Blockade of airway inflammation and hyperresponsiveness by HIV-TAT-dominant negative Ras

We have reported previously that HIV-TAT-dominant negative (dn) Ras inhibits eosinophil adhesion to ICAM-1 after activation by IL-5 and eotaxin. In this study, we evaluated the role of Ras in Ag-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of dnRas, which was fused to an HIV-TAT protein transduction domain (TAT-dnRas). Uptake of TAT-dnRas (t(1/2) = 12 h) was demonstrated in leukocytes after i.p. administration. OVA-sensitization significantly increased eosinophil and lymphocyte numbers in bronchoalveolar lavage fluid 24 h after final challenge. Treatment of animals with 3-10 mg/kg TAT-dnRas blocked the migration of eosinophils from 464 +/- 91 x 10(3)/ml to 288 +/- 79 x 10(3)/ml with 3 mg/kg of TAT-dnRas (p < 0.05), and further decreased to 116 +/- 63 x 10(3)/ml after 10 mg/kg TAT-dnRas (p < 0.01). Histological examination demonstrated that inflammatory cell infiltration (largely eosinophils and mononuclear cells) and mucin production around the airways caused by OVA were blocked by TAT-dnRas. OVA challenge also caused airway hyperresponsiveness to methacholine, which was dose dependently blocked by treatment with TAT-dnRas. TAT-dnRas also blocked Ag-induced IL-4 and IL-5, but not IFN-gamma, production in lung tissue. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by pretreatment with TAT-dnRas. By contrast, TAT-green fluorescent protein or dnRas lacking the TAT protein transduction domain did not block airway inflammation, cytokine production, or airway hyperresponsiveness. We conclude that Ras mediates Th2 cytokine production, airway inflammation, and airway hyperresponsiveness in immune-sensitized mice.     

Planting Design in the Ecuadorian Andes: Memory,Resilience, and Re-Connection

Modern cities are constantly growing and evolving. This expansion of urban development bleeds into the surrounding landscapes, causing the displacement and disturbance of native plant and animal species to remote areas where topography limits human access. As a result, metropolitan areas often become gray places with low biodiversity, elevated temperatures, poor air quality, flood issues, and lack of a local identity. Quito, Ecuador is one of the cities facing this important challenge. Perched high in the Andes, Quito is a place of great biodiversity, nevertheless the constructed landscapes are dominated by introduced species due to colonization and to the lack of availability of native species in the nursery trade. This article walks through the creation of a native nursery in Quito and the implementation of initial trial plots, a green roof, and a garden. It explains the discoveries made during the process and provides directions for future goals to reintroduce native plant species into urban environments and contemporary landscapes in order to create more sustainable cities. The goal is to help people reconnect with their natural heritage and to learn about native plants to ensure the continuity of ancestral knowledge of the natural world for future generations.     

NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex

NMDA receptors play important roles in learning and memory and in sculpting neural connections during development. After the period of peak cortical plasticity, NMDA receptor-mediated EPSCs (NMDAR EPSCs) decrease in duration. A likely mechanism for this change in NMDA receptor properties is the molecular alteration of NMDA receptor structure by regulation of NMDA receptor subunit gene expression. The four modulatory NMDAR2A-D (NR2A-D) NMDA receptor subunits are known to alter NMDA receptor properties, and the expression of these subunits is regulated developmentally. It is unclear, however, how the four NR2 subunits are expressed in individual neurons and which NR2 subunits are important to the regulation of NMDA receptor properties during development in vivo. Analysis of NR2 subunit gene expression in single characterized neurons of postnatal neocortex revealed that cells expressing NR2A subunit mRNA had faster NMDAR EPSCs than cells not expressing this subunit, regardless of postnatal age. Expression of NR2A subunit mRNA in cortical neurons at even low levels seemed sufficient to alter the NMDA receptor time course. The proportion of cells expressing NR2A and displaying fast NMDAR EPSCs increased developmentally, thus providing a molecular basis for the developmental change in mean NMDAR EPSC duration.     

Differential effect of ethanol on PC12 cell death

Our goal was to examine the effects of ethanol on cell death using rat pheochromocytoma (PC12) cells as a neuronal model. Withdrawal of serum for 24 hr increased the number of PC12 cells labeled with ethidium homodimer indicating an increase in cell death. Inclusion of 50 mM ethanol during the serum deprivation further increased the amount of ethidium fluorescence by 39%. Although reducing the serum concentration from the usual 15 to 4% did not alter cellular viability, a significant increase in the amount of ethidium fluorescence was observed in PC12 cells incubated for 24 hr in the presence of 4% serum and 150 mM ethanol. No change in viability was observed in cells exposed to either 150 mM ethanol in the presence of 15% serum or 50 mM ethanol in the presence of 4% serum. Inclusion of ethanol during serum deprivation increased the amount of soluble DNA found in the 15,000 x g supernatant. Similarly, using the terminal deoxynucleotidyl transferase dUTP nick-end labeling method to visualize DNA fragmentation in situ, ethanol caused a 213% increase in the number of PC12 cells labeled during serum withdrawal. Agarose gel electrophoresis of the DNA isolated from cells maintained in the absence of serum yielded the classical DNA laddering pattern of 180 to 200 bp fragments suggestive of apoptosis. Ethanol caused a concentration-dependent increase in the amount of DNA laddering in cells deprived of serum. Furthermore, ethanol significantly potentiated the DNA laddering of cells maintained in 4% serum. In contrast to the ethanol-induced increase in cell death when serum factors were reduced or withdrawn, 150 mM ethanol lowered by 34% the number of ethidium-labeled PC12 cells observed after a 30-min exposure to 2 mM H2O2. Similarly, agarose gel electrophoresis of the DNA from H2O2-treated cells did not display DNA laddering. This study demonstrates that: 1) ethanol antagonizes the trophic action of serum factors; 2) pharmacologically relevant ethanol concentrations significantly potentiate apoptosis after serum withdrawal and 3) this enhancement appears specific for apoptosis.     

Folding intermediates of wild-type and mutants of barnase. I. Use of phi-value analysis and m-values to probe the cooperative nature of the folding pre-equilibrium

It is difficult to determine whether transient folding intermediates have a cooperative (or first-order) folding transition without measuring their rates of formation directly. An intermediate I could be formed by a second-order transition from a denatured state D that is progressively changed into I as conditions are changed. We have not been able to monitor the rate of formation of the folding intermediate of barnase directly, but have analysed its reactivity and the equilibrium constant for its formation over a combination of wide ranges of temperature, concentration of denaturant and structural variation. Phase diagrams have been constructed for wild-type and 16 mutant proteins to map out the nature of the energy landscape of the denatured state. The free energy of unfolding of I, delta GD-I, changes with [urea] according to a highly cooperative transition. Further, mD-I (= delta delta GD-I/delta [urea]) for wild-type and several mutants is relatively insensitive to temperature, as would be expected for an intermediate that is formed cooperatively, rather than one that melts out according to a second-order transition. The phi-values for the formation of I change abruptly through the folding transitions rather than have the smooth changes expected for a second-order transition. There is a subset of mutants for which both mD-I and phi-value analysis indicate that a second intermediate becomes populated close to the melting temperatures of the native proteins. The folding intermediate of barnase is, thus, a relatively discrete and compact entity which is formed cooperatively.     

Phosphorylation of triciribine is necessary for activity against HIV type 1

Triciribine (TCN) is a tricyclic nucleoside with known antineoplastic and antiviral activity. It is a potent and selective inhibitor of HIV-1 and HIV-2, including strains known to be resistant to AZT or TIBO. TCN is phosphorylated to its 5'-monophosphate (TCN-P) by intracellular adenosine kinase (AK), but is not converted to di- or triphosphates. We now report that 5'-phosphorylation is requisite for the activity of TCN against HIV-1. CEM cells incubated with TCN at concentrations ranging from 0.1 to 330 microM gave intracellular TCN-P concentrations from 27 to 775 microM, respectively. There was no difference in the amount of intracellular TCN-P detected in uninfected compared with HIV-1-infected CEM cells. The antiviral effect of TCN against HIV-1 was strongly antagonized by the AK inhibitor 5-iodotubercidin (ITu). In contrast, TCN and ITu only exhibited additive cytotoxicity. The 5'-deoxy analog of TCN, which cannot be phosphorylated, had no antiviral effect against HIV-1 at a concentration more than 100 times higher than the IC50 of TCN. Similarly, TCN was not active against HIV-1 in an AK-deficient cell line (AA-2) at concentrations shown to inhibit the virus by >95% in CEM cells. Consistent with its AK-deficient phenotype, this cell line phosphorylated TCN to only 3% of the extent observed in CEM cells. We conclude that TCN must be phosphorylated to TCN-P for activity against HIV-1.     

The spin trap reagent PBN attenuates degeneration of 5-HT neurones in rat brain induced by p-chloroamphetamine but not fenfluramine

Dark Agouti rats injected with either p-chloroamphetamine (PCA; 2.5 mg/kg i.p.) or fenfluramine (15 mg/kg i.p.) had substantial decreases (approximately 50%) in the concentration of 5-HT and 5-HIAA and binding of [3H]paroxetine in the cerebral cortex 7 days later. This indicates that both compounds had produced neurodegeneration of 5-HT axon terminals. Two doses of alpha-phenyl-N-tert-butyl nitrone (PBN; 150 mg/kg i.p.) 130 min apart had no effect on cortical 5-HT content or [3H]paroxetine binding. However, when PBN (150 mg/ kg) was given 10 min before and 120 min after PCA (2.5 mg/kg) it attenuated the PCA-induced neurodegeneration. In contrast, PBN was without significant effect on the fenfluramine-induced damage. Changes in rectal temperature following either the neurotoxins or neurotoxins+ PBN were no more than +/-1 degree C of saline-injected control rats. These data indicate that PCA, like MDMA, probably induces neurotoxic degeneration because of the formation of catechol or quinone metabolites and subsequent reactive tree radical formation. Such a mechanism does not appear to explain fenfluramine-induced damage to 5-HT neurones.