Characterization of Phosphorylation- and RNA-Dependent UPF1 Interactors by Quantitative Proteomics

Human up-frameshift 1 (UPF1) is an ATP-dependent RNA helicase and phosphoprotein implicated in several biological processes but is best known for its key function in nonsense-mediated mRNA decay (NMD). Here we employed a combination of stable isotope labeling of amino acids in cell culture experiments to determine by quantitative proteomics UPF1 interactors. We used this approach to distinguish between RNA-mediated and protein-mediated UPF1 interactors and to determine proteins that preferentially bind the hypo- or the hyper-phosphorylated form of UPF1. Confirming and expanding previous studies, we identified the eukaryotic initiation factor 3 (eIF3) as a prominent protein-mediated interactor of UPF1. However, unlike previously reported, eIF3 binds to UPF1 independently of UPF1’s phosphorylation state. Furthermore, our data revealed many nucleus-associated RNA-binding proteins that preferentially associate with hyper-phosphorylated UPF1 in an RNase-sensitive manner, suggesting that UPF1 gets recruited to mRNA and becomes phosphorylated before being exported to the cytoplasm as part of the mRNP.

In human basophils, IL-3 selectively induces RANKL expression that is modulated by IgER-dependent and IgER-independent stimuli

BACKGROUND Receptor activator of NF-κB ligand (RANKL) is expressed as either surface (hRANKL1, hRANKL2) or soluble (hRANKL3) form. RANKL is involved in multifaceted processes of immunoregulation and bone resorption such as they occur in rheumatoid arthritis (RA). Interestingly, activated basophils, which are effector cells in allergic inflammation, contribute to the progress of collagen-induced arthritis (CIA), a mouse model for RA. Here, we investigate under which conditions human basophils express RANKL. METHODS Among other stimuli, basophils were cultured with IL-3 alone. Alternatively, as a secondary stimulus, IgER-dependent or IgER-independent agents were added simultaneously either with IL-3 or after prolonged IL-3 culturing. Expression of RANKL protein and mRNA was analyzed by flow cytometry, ELISA, and real-time PCR. A coculture system was applied to investigate biological activity of basophil-derived RANKL. RESULTS We show that in human basophils, IL-3 but no other stimulus induces de novo expression of soluble and surface RANKL, of which the latter enhances survival of MoDC. Upon simultaneous stimulation, IgER cross-linking reduces surface RANKL expression, while IgER-independent stimuli have no effect. This is in contrast to consecutive stimulation, as triggering with both IgER-dependent and IgER-independent stimuli enhances RANKL expression, particularly in its soluble form. Real-time PCR analysis shows that RANKL expression is mainly regulated at the mRNA level. CONCLUSION This study identifies IL-3 as a potent inducer of RANKL expression in human basophils, suggesting them to interact with bone physiology and activation of immune cells. IgER-dependent and IgER-independent stimuli modulate the IL-3-mediated RANKL expression in a time- and stimulus-dependent fashion.

Chlorophyll content and temperature-dependent net photosynthesis and respiration rate in the lichen Cetraria aculeata

We studied polar and temperate samples of the lichen Cetraria aculeata to investigate whether genetical differences between photobionts are correlated with physiological properties of the lichen holobiont. Net photosynthesis and dark respiration (DR) at different temperatures (from 0 to 30 °C) and photon flux densities (from 0 to 1,200 ?mol/m**2/s) were studied for four populations of Cetraria aculeata. Samples were collected from maritime Antarctica, Svalbard, Germany and Spain, representing different climatic situations. Sequencing of the photobiont showed that the investigated samples fall in the polar and temperate clade described in Fernández-Mendoza et al. (2011, doi:10.1111/j.1365-294X.2010.04993.x). Lichens with photobionts from these clades differ in their temperature optimum for photosynthesis, maximal net photosynthesis, maximal DR and chlorophyll content. Maximal net photosynthesis was much lower in Antarctica and Svalbard than in Germany and Spain. The difference was smaller when rates were expressed by chlorophyll content. The same is true for the temperature optima of polar (11 °C) and temperate (15 and 17 °C) lichens. Our results indicate that lichen mycobionts may adapt or acclimate to local environmental conditions either by selecting algae from regional pools or by regulating algal cell numbers (chlorophyll content) within the thallus.

Haemolymph inorganic ion composition, physiology and behaviour dependent on temperature and magnesium in adult Paralomis granulosa (Decapoda, Anomura, Lithodidae)

A low capacity for regulation of extracellular Mg2+ has been proposed to exclude reptant marine decapod crustaceans from temperatures below 0°C and thus to exclude them from the high Antarctic. To test this hypothesis and to elaborate the underlying mechanisms in the most cold-tolerant reptant decapod family of the sub-Antarctic, the Lithodidae, thermal tolerance was determined in the crab Paralomis granulosa (Decapoda, Anomura, Lithodidae) using an acute stepwise temperature protocol (-1°, 1°, 4°, 7°, 10°, and 13°C). Arterial and venous oxygen partial pressures (Po2) in hemolymph, heartbeat and ventilation beat frequencies, and hemolymph cation composition were measured at rest and after a forced activity (righting) trial. Scopes for heartbeat and ventilation beat frequencies and intermittent heartbeat and scaphognathite beat rates at rest were evaluated. Hemolymph [Mg2+] was experimentally reduced from 30 mmol/L to a level naturally observed in Antarctic caridean shrimps (12 mmol/L) to investigate whether the animals remain more active and tolerant to cold (-1°, 1°, and 4°C). In natural seawater, righting speed was significantly slower at -1° and 13°C, compared with acclimation temperature (4°C). Arterial and venous hemolymph Po2 increased in response to cooling even though heartbeat and ventilation beat frequencies as well as scopes decreased. At rest, ionic composition of the hemolymph was not affected by temperature. Activity induced a significant increase in hemolymph [K+] at -1° and 1°C. Reduction of hemolymph [Mg2+] did not result in an increase in activity, an increase in heartbeat and ventilation beat frequencies, or a shift in thermal tolerance to lower temperatures. In conclusion, oxygen delivery in this cold-water crustacean was not acutely limiting cold tolerance, and animals may have been constrained more by their functional capacity and motility. In contrast to earlier findings in temperate and subpolar brachyuran crabs, these constraints remained insensitive to changing Mg2+ levels.

Modal contribution and state space order selection in operational modal analysis

The estimation of modal parameters of a structure from ambient measurements has attracted the attention of many researchers in the last years. The procedure is now well established and the use of state space models, stochastic system identification methods and stabilization diagrams allows to identify the modes of the structure. In this paper the contribution of each identified mode to the measured vibration is discussed. This modal contribution is computed using the Kalman filter and it is an indicator of the importance of the modes. Also the variation of the modal contribution with the order of the model is studied. This analysis suggests selecting the order for the state space model as the order that includes the modes with higher contribution. The order obtained using this method is compared to those obtained using other well known methods, like Akaike criteria for time series or the singular values of the weighted projection matrix in the Stochastic Subspace Identification method. Finally, both simulated and measured vibration data are used to show the practicability of the derived technique. Finally, it is important to remark that the method can be used with any identification method working in the state space model.

Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human β-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked β-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human β-globin and the green fluorescent protein in 20–95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of β-locus control region hypersensitive site 2 alone, human β-globin mRNA expression levels ranged from 0.15% to 20% with human β-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.

The ATPase and protease domains of yeast mitochondrial Lon: Roles in proteolysis and respiration-dependent growth

The ATP-dependent Lon protease of Saccharomyces cerevisiae mitochondria is required for selective proteolysis in the matrix, maintenance of mitochondrial DNA, and respiration-dependent growth. Lon may also possess a chaperone-like function that facilitates protein degradation and protein-complex assembly. To understand the influence of Lon’s ATPase and protease activities on these functions, we examined several Lon mutants for their ability to complement defects of Lon-deleted yeast cells. We also developed a rapid procedure for purifying yeast Lon to homogeneity to study the enzyme’s activities and oligomeric state. A point mutation in either the ATPase or the protease site strongly inhibited the corresponding activity of the pure protein but did not alter the protein’s oligomerization; when expressed at normal low levels neither of these mutant enzymes supported respiration-dependent growth of Lon-deleted cells. When the ATPase- or the protease-containing regions of Lon were expressed as separate truncated proteins, neither could support respiration-dependent growth of Lon-deleted cells; however, coexpression of these two separated regions sustained wild-type growth. These results suggest that yeast Lon contains two catalytic domains that can interact with one another even as separate proteins, and that both are essential for the different functions of Lon.

Glucocorticoids have state-dependent stimulant effects on the mesencephalic dopaminergic transmission.

An increase in the activity of mesencephalic dopaminergic neurons has been implicated in the appearance of pathological behaviors such as psychosis and drug abuse. Several observations suggest that glucocorticoids might contribute to such an increase in dopaminergic activity. The present experiments therefore analyzed the effects of corticosterone, the major glucocorticoid in the rat, both on dopamine release in the nucleus accumbens of freely moving animals by means of microdialysis, and on locomotor activity, a behavior dependent on accumbens dopamine. Given that glucocorticoids have certain state-dependent neuronal effects, their action on dopamine was studied in situations differing in dopaminergic tonus, including during the light and dark phases of the circadian cycle, during eating, and in groups of animals differing in their locomotor reactivity to novelty. Dopaminergic activity is increased in the dark period, further increased during food-intake, and is higher in rats defined as high responders to novelty than in low responders. Corticosterone, peripherally administered in a dose that approximates stress-induced plasma concentrations, increased extracellular concentrations of dopamine, and this increase was augmented in the dark phase, during eating, and in high responder rats. Corticosterone had little or no effects in the light phase and in low responder rats. Corticosterone also stimulated locomotor activity, an effect that paralleled the release of dopamine and was abolished by neurochemical (6-hydroxydopamine) depletion of accumbens dopamine. In conclusion, glucocorticoids have state-dependent stimulant effects on mesencephalic dopaminergic transmission, and an interaction between these two factors might be involved in the appearance of behavioral disturbances.

Oral tolerance in myelin basic protein T-cell receptor transgenic mice: suppression of autoimmune encephalomyelitis and dose-dependent induction of regulatory cells.

Orally administered antigens induce a state of immunologic hyporesponsiveness termed oral tolerance. Different mechanisms are involved in mediating oral tolerance depending on the dose fed. Low doses of antigen generate cytokine-secreting regulatory cells, whereas high doses induce anergy or deletion. We used mice transgenic for a T-cell receptor (TCR) derived from an encephalitogenic T-cell clone specific for the acetylated N-terminal peptide of myelin basic protein (MBP) Ac-1-11 plus I-Au to test whether a regulatory T cell could be generated from the same precursor cell as that of an encephalitogenic Th1 cell and whether the induction was dose dependent. The MBP TCR transgenic mice primarily have T cells of a precursor phenotype that produce interleukin 2 (IL-2) with little interferon gamma (IFN-gamma), IL-4, or transforming growth factor beta (TGF-beta). We fed transgenic animals a low-dose (1 mg x 5) or high-dose (25 mg x 1) regimen of mouse MBP and without further immunization spleen cells were tested for cytokine production. Low-dose feeding induced prominent secretion of IL-4, IL-10, and TGF-beta, whereas minimal secretion of these cytokines was observed with high-dose feeding. Little or no change was seen in proliferation or IL-2/IFN-gamma secretion in fed animals irrespective of the dose. To demonstrate in vivo functional activity of the cytokine-secreting cells generated by oral antigen, spleen cells from low-dose-fed animals were adoptively transferred into naive (PLJ x SJL)F1 mice that were then immunized for the development of experimental autoimmune encephalomyelitis (EAE). Marked suppression of EAE was observed when T cells were transferred from MBP-fed transgenic animals but not from animals that were not fed. In contrast to oral tolerization, s.c. immunization of transgenic animals with MBP in complete Freund's adjuvant induced IFN-gamma-secreting Th1 cells in vitro and experimental encephalomyelitis in vivo. Despite the large number of cells reactive to MBP in the transgenic animals, EAE was also suppressed by low-dose feeding of MBP prior to immunization. These results demonstrate that MBP-specific T cells can differentiate in vivo into encephalitogenic or regulatory T cells depending upon the context by which they are exposed to antigen.

Time and cost of vocational rehabilitation, comparing social security trust fund rehabilitations with all other rehabilitated clients /

"July 1, 1973."

Workmen's compensation law and Industrial board rules, with amendments, additions and annotations to March 1, 1936. Issued under the direction of Elmer F. Andrews, industrial commissioner.

Includes index.

Guide book of the Pacific : containing the time and distance tables, rates, fares and freight tariff, of all steamship, steamboat, railway, stage and express lines, on or connecting with the Pacific coast and the interior ...

At head of title: State, territorial and ocean.

The Mestizo State: Violence and State Formation in Peru (1980 – 2000)

Thesis (Master's)--University of Washington, 2016-07

Bottom-up risk regulation? How nanotechnology risk knowledge gaps challenge federal and state environmental agencies

Nanotechnologies have been called the "Next Industrial Revolution." At the same time, scientists are raising concerns about the potential health and environmental risks related to the nano-sized materials used in nanotechnologies. Analyses suggest that current U.S. federal regulatory structures are not likely to adequately address these risks in a proactive manner. Given these trends, the premise of this paper is that state and local-level agencies will likely deal with many "end-of-pipe" issues as nanomaterials enter environmental media without prior toxicity testing, federal standards, or emissions controls. In this paper we (1) briefly describe potential environmental risks and benefits related to emerging nanotechnologies; (2) outline the capacities of the Toxic Substances Control Act, the Clean Air Act, the Clean Water Act, and the Resources Conservation and Recovery Act to address potential nanotechnology risks, and how risk data gaps challenge these regulations; (3) outline some of the key data gaps that challenge state-level regulatory capacities to address nanotechnologies' potential risks, using Wisconsin as a case study; and (4) discuss advantages and disadvantages of state versus federal approaches to nanotechnology risk regulation. In summary, we suggest some ways government agencies can be better prepared to address nanotechnology risk knowledge gaps and risk management.

State-dependent microstructural white matter changes in bipolar I depression

Abnormalities in fronto-limbic-striatal white matter (WM) have been reported in bipolar disorder (BD), but results have been inconsistent across studies. Furthermore, there have been no detailed investigations as to whether acute mood states contribute to microstructural changes in WM tracts. In order to compare fiber density and structural integrity within WM tracts between BD depression and remission, whole-brain fractional anisotropy (FA) and mean diffusivity (MD) were assessed in 37 bipolar I disorder (BD-I) patients (16 depressed and 21 remitted), and 26 healthy individuals with diffusion tensor imaging. Significantly decreased FA and increased MD in bilateral prefronto-limbic-striatal white matter and right inferior fronto-occipital, superior and inferior longitudinal fasciculi were shown in all BD-I patients versus controls, as well as in depressed BD-I patients compared to both controls and remitted BD-I patients. Depressed BD-I patients also exhibited increased FA in the ventromedial prefrontal cortex. Remitted BD-I patients did not differ from controls in FA or MD. These findings suggest that BD-I depression may be associated with acute microstructural WM changes.