RGS expression rate-limits recovery of rod photoresponses

Signaling through G protein-coupled receptors (GPCRs) underlies many cellular processes, yet it is not known which molecules determine the duration of signaling in intact cells. Two candidates are G protein-coupled receptor kinases (GRKs) and Regulators of G protein signaling (RGSs), deactivation enzymes for GPCRs and G proteins, respectively. Here we investigate whether GRK or RGS governs the overall rate of recovery of the light response in mammalian rod photoreceptors, a model system for studying GPCR signaling. We show that overexpression of rhodopsin kinase (GRK1) increases phosphorylation of the GPCR rhodopsin but has no effect on photoresponse recovery. In contrast, overexpression of the photoreceptor RGS complex (RGS9-1.Gbeta5L.R9AP) dramatically accelerates response recovery. Our results show that G protein deactivation is normally at least 2.5 times slower than rhodopsin deactivation, resolving a long-standing controversy concerning the mechanism underlying the recovery of rod visual transduction.     

Simvastatin causes endothelial cell apoptosis and attenuates severe pulmonary hypertension

Severe pulmonary hypertension (SPH) is characterized by precapillary arteriolar lumen obliteration, dramatic right ventricular hypertrophy, and pericardial effusion. Our recently published rat model of SPH recapitulates major components of the human disease. We used this model to develop new treatment strategies for SPH. SPH in rats was induced using VEGF receptor blockade in combination with chronic hypoxia. A large variety of drugs used in this study, including anticancer drugs (cyclophosphamide and paclitaxel), the angiotensin-converting enzyme inhibitor lisinopril, the antiangiogenic agent thalidomide, and the peroxisome proliferator-actived receptor-gamma agonist PGJ2, failed to decrease mean pulmonary artery pressure (PAP) or right ventricular hypertrophy. In contrast, treatment of rats with established SPH with simvastatin markedly reduced mean PAP and right ventricular hypertrophy, and this reduction was associated with caspase-3 activation and pulmonary microvascular endothelial cell apoptosis. Simvastatin partially restored caveolin-1, caveolin-2, and phospho-caveolin expression in vessel walls. In rat primary pulmonary microvascular endothelial cells, simvastatin induced caspase 3 activation and Rac 1 expression while suppressing Rho A and attenuated levels of Akt and ERK phosphorylation. We conclude that simvastatin is effective in inducing apoptosis in hyperproliferative pulmonary vascular lesions and could be considered as a potential drug for treatment of human SPH.     

Evaluation of agrobacterium-mediated transformation of agricus bisporus using a range of promoters linked to hygromycin resistance

There is interest in establishing genetic modification technologies for the cultivated mushroom Agaricus bisporus, both for improved crop characteristics and for molecular pharming. For these methods to be successful, it is necessary to establish a set of transformation systems that include robust and reliable vectors for gene manipulation. In this article, we report the evaluation of a series of promoters for driving expression of the Escherichia coli hph gene encoding hygromycin phosphotransferase. This was achieved using the Aspergillus nidulans gpdA and the A. bisporus gpdII and trip2 promoters. The Coprinus cinereus β-tubulin promoter gave contrasting results depending on the size of promoter used, with a 393-bp region being effective, whereas the longer 453-bp fragment failed to yield any hygromycin-resistant transformants. The C. cinereus trp 1 and the A. bisporus lcc1 promoters both failed to yield transformants. We also show that transformation efficiency may be improved by careful selection of both appropriate Agrobacterium strains, with ALG-1 yielding more than LBA1126 and by the choice of the binary vectors used to mobilize the DNA, with pCAMBIA vectors appearing to be more efficient than either pBIN19- or pGREEN-based systems.     

An ultraefficient affinity-based high-throughout screening process: application to bacterial cell wall biosynthesis enzyme MurF

The authors describe the discovery of a new class of inhibitors to an essential Streptococcus pneumoniae cell wall biosyn-thesis enzyme, MurF, by a novel affinity screening method. The strategy involved screening very large mixtures of diverse small organic molecules against the protein target on the basis of equilibrium binding, followed by iterative ultrafiltration steps and ligand identification by mass spectrometry. Hits from any affinity-based screening method often can be relatively nonselective ligands, sometimes referred to as "nuisance" or "promiscuous" compounds. Ligands selective in their binding affinity for the MurF target were readily identified through electronic subtraction of an empirically determined subset of promiscuous compounds in the library without subsequent selectivity panels. The complete strategy for discovery and identification of novel specific ligands can be applied to all soluble protein targets and a wide variety of ligand libraries.     

Identification of small-molecule inhibitors of protein kinase B (PKB/AKT) in an AlphaScreenTM high-throughput screen

Protein kinase B (PKB/AKT) has been identified as a promising cancer drug target downstream of PI3 kinase. To find novel inhibitors of PKB/AKT kinase activity for progression as anticancer agents, the authors have used a high-throughput screen based on AlphaScreentrade mark technology. A known kinase inhibitor, the isoquinoline H8, was used as a positive control with mean inhibition in the screen of 43.4% +/- 13.1%. The performance of the screen was highly acceptable with Z' and Z factors of 0.83 +/- 0.07 and 0.75 +/- 0.04, respectively. A number of confirmed hits ( approximately 0.1% hit rate) were identified from 63,500 compounds screened. Five compounds have previously been described as PKB inhibitors, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. Five hits had the potential to interfere with the assay signal and were deemed to be false positives. Two compounds were nonspecific inhibitors of PKB as enzyme inhibition in a filter-based assay was markedly reduced in the presence of 0.01% Triton X100. The authors now include an interference assay during hit confirmation procedures and check compound activity in the presence of Triton X100 in an attempt to eliminate nonspecific aggregators at an early stage.     

The effect of simulated censored data on estimates of heritability of longevity in the Thoroughbred racing industry

We examined the impact of censored data on estimates of heritability of longevity. Longevity, defined as the length of productive racing life of an individual, is influenced by many factors. A simulated data set, modelled on the Irish Thoroughbred industry, was used to estimate heritabilities of longevity. Several scenarios representing various levels of censoring of performance data were created. The heritability of longevity was estimated for each scenario and compared to the estimated heritability of 0.120 for the complete data set. It was found that the estimates of heritability (0.107, 0.106, 0.082) were biased downwards with (10, 20, and 25%, respectively) censoring of data from poor-performing animals. We found that for a complete reporting it is necessary to reduce bias in the estimation of heritability of longevity.     

Diabetes mellitus: a potential target for stem cell therapy

Type 1 diabetes mellitus has received much attention recently as a potential target for the emerging science of stem cell medicine. In this autoimmune disease, the insulin-secreting beta-cells of the pancreas are selectively and irreversibly destroyed by autoimmune assault. Advances in islet transplantation procedures now mean that patients with the disease can be cured by transplantation of primary human islets of Langerhans. A major drawback in this therapy is the availability of donor islets, and the search for substitute transplant tissues has intensified in the last few years. This review will describe the essential requirements of a material designed as a replacement beta-cell and will look at the potential sources of such replacements. These include embryonic stem (ES) cells and multipotent adult stem/progenitor cells from a range of tissues including the pancreas, intestine, liver, bone marrow and brain. These stem cell populations will be evaluated and the different experimental approaches that have been employed to derive functional insulin-expressing cells will be discussed. The review will also look at the capability of human ES (hES) cells generated by somatic cell nuclear transfer and some adult stem cell populations such as bone marrow-derived stem cells, to offer autologous transplant material that would remove the need for immunosuppression. In patients with Type 1 diabetes, auto-reactive T-cells are programmed to recognise the insulin-producing beta-cells. As a result, for therapeutic replacement tissues, it may be more sensible to derive cells that behave like beta-cells but are immunologically distinct. Thus, the potential of cells derived from non-beta-cell origin to avoid the autoimmune response will also be discussed. Finally, the review will summarise the future prospects for stem cell therapies for diabetes and will highlight some of the problems that may be faced by researchers working in this area, such as malignancy, irreproducible differentiation strategies, immune-system rejection and social and ethical concerns over the use of hES cells.     

WIP regulates the stability and localization of WASP to podosomes in migrating dendritic cells

The Wiskott-Aldrich Syndrome protein (WASP) is an adaptor protein that is essential for podosome formation in hematopoietic cells. Given that 80% of identified Wiskott-Aldrich Syndrome patients result from mutations in the binding site for WASP-interacting-protein (WIP), we examined the possible role of WIP in the regulation of podosome architecture and cell motility in dendritic cells (DCs). Our results show that WIP is essential both for the formation of actin cores containing WASP and cortactin and for the organization of integrin and integrin-associated proteins in circular arrays, specific characteristics of podosome structure. We also found that WIP is essential for the maintenance of the high turnover of adhesions and polarity in DCs. WIP exerts these functions by regulating calpain-mediated cleavage of WASP and by facilitating the localization of WASP to sites of actin polymerization at podosomes. Taken together, our results indicate that WIP is critical for the regulation of both the stability and localization of WASP in migrating DCs and suggest that WASP and WIP operate as a functional unit to control DC motility in response to changes in the extracellular environment.     

Methylation-dependent fragment separation: direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA

Fundamental to understanding the role of cytosine (C) methylation in genomic DNA (gDNA) is the need for robust analysis methods to determine the location and degree of this modification. We report a novel method for methylation detection by denaturing capillary electrophoresis (CE) using standard fragment analysis conditions. Bisulfite treatment of gDNA will selectively deaminate C but not 5-methylcytosine (5mC). Amplicons generated from bisulfite-converted gDNA are analyzed immediately after PCR using a 6-carboxy fluorescein (6-FAM) dye-labeled primer. The amplicons from methylated and unmethylated gDNA separate based solely on base composition due to the presence of multiple C versus thymine (T) differences. By direct detection of PCR amplicons following PCR using primers that anneal independent of methylation status, the overall workflow from gDNA sample input to data analysis is relatively simple. Furthermore, the same PCR product is suitable for additional analyses such as direct sequencing, cloning and sequencing, single-base extension, and post-PCR incorporation of a modified dCTP, the latter of which allows resolution of amplicons with as little as a single C/T difference. We show the utility of this novel CE detection assay by analyzing the hypermethylated region of the fragile-X FMR1 locus.     

Gbetagamma that interacts with adenylyl cyclase in opioid tolerance originates from a Gs protein

We previously demonstrated that chronic morphine induces a change in G protein coupling by the mu opioid receptor (MOR) from Gi/o to Gs, concurrent with the instatement of an interaction between Gbetagamma and adenylyl cyclase types II and IV. These two signaling changes confer excitatory effects on the cell in place of the typical inhibition by opioids and are associated with morphine tolerance and dependence. Both signaling changes and these behavioral manifestations of chronic morphine are attenuated by cotreatment with ultra-low-dose naloxone. In the present work, using striatum from chronic morphine-treated rats, we isotyped the Gbeta within Gs and Go heterotrimers that coupled to MOR and compared these to the Gbeta isotype of the Gbetagamma that interacted with adenylyl cyclase II or IV after chronic morphine treatment. Isotyping results show that chronic morphine causes a Gs heterotrimer associated with MOR to release its Gbetagamma to interact with adenylyl cyclase. These data suggest that the switch to Gs coupling by MOR in response to chronic morphine, which is attenuated by ultra-low-dose opioid antagonist cotreatment, leads to a two-pronged stimulation of adenylyl cyclase utilizing both Galpha and Gbetagamma subunits of the Gs protein novel to this receptor.