RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity

Regulator of G-protein signaling (RGS) proteins are GTPase activating proteins (GAPs) of heterotrimeric G-proteins that alter the amplitude and kinetics of receptor-promoted signaling. In this study we defined the G-protein alpha-subunit selectivity of purified Sf9 cell-derived R7 proteins, a subfamily of RGS proteins (RGS6, -7, -9, and -11) containing a Ggamma-like (GGL) domain that mediates dimeric interaction with Gbeta(5). Gbeta(5)/R7 dimers stimulated steady state GTPase activity of Galpha-subunits of the G(i) family, but not of Galpha(q) or Galpha(11), when added to proteoliposomes containing M2 or M1 muscarinic receptor-coupled G-protein heterotrimers. Concentration effect curves of the Gbeta(5)/R7 proteins revealed differences in potencies and efficacies toward Galpha-subunits of the G(i) family. Although all four Gbeta(5)/R7 proteins exhibited similar potencies toward Galpha(o), Gbeta(5)/RGS9 and Gbeta(5)/RGS11 were more potent GAPs of Galpha(i1), Galpha(i2), and Galpha(i3) than were Gbeta(5)/RGS6 and Gbeta(5)/RGS7. The maximal GAP activity exhibited by Gbeta(5)/RGS11 was 2- to 4-fold higher than that of Gbeta(5)/RGS7 and Gbeta(5)/RGS9, with Gbeta(5)/RGS6 exhibiting an intermediate maximal GAP activity. Moreover, the less efficacious Gbeta(5)/RGS7 and Gbeta(5)/RGS9 inhibited Gbeta(5)/RGS11-stimulated GTPase activity of Galpha(o). Therefore, R7 family RGS proteins are G(i) family-selective GAPs with potentially important differences in activities.     

Dynamic Modeling of In‐Use Cement Stocks in the United States

A dynamic substance‐flow model is developed to characterize the stocks and flows of cement utilized during the 20th century in the United States, using the generic cement life cycle as a systems boundary. The motivation for estimating historical inventories of cement stocks and flows is to provide accurate estimates of contemporary cement in‐use stocks in U.S. infrastructure and future discards to relevant stakeholders in U.S. infrastructure, such as the federal and state highway administrators, departments of transportation, public and private utilities, and the construction and cement industries. Such information will assist in planning future rehabilitation projects and better life cycle management of infrastructure systems. In the present policy environment of climate negotiations, estimates of in‐use cement infrastructure can provide insights about to what extent built environment can act as a carbon sink over its lifetime. The rate of addition of new stock, its composition, and the repair of existing stock are key determinants of infrastructure sustainability. Based upon a probability of failure approach, a dynamic stock and flow model was developed utilizing three statistical lifetime distributions—Weibull, gamma, and lognormal—for each cement end‐use. The model‐derived estimate of the “in‐use” cement stocks in the United States is in the range of 4.2 to 4.4 billion metric tons (gigatonnes, Gt). This indicates that 82% to 87% of cement utilized during the last century is still in use. On a per capita basis, this is equivalent to 14.3 to 15.0 tonnes of in‐use cement stock per person. The in‐use cement stock per capita has doubled over the last 50 years, although the rate of growth has slowed.     

Contrasting effects of low-dose IL-2 on vaccine-boosted simian immunodeficiency virus (SIV)-specific CD4+ and CD8+ T cells in macaques chronically infected with SIVmac251

IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1 infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we investigated how continuous low-dose IL-2 affected the CD4+ and CD8+ T cell response induced by two inoculations of a canarypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had normal levels of CD4+ T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly augmented Gag-specific CD8+ T cell responses, but actually reduced Gag-specific CD4+ T cell responses. Although IL-2 at low doses did not change the overall concentration of circulating CD4+ or CD8+ T cells, it expanded the frequency of CD4+CD25+ T cells. Depletion of the CD4+CD25+ T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4+ responses or augmentation of SIV-specific CD8+ T cell responses. Thus, we conclude that the decrease in virus-specific CD4+ T cell response may be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T regulatory population.     

Microarray analysis of bacterial gene expression: towards the regulome

Microarray technology allows co-regulated genes to be identified. In order to identify genes that are controlled by specific regulators, gene expression can be compared in mutant and wild-type bacteria. However, there are a number of pitfalls with this approach; in particular, the regulator may not be active under the conditions in which the wild-type strain is cultured. Once co-regulated genes have been identified, proteinbinding motifs can be identified. By combining these data with a map of promoters, or operons (the operome), the regulatory networks in the cell (the regulome) can start to be built up.     

Location of two antioxidants in oriented model membranes. Small-angle x-ray diffraction study.

Small-angle x-ray diffraction has been applied in locating either butylated hydroxytoluene (BHT) or delta-tocopherol and their brominated analogues at a concentration of 40 mol% in oriented bilayers of dipalmitoylphosphatidylcholine (DPPC) or DPPC + 15 mol% cholesterol at 20 degrees C. Phases were determined using swelling experiments with structure factors plotted in reciprocal space, creating a relatively smooth curve as the amount of water between the bilayers was changed. Continuous Fourier transforms were also calculated using sampling theory (Shannon, C. E. 1949. Proc. Inst. Radio Engrs. NY. 37:10-21) to further test the consistency of the phase assignments. Fourier synthesis of structure factors resulted in absolute electron density profiles for different bilayers to a resolution of 5-6 A. In addition, difference Patterson maps were constructed to confirm the positions of the bromine atoms in the unit cell. Analysis of the data indicates the following: (a) The BHT molecules are dispersed throughout the alkyl-chain region in DPPC samples with and without cholesterol. (b) The chromanol ring of delta-tocopherol is in the vicinity of the glycerol backbone-headgroup region in samples of DPPC or DPPC + 15 mol% cholesterol. (c) Difference Patterson maps confirm the localization of bromine atoms in the various delta-tocopherol samples and lack of bromine localization in the various BHT samples.     

Ablation of rat TRPV1-expressing Adelta/C-fibers with resiniferatoxin: analysis of withdrawal behaviors, recovery of function and molecular correlates

Microglia are the resident macrophages in the central nervous system. In the spinal cord dorsal horn, microglia stay in resting condition during physiological sensory processing, and are activated under pathological conditions such as peripheral nerve injury. In cases such as this, the nearby resting microglia increase their motility and accumulate at the site of injury. However, direct evidence to support that nerve activity can enhance the motility of microglia has not yet to be reported. In this study we investigated whether the activation of spinal microglia under in vivo nerve injury may be mimicked by neuronal activity in the spinal cord slice preparation. We found that local application of spinal excitatory neurotransmitters, such as glutamate and substance P did not cause any change in the motility of microglial cells in the spinal cord dorsal horn. The motility of microglial cells is unlikely modulated by other transmitters, neuromodulators and chemokines, because similar applications such as GABA, serotonin, noradrenaline, carbachol, fractalkine or interleukin did not produce any obvious effect. Furthermore, low or high frequency stimulation of spinal dorsal root fibers at noxious intensities failed to cause any enhanced extension or retraction of the microglia processes. By contrast, focal application of ATP triggered rapid and robust activation of microglial cells in the spinal dorsal horn. Our results provide the first evidence that the activation of microglia in the spinal cord after nerve injury is unlikely due solely to neuronal activity, non-neuronal factors are likely responsible for the activation of nerve injury-related microglial cells in the spinal dorsal horn.     

Small Molecule Inhibitors of Phospholipase C from a Novel High-throughput Screen

Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.