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Inflammatory processes are activated following ischemic stroke that lead to increased tissue damage for weeks following the ischemic insult, but there are no approved therapies that target this inflammation-induced secondary injury. Here, we report that SynB1-ELP-p50i, a novel protein inhibitor of the nuclear factor kappa B (NF-κB) inflammatory cascade bound to the drug carrier elastin-like polypeptide (ELP), decreases NF-κB induced inflammatory cytokine production in cultured macrophages, crosses the plasma membrane and accumulates in the cytoplasm of both neurons and microglia in vitro, and accumulates at the infarct site where the blood-brain barrier (BBB) is compromised following middle cerebral artery occlusion (MCAO) in rats. Additionally, SynB1-ELP-p50i treatment reduces infarct volume by 11.86% compared to saline-treated controls 24 h following MCAO. Longitudinally, SynB1-ELP-p50i treatment improves survival for 14 days following stroke with no effects of toxicity or peripheral organ dysfunction. These results show high potential for ELP-delivered biologics for therapy of ischemic stroke and other central nervous system disorders and further support targeting inflammation in ischemic stroke., (© 2023 Federation of American Societies for Experimental Biology.)

Inflammatory processes are activated following ischemic strokes and lead to increased tissue damage for weeks following the ischemic insult, but there are no approved therapies that target this inflammation-induced secondary injury. Here, we report that SynB1-ELP-p50i, a novel protein inhibitor of the nuclear factor kappa B (NF-κB) inflammatory cascade bound to drug carrier elastin-like polypeptide (ELP), is able to enter both neurons and microglia, cross the blood-brain barrier, localize exclusively in the ischemic core and penumbra in Wistar-Kyoto and spontaneously hypertensive rats (SHRs), and reduce infarct volume in male SHRs. Additionally, in male SHRs, SynB1-ELP-p50i treatment improves survival for 14 days following stroke with no effects of toxicity or peripheral organ dysfunction. These results show high potential for ELP-delivered biologics for therapy of ischemic stroke and other central nervous system disorders and further support targeting inflammation in ischemic stroke.

Vascular Endothelial Growth Factor (VEGF), a key mediator of angiogenesis and vascular repair, is reduced in chronic ischemic renal diseases, leading to microvascular rarefaction and deterioration of renal function. We developed a chimeric fusion of human VEGF-A 121 with the carrier protein Elastin-like Polypeptide (ELP-VEGF) to induce therapeutic angiogenesis via targeted renal VEGF therapy. We previously showed that ELP-VEGF improves renal vascular density, renal fibrosis, and renal function in swine models of chronic renal diseases. However, VEGF is a potent cytokine that induces angiogenesis and increases vascular permeability, which could cause undesired off-target effects or be deleterious in a patient with a solid tumor. Therefore, the current study aims to define the toxicological profile of ELP-VEGF and assess its risk for exacerbating tumor progression and vascularity using rodent models. A dose escalating toxicology assessment of ELP-VEGF was performed by administering a bolus intravenous injection at doses ranging from 0.1 to 200 mg/kg in Sprague Dawley (SD) rats. Blood pressure, body weight, and glomerular filtration rate (GFR) were quantified longitudinally, and terminal blood sampling and renal vascular density measurements were made 14 days after treatment. Additionally, the effects of a single administration of ELP-VEGF (0.1-10 mg/kg) on tumor growth rate, mass, and vascular density were examined in a mouse model of breast cancer. At doses up to 200 mg/kg, ELP-VEGF had no effect on body weight, caused no changes in plasma or urinary markers of renal injury, and did not induce renal fibrosis or other histopathological findings in SD rats. At the highest doses (100-200 mg/kg), ELP-VEGF caused an acute, transient hypotension (30 min), increased GFR, and reduced renal microvascular density 14 days after injection. In a mouse tumor model, ELP-VEGF did not affect tumor growth rate or tumor mass, but analysis of tumor vascular density by micro-computed tomography (μCT) revealed significant, dose dependent increases in tumor vascularity after ELP-VEGF administration. ELP-VEGF did not induce toxicity in the therapeutic dosing range, and doses one hundred times higher than the expected maximum therapeutic dose were needed to observe any adverse signs in rats. In breast tumor-bearing mice, ELP-VEGF therapy induced a dose-dependent increase in tumor vascularity, demanding caution for potential use in a patient suffering from kidney disease but with known or suspected malignancy.

TNF-related apoptosis-inducing ligand (TRAIL) was initially described to induce apoptosis of tumor cells and/or virally infected cells, although sparing normal cells, and has been implicated in the pathogenesis of HIV disease. We previously identified TRAILshort, a TRAIL splice variant, in HIV-infected patients and characterized it as being a dominant negative ligand to subvert TRAIL-mediated killing. Herein, using single-cell genomics we demonstrate that TRAILshort is produced by HIV-infected cells, as well as by uninfected bystander cells, and that the dominant stimulus which induces TRAILshort production are type I IFNs and TLR7, TLR8, and TLR9 agonists. TRAILshort has a short t 1/2 by virtue of containing a PEST domain, which targets the protein toward the ubiquitin proteasome pathway for degradation. Further we show that TRAILshort binds preferentially to TRAIL receptors 1 and 2 with significantly reduced interaction with the decoy TRAIL receptors 3 and 4. Recombinant TRAILshort is sufficient to protect cells against TRAIL-induced killing, whereas immunodepletion of TRAILshort with a specific Ab restores TRAIL sensitivity. Importantly we show that TRAILshort is shed in microvesicles into the cellular microenvironment and therefore confers TRAIL resistance not only on the cell which produces it, but also upon neighboring bystander cells. These results establish a novel paradigm for understanding and overcoming TRAIL resistance, in particular how HIV-infected cells escape immune elimination by the TRAIL:TRAILshort receptor axis., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Importance: Recent discussion has focused on questions related to the repeal and replacement of portions of the Affordable Care Act (ACA). However, issues central to the future of health and health care in the United States transcend the ACA provisions receiving the greatest attention. Initiatives directed to certain strategic and infrastructure priorities are vital to achieve better health at lower cost., Objectives: To review the most salient health challenges and opportunities facing the United States, to identify practical and achievable priorities essential to health progress, and to present policy initiatives critical to the nation's health and fiscal integrity., Evidence Review: Qualitative synthesis of 19 National Academy of Medicine-commissioned white papers, with supplemental review and analysis of publicly available data and published research findings., Findings: The US health system faces major challenges. Health care costs remain high at $3.2 trillion spent annually, of which an estimated 30% is related to waste, inefficiencies, and excessive prices; health disparities are persistent and worsening; and the health and financial burdens of chronic illness and disability are straining families and communities. Concurrently, promising opportunities and knowledge to achieve change exist. Across the 19 discussion papers examined, 8 crosscutting policy directions were identified as vital to the nation's health and fiscal future, including 4 action priorities and 4 essential infrastructure needs. The action priorities-pay for value, empower people, activate communities, and connect care-recurred across the articles as direct and strategic opportunities to advance a more efficient, equitable, and patient- and community-focused health system. The essential infrastructure needs-measure what matters most, modernize skills, accelerate real-world evidence, and advance science-were the most commonly cited foundational elements to ensure progress., Conclusions and Relevance: The action priorities and essential infrastructure needs represent major opportunities to improve health outcomes and increase efficiency and value in the health system. As the new US administration and Congress chart the future of health and health care for the United States, and as health leaders across the country contemplate future directions for their programs and initiatives, their leadership and strategic investment in these priorities will be essential for achieving significant progress.

Nitrate water pollution, which is mainly caused by agricultural activities, remains an international problem. It can cause serious long-term environmental and human health issues due to nitrate time-lag in the groundwater system. However, the nitrate subsurface legacy issue has rarely been considered in environmental water management. We have developed a simple catchment-scale approach to investigate the impact of historical nitrate loading from agricultural land on the nitrate-concentration trends in sandstones, which represent major aquifers in the Eden Valley, UK. The model developed considers the spatio-temporal nitrate loading, low permeability superficial deposits, dual-porosity unsaturated zones, and nitrate dilution in aquifers. Monte Carlo simulations were undertaken to analyse parameter sensitivity and calibrate the model using observed datasets. Time series of annual average nitrate concentrations from 1925 to 2150 were generated for four aquifer zones in the study area. The results show that the nitrate concentrations in 'St Bees Sandstones', 'silicified Penrith Sandstones', and 'non-silicified Penrith Sandstones' keep rising or stay high before declining to stable levels, whilst that in 'interbedded Brockram Penrith Sandstones' will level off after a slight decrease. This study can help policymakers better understand local nitrate-legacy issues. It also provides a framework for informing the long-term impact and timescale of different scenarios introduced to deliver water-quality compliance. This model requires relatively modest parameterisation and is readily transferable to other areas., (Copyright © 2016. Published by Elsevier B.V.)

Background and Objective: To determine macular retinal sublayer thickness changes in G11778A Leber hereditary optic neuropathy (LHON)., Patients and Methods: The authors performed a cross-sectional study by segmenting spectral-domain Cirrus OCT (Carl Zeiss Meditec, Dublin, CA) 512 × 128 macular cube scans from a prospective, observational study of G11778A LHON. The thickness of the retinal sublayers of LHON affected subjects and asymptomatic carriers were compared to those of a normal group., Results: The study included 20 LHON-affected subjects (13 males; age: 31 years ± 14 years, range: 10 years to 61 years; time since onset of visual loss: 5.9 years ± 8.7 years; 0.4-29.8), 31 asymptomatic LHON carriers (five males; age: 38 years ± 18 years, range: 9 years to 65 years), and 14 normal subjects (five males; age: 39 years ± 13 years, range: 23 years to 61 years). The retinal sublayer thickness parameters were not significantly correlated with age in any of the groups. There were no differences between carriers and normal subjects for thickness of total retina or any sublayer. Affected LHON retinal nerve fiber layer (RNFL) and ganglion cell layer plus inner plexiform layer (GCL+IPL) were thinner, whereas the photoreceptor outer segment (OS) layer was thicker than carriers and normal subjects (P values ranged from .042 to < .001), except for the OS layer in the inferior inner ring and temporal outer ring. Differences between groups were not significant in the inner nuclear layer plus outer plexiform layer (INL+OPL). The affected LHON outer nuclear layer plus inner segment layer was thicker in some quadrants, and the affected LHON choroid layer was generally thinner than carriers and normal subjects; however, these differences were not significant after accounting for age., Conclusion: LHON-affected patients have thickened photoreceptor OS layers in spite of having thinner RNFL and GCL+IPL layers. The findings indicate LHON also has an effect on the morphology of the photoreceptors. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:802-810.]., (Copyright 2016, SLACK Incorporated.)

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.

The four residues at the amino-terminus of mature Smac/DIABLO are an IAP binding motif (IBM). Upon exit from mitochondria, mature Smac interacts with inhibitor of apoptosis proteins (IAPs), abrogating caspase inhibition. We used the ubiquitin fusion model to express mature Smac in the cytosol. Transiently expressed mature Smac56-239 (called Smac56) and Smac60-239 (called Smac60), which lacks the IBM, interacted with X-linked inhibitor of apoptosis protein (XIAP). However, stable expression produced wild type Smac56 that failed to homodimerize, interact with XIAP, and potentiate caspase activation. Cytosolic Smac60 retained these functions. Cytosolic Smac56 apparently becomes posttranslationally modified at the dimer interface region, which obliterated the epitope for a monoclonal antibody. Cytosolic Smacδ, which has the IBM but lacks amino acids 62-105, homodimerized and weakly interacted with XIAP, but failed to potentiate apoptosis. These findings suggest that the IBM of Smac is a recognition point for a posttranslational modification(s) that blocks homodimerization and IAP interaction, and that amino acids 62-105 are required for the proapoptotic function of Smac.

Although early studies of inhibitor of apoptosis proteins (IAPs) suggested that cIAP1 directly binds and inhibits caspases similarly to X-linked IAP (XIAP), a recent one found that micromolar concentrations of cIAP1 only weakly inhibit caspase-3, -7, or -9. Here, we show that cIAP1 specifically and cooperatively blocks the cytochrome c-dependent apoptosome in vitro. Hence, cIAP1 prevented the activation of procaspase-3 but had no effect on the processing of procaspase-9 or the activity of prior activated caspase-3. Like cIAP1, XIAP had no effect on procaspase-9 processing and was a more potent inhibitor of procaspase-3 activation than of already activated caspase-3 activity. Inhibition of procaspase-3 activation depended on BIR2 and BIR3 of cIAP1 and was independent of BIR1, RING, CARD, and UBA domains. Smac prevented cIAP1 from inhibiting procaspase-3 activation and reversed the inhibition by prior addition of cIAP1. A procaspase-9 mutant (D315A) that cannot produce the p12 subunit was resistant to inhibition by cIAP1. Therefore, the N-terminal Ala-Thr-Pro-Phe motif of the p12 subunit of the caspase-9 apoptosome facilitates apoptosome blockade. Consequently, cIAP1 cooperatively interacts with oligomerized processed caspase-9 in the apoptosome and blocks procaspase-3 activation.

Although registered nurses rank similarly with physicians in the public's esteem, physicians are more visible than nurses in media coverage, public policy, and political spheres. Thus, nursing workforce issues are overshadowed by those of other health priorities, including Medicare and health reform. The purpose of this research was to understand the visibility and salience of the health workforce in general, gain an understanding about the effectiveness of messages concerning the nursing workforce in particular, and to understand why nursing workforce issues do not appear to have gained more traction in national health care policymaking. The National Survey of Thoughtleaders about the Health Workforce was administered via mail, telephone and online to health workforce and policy thoughtleaders from August 2009-October 2009. Of 301 thoughtleaders contacted, 123 completed questionnaires for a response rate of 41%. Thoughtleaders agree that nurses are critical to the quality and safety of our healthcare system, that there are current nursing shortages, and that nursing shortages will be intensified by health reform. Thoughtleaders reported that while they do hear about nursing issues frequently, they do not view most sources of information as proposing effective policy solutions. This study highlights a critical gap in effective policy advocacy and leadership to advance nurse workforce issues higher on the national health agenda., (Copyright 2010 Mosby, Inc. All rights reserved.)

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.

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.

Previous molecular analyses of human astrocytomas have identified many genetic changes associated with astrocytoma formation and progression. In an effort to identify novel gene expression changes associated with astrocytoma formation, which might reveal new potential targets for glioma therapeutic drug design, we used the B8-RAS-transgenic mouse astrocytoma model. Using multiplex gene expression profiling, we found that growth-associated protein 43 (GAP43) RNA and protein expression were lost in select human and mouse glioma cell lines. In this study, we demonstrate that re-expression of GAP43 in deficient C6 glioma cells results in growth suppression in clonogenic assays, as well as in multiple independently derived C6 glioma cell lines in vitro. GAP43-expressing C6 cells also exhibit reduced tumor growth as s.c. explants in immunocompromised mice in vivo. In addition, GAP43-expressing C6 clones demonstrate impaired cell motility and increased homophilic aggregation. GAP43 re-expression is also associated with reduced mitogen-activated protein kinase and AKT activation in C6 cells, suggesting that GAP43 functions as a novel glioma growth suppressor by modulating mitogenic signaling pathways.

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor predisposition syndrome characterized by benign proliferations (hamartomas). In the brain, individuals with TSC develop autism, mental retardation and seizures associated with focal cortical dysplasias, subependymal nodules, and subependymal giant cell astrocytomas (SEGAs). We hypothesize that dysregulated astrocyte function due to mutations in the tumor suppressor genes, TSC1 and TSC2, may contribute to the pathogenesis of these brain abnormalities. In this report, we demonstrate that mice heterozygous for a targeted defect in either the Tsc1 or Tsc2 genes(Tsc1+/- and Tsc2+/- mice) exhibit a 1.5-fold increase in the number of astrocytes in vivo. Whereas increased astrocyte numbers in vivo were suggestive of a proliferative advantage, Tsc2+/- primary astrocyte cultures did not show a cell-autonomous growth advantage, anchorage-independent growth, increased saturation density, or increased fluid-phase endocytosis compared to wild type astrocytes. Tsc2 null mouse embryonic fibroblasts (MEFs) however, did exhibit increased saturation density compared to Tsc2 wild type controls. In both Tsc2+/- astrocytes and Tsc2 null mouse embryonic fibroblasts, p27-Kip1 expression was decreased compared to wild type cells, and was reversed by tuberin re-expression in Tsc2-/- MEFs. In contrast, no change in endocytosis was observed upon tuberin re-expression in Tsc2-/- MEFs. Collectively, these results suggest Tsc heterozygosity may provide a non-cell-autonomous growth advantage for astrocytes that may involve p27-Kip1 expression.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.