An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G beta gamma subunits.

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.

Denitrification and inference of nitrogen sources in the karstic Floridan Aquifer

Aquifer denitrification is among the most poorly constrained fluxes in global and regional nitrogen budgets. The few direct measurements of denitrification in groundwaters provide limited information about its spatial and temporal variability, particularly at the scale of whole aquifers. Uncertainty in estimates of denitrification may also lead to underestimates of its effect on isotopic signatures of inorganic N, and thereby confound the inference of N source from these data. In this study, our objectives are to quantify the magnitude and variability of denitrification in the Upper Floridan Aquifer (UFA) and evaluate its effect on N isotopic signatures at the regional scale. Using dual noble gas tracers (Ne, Ar) to generate physical predictions of N2 gas concentrations for 112 observations from 61 UFA springs, we show that excess (i.e. denitrification-derived) N2 is highly variable in space and inversely correlated with dissolved oxygen (O2). Negative relationships between O2 and δ15N NO3 across a larger dataset of 113 springs, well-constrained isotopic fractionation coefficients, and strong 15N:18O covariation further support inferences of denitrification in this uniquely organic-matter-poor system. Despite relatively low average rates, denitrification accounted for 32 % of estimated aquifer N inputs across all sampled UFA springs. Back-calculations of source δ15N NO3 based on denitrification progression suggest that isotopically-enriched nitrate (NO3-) in many springs of the UFA reflects groundwater denitrification rather than urban- or animal-derived inputs. © Author(s) 2012.

Life scripts help to maintain autobiographical memories of highly positive, but not highly negative, events.

A representative sample of 1,307 respondents between the ages of 20 and 94 was asked how old they were when they felt most afraid, most proud, most jealous, most in love, and most angry. They were also asked when they had experienced their most important event and whether this event was positive or negative. In general, there was a reminiscence "bump" for positive but not negative events. To provide data on life scripts, 87 psychology students answered the same questions for a hypothetical 70-year-old. The undergraduates were more confident in dating positive than in dating negative events, and when they were confident, the distribution of responses predicted the survey data. The results support the idea of culturally shared life scripts for positive but not negative events, which structure retrieval processes and spaced practice.

Age-related immune reconstitution during highly active antiretroviral therapy in human immunodeficiency virus type 1-infected children.

OBJECTIVES: To evaluate the immune reconstitution in HIV-1-infected children in whom highly active antiretroviral therapy (HAART) controlled viral replication and to assess the existence of a relation between the magnitude of this restoration and age. METHODS: All HIV-1-infected children in whom a new HAART decreased plasma viral load below 400 copies/ml after 3 months of therapy were prospectively enrolled in a study of their immune reconstitution. Viral load, lymphocyte phenotyping, determination of CD4+ and CD8+ T cell receptor repertoires and proliferative responses to mitogens and recall antigens were assessed every 3 months during 1 year. RESULTS: Nineteen children were evaluated. Naive and memory CD4+ percentages were already significantly increased after 3 months of HAART. In contrast to memory CD4+ percentages, naive CD4+ percentages continued to rise until 12 months. Age at baseline was inversely correlated with the magnitude of the rise in naive CD4+ cells after 3, 6 and 9 months of therapy but not after 12 months. Although memory and activated CD8+ cells were already decreasing after 3 months, abnormalities of the CD8 T cell receptor repertoire and activation of CD8+ cells persisted at 1 year. HAART increased the response to mitogens as early as 3 months after starting therapy. CONCLUSIONS: In children the recovery of naive CD4+ cells occurs more rapidly if treatment is started at a younger age, but after 1 year of viral replication control, patients of all ages have achieved the same level of restoration. Markers of chronic activation in CD8+ cells persist after 1 year of HAART.

Cellular immune responses in human immunodeficiency virus (HIV)-1-infected children: Is immune restoration by highly active anti-retroviral therapy comparable to non-progression?

The objective of this study was to investigate whether the restored immune functions of vertically human immunodeficiency virus (HIV)-infected children who were severely immunodeficient before the initiation of highly active anti-retroviral therapy (HAART) are comparable to those of untreated slow progressors. We therefore assessed T cell proliferation and cytokine [interferon (IFN)-γ, interleukin (IL)-5 and IL-13] secretions after mitogen, recall antigens and HIV-1-specific stimulation in 12 untreated slow progressors, 16 untreated progressors and 18 treated patients. Treated children were profoundly immunodeficient before the initiation of HAART and had long-lasting suppression of viral replication on treatment. We demonstrated that slow progressors are characterized not only by the preservation of HIV-1-specific lymphoproliferative responses but also by the fact that these responses are clearly T helper type 1 (Th1)-polarized. Children on HAART had proliferative responses to HIV-1 p24 antigen, purified protein derivative (PPD) and tetanus antigen similar to slow progressors and higher than those of progressors. However, in contrast to slow progressors, most treated children exhibited a release of Th2 cytokines accompanying the IFN-γ secretion in response to the HIV-1 p24 antigen. Moreover, despite higher proliferative responses to phytohaemagglutinin (PHA) than the two groups of untreated children, treated children had lower levels of IFN-γ secretion in response to PHA than slow progressors. These data show that in severely immunodeficient vertically HIV-infected children, a long-lasting HAART allows recovering lymphoproliferative responses similar to untreated slow progressors. However, alterations in IFN-γ secretion in response to the mitogen PHA persisted, and their cytokine release after HIV-specific stimulation was biased towards a Th2 response. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

Age-related immune reconstitution during highly active antiretroviral therapy in human immunodeficiency virus type 1-infected children [2] (multiple letters)

SCOPUS: le.j

Observation of C60 film formation on a highly oriented pyrolitic graphite substrate via scanning tunnelling microscopy

We have investigated the early stages in the adsorption process of C60 molecules on a highly oriented pyrolitic graphite (HOPG) substrate. C60 powder was thermally evaporated in UHV of 10−8 Pa conditions onto a freshly cleaved HOPG surface. We did not observe individual fullerenes on the substrate for the case of short deposition times and low evaporation rates. However, small islands of C60 molecules with an fcc structure could be observed when the deposition rate was about 0.2 nm/min and the total thickness was above 1 nm. The islands did not grow in the vicinity of the HOPG steps. The typical lateral dimensions of these islands were of the order of a few hundred square nanometers, having thickness of up to five monolayers. We modified the shapes and positions of these islands by the STM tip, using a small (less than 1 V) bias voltage.

SOFT – generating highly flexible object code from XML specifications

Many code generation tools exist to aid developers in carrying out common mappings, such as from Object to XML or from Object to relational database. Such generated code tends to possess a high binding between the Object code and the target mapping, making integration into a broader application tedious or even impossible. In this paper we suggest XML technologies and the multiple inheritance capabilities of interface based languages such as Java, offer a means to unify such executable specifications, thus building complete, consistent and useful object models declaratively, without sacrificing component flexibility.

Inclusion of poorly soluble drugs in highly ordered mesoporous silica nanoparticles

Silica nanoparticles (MSNs) with a highly ordered mesoporous structures (103A) with cubic Im3 m have been synthesized using triblock copolymers with high poly(alkylene oxide) (EO) segments in acid media. The produced nanoparticles displayed large specific surface area (approximately 765 cm(2)/g) with an average particles size of 120 nm. The loading efficiency was assessed by incorporating three major antiepileptic active substances via passive loading and it was found to varying from 17 to 25%. The state of the adsorbed active agents was further analyzed using differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD). Dissolution studies revealed rapid release profiles within the first 3 h. The viability of 3T3 endothelial cells was not affected in the presence of MSNs indicating negligible cytotoxicity. 2009 Elsevier B.V. All rights reserved.

Aspects Of Microbial Heterotrophic Production In A Highly Turbid Estuary

The uptake of 14C glucose by natural microbial populations has been studied in the Severn Estuary and Bristol Channel, U.K.; the turbidity (suspended solids) in the estuary varied between < 5 mg · 1−1 at the seaward extremity to >800 mg · 1−1 in the estuary proper. The heterotrophic potential, Vm, was found to correlate with turbidity and particulate organic carbon but there was no correlation between microbial biomass, as assessed by plate counts, and turbidity or Vm; measurement of Vm ranged from 0.9 × 10−4 to 288 × 10−4μgC·1−1·h−1 and turnover time from <2 to >100 h. In 17 out of 42 experiments, the uptake of 14C glucose did not conform to Michaelis kinetics and in five of these experiments the data suggested that there may be a threshold of glucose concentration below which there is no uptake.

Filtration Unit For Use With Continuous Autoanalytical Systems Applied To Highly Turbid Waters

A simple unit for filtration prior to continuous autoanalysis of highly turbid waters is described. Seawater can be supplied at a rate of 10 ml min−1, after filtration through a 0.45 μm pore-sized membrane filter (47 mm diameter), for at least 45 min from sea water containing 1000 parts/106 of suspended solids.