the rat hippocampal formation in vitro. Compared with 1 or 10 mM glucose, superfusion of CA1 slices with 0.1 mM glucose enhanced the K+-evoked release of both glutamate and aspartate, increased the ratio of aspartate release to glutamate release and did not affect the release of GABA. With both high and low glucose concentrations, the K+-evoked release of glutamate and aspartate originated predominantly from a Ca2+-sensitive store associated with the SCCIA projection. Superfusion with glucose-deficient medium abolished the inhibitory effect of adenosine on glutamate and aspartate release, but augmented the enhancing effect of the adenosine antagonist 8-phenyltheophylline. These results suggest that enough endogenous adenosine was released from the slices under these conditions to saturate the presynaptic A1 receptors. Despite its facilitatory effect on excitatory transmitter release, glucose-deficient medium inhibited transmission at Schaffer collateral-commissural synapses. Even when the postsynaptic response to a single electrical pulse was abolished, however, a substantial response could still be evoked through paired-pulse or frequency potentiation and the inhibition promptly reversed upon superfusion with 10 mM glucose. The increased ratio of aspartate release to glutamate release appears to reflect changes in the tissue content of these amino acids. The enhanced release of both excitants is suggested to result partly from a rise in intraterminal Ca2+ concentration and partly from inhibition of glutamate/aspartate uptake. Enhanced aspartate release may be particularly relevant to hypoglycemic damage in the CA1 area, because aspartate is a more potent hippocampal excitotoxin than glutamate.

Regulation of glutamate and aspartate release from slices of the hippocampal CA1 area: effects of adenosine and baclofen.

Glutamate and/or aspartate is the probable transmitter released from synaptic terminals of the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers in area CA1 of the rat hippocampal formation. Slices of the CA1 area were employed to test the effects of adenosine- and gamma-aminobutyrate (GABA)-related compounds on the release of glutamate and aspartate from this projection. Under the conditions of these experiments, the release of glutamate and aspartate evoked by 50 mM K+ was more than 90% Ca2+-dependent and originated predominantly from the CA3-derived pathways. Adenosine reduced the K+-evoked release of glutamate and aspartate by a maximum of about 60%, but did not affect the release of GABA. This action was reversed by 1 microM 8-phenyltheophylline. The order of potency for adenosine analogues was as follows: L-N6-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than D-N6-phenylisopropyladenosine approximately equal to 2-chloroadenosine greater than adenosine much greater than 5'-N-ethylcarboxamidoadenosine. 8-Phenyltheophylline (10 microM) by itself enhanced glutamate/aspartate release, whereas dipyridamole alone depressed release. These results support the view that adenosine inhibits transmission at Schaffer collateral-commissural-ipsilateral associational synapses mainly by reducing transmitter release and that these effects involve the activation of an A1 receptor. Neither adenosine, L-N6-phenylisopropyladenosine, nor 8-phenyltheophylline affected the release of glutamate or aspartate evoked by 10 microM veratridine. The differing effects of adenosine compounds on release evoked by K+ and veratridine suggest that A1 receptor activation either inhibits Ca2+ influx through the voltage-sensitive channels or interferes with a step subsequent to Ca2+ entry that is coupled to the voltage-sensitive Ca2+ channels in an obligatory fashion. Neither baclofen nor any other agent active at GABAB or GABAA receptors affected glutamate or aspartate release evoked by elevated K+ or veratridine. Therefore, either baclofen does not inhibit transmission at these synapses by depressing transmitter release or else it does so in a way that cannot be detected when a chemical depolarizing agent is employed.

Maturation of sympathetic neurotransmission in the rat heart. IX. Development of transsynaptic regulation of cardiac adrenergic sensitivity.

The number of beta adrenoceptors and the cardiac sensitivity to adrenergic stimulation increase substantially in the immediate postnatal period of the rat. To determine whether transsynaptic input influences this developmental process, the effects of a sympathomimetic and of agents which destroy noradrenergic nerve terminals on regulation of adrenergic postsynaptic sensitivity were compared in hearts from adult and developing rats. In mature animals, chronic exposure to the beta agonist isoproterenol (2.5 mg/kg s.c.) led to rapid onset (3-5 days) of chronotropic adrenergic subsensitivity accompanied by a loss of beta adrenoceptor binding sites; chemical sympathectomy by daily administration of guanethidine (50 mg/kg s.c.) or by 6-hydroxydopamine (100 mg/kg s.c. given once daily for 3 days) resulted in chronotropic adrenergic supersensitivity and increases in binding sites. These data in the adult agree with classical transsynaptic modulation of adrenergic postsynaptic reactivity. In contrast, identical drug treatments of immature rats beginning 1 day after birth failed to evoke changes in either chronotropic adrenergic sensitivity or in numbers of beta adrenoceptor binding sites until the 3rd to 4th week. Consequently, the initial development of beta adrenoceptors and responsiveness to catecholamines in the neonatal myocardium are not transsynaptically regulated; rather, other (e.g. hormonal) factors appear to control early maturation of cardiac adrenergic sensitivity.