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Insulin-stimulated translocation of the glucose transporter GLUT4 to the cell surface in fat and muscle cells is the basis for insulin-stimulated glucose transport. Studies in adipocytes strongly support the following molecular mechanism for this process. Insulin-elicited phosphorylation of the GTPase-activating protein TBC1D4 (AS160) suppresses its activity toward Rab10 and thereby leads to an increase in the GTP-bound form of Rab10, which in turn triggers movement of vesicles containing GLUT4 to the plasma membrane and their fusion with the membrane. This process is expected to require the participation of a guanine nucleotide exchange factor (GEF) to generate the GTP-bound form of Rab10, but this GEF has not hitherto been identified. The present study identifies Dennd4C, a recently described GEF for Rab10, as the primary GEF required for GLUT4 translocation. Knockdown of Dennd4C markedly inhibited GLUT4 translocation, and ectopic expression of Dennd4C slightly stimulated it. Dennd4C was found in isolated GLUT4 vesicles. This study thus identifies another key component in the machinery of GLUT4 translocation. Moreover, it provides a potential explanation for the moderate association of a variant in the Dennd4C gene with type 2 diabetes.

Protein phosphorylation is a reversible post-translational modification known to regulate protein function, subcellular localization, complex formation, and protein degradation. Detailed phosphoproteomic information is critical to kinomic studies of signal transduction and for elucidation of cancer biomarkers, such as in non-small-cell lung adenocarcinoma, where phosphorylation is commonly dysregulated. However, the collection and analysis of phosphorylation data remains a difficult problem. The low concentrations of phosphopeptides in complex biological mixtures as well as challenges inherent in their chemical nature have limited phosphoproteomic characterization and some phosphorylation sites are inaccessible by traditional workflows. We developed a sequential digestion method using complementary proteases, Glu-C and trypsin, to increase phosphoproteomic coverage and supplement traditional approaches. The sequential digestion method is more productive than workflows utilizing only Glu-C and we evaluated the orthogonality of the sequential digestion method relative to replicate trypsin-based analyses. Finally, we demonstrate the ability of the sequential digestion method to access new regions of the phosphoproteome by comparison to existing public phosphoproteomic databases. Our approach increases coverage of the human lung cancer phosphoproteome by accessing both new phosphoproteins and novel phosphorylation site information.

Was the United States founded to cultivate virtue or to protect rights? Republican interpretations argue the first, liberal ones the second, and the former has largely displaced the latter as the leading interpretation of the American Revolution. This article revisits the republican-liberal debate by addressing the revisionists' conspicuous neglect of the Declaration of Independence. The author concludes that the essential political premise of the American Revolution is that government exists to secure natural rights, not to cultivate virtue.

Background Widely accessible small animal models suitable for the study of hepatitis C virus (HCV) in vivo are lacking, primarily because rodent hepatocytes cannot be productively infected and because human hepatocytes are not easily engrafted in immunodeficient mice. Methodology/Principal Findings We report here on a novel approach for human hepatocyte engraftment that involves subcutaneous implantation of primary human fetal hepatoblasts (HFH) within a vascularized rat collagen type I/human fibronectin (rCI/hFN) gel containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC) in severe combined immunodeficient X beige (SCID/bg) mice. Maturing hepatic epithelial cells in HFH/Bcl-2-HUVEC co-implants displayed endocytotic activity at the basolateral surface, canalicular microvilli and apical tight junctions between adjacent cells assessed by transmission electron microscopy. Some primary HFH, but not Huh-7.5 hepatoma cells, appeared to differentiate towards a cholangiocyte lineage within the gels, based on histological appearance and cytokeratin 7 (CK7) mRNA and protein expression. Levels of human albumin and hepatic nuclear factor 4α (HNF4α) mRNA expression in gel implants and plasma human albumin levels in mice engrafted with HFH and Bcl-2-HUVEC were somewhat enhanced by including murine liver-like basement membrane (mLBM) components and/or hepatocyte growth factor (HGF)-HUVEC within the gel matrix. Following ex vivo viral adsorption, both HFH/Bcl-2-HUVEC and Huh-7.5/Bcl-2-HUVEC co-implants sustained HCV Jc1 infection for at least 2 weeks in vivo, based on qRT-PCR and immunoelectron microscopic (IEM) analyses of gel tissue. Conclusion/Significance The system described here thus provides the basis for a simple and robust small animal model of HFH engraftment that is applicable to the study of HCV infections in vivo.

The signal transduction pathway leading from the insulin receptor to stimulate the fusion of vesicles containing the glucose transporter GLUT4 with the plasma membrane in adipocytes and muscle cells is not completely understood. Current evidence suggests that in addition to the Rab GTPase-activating protein AS160, at least one other substrate of Akt (also called protein kinase B), which is as yet unidentified, is required. Sec8 is a component of the exocyst complex that has been previously implicated in GLUT4 trafficking. In the present study, we report that insulin stimulates the phosphorylation of Sec8 on Ser-32 in 3T3-L1 adipocytes. On the basis of the sequence around Ser-32 and the finding that phosphorylation is inhibited by the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, it is likely that Akt is the kinase for Ser-32. We examined the possible role of Ser-32 phosphorylation in the insulin-stimulated trafficking of GLUT4, as well as the TfR (transferrin receptor), to the plasma membrane by determining the effects of overexpression of the non-phosphorylatable S32A mutant of Sec8 and the phosphomimetic S32E mutant of Sec8. Substantial overexpression of both mutants had no effect on the amount of GLUT4 or TfR at the cell surface in either the untreated or insulin-treated states. These results indicate that insulin-stimulated phosphorylation of Sec8 is not part of the mechanism by which insulin enhances the fusion of vesicles with the plasma membrane.

Background: Cell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process. Methodology/Principal Findings: Here, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology. Conclusions/Significance: We conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization.

The omentum, an important peritoneal tissue, is studded with a high number of immune aggregates, or “milky spots,” the number, function, and phenotype of which is largely unknown. We have analyzed the immune composition on the normal omentum and also have shown that both free immune cells and tumor cells in the peritoneal fluid bind preferentially to these immune aggregates. This binding may be mediated by the network of collagen I fibers, which overlay these areas. In addition, we have shown that not only do omental vessels express vascular endothelial growth factor receptor 3 (VEGFR3), a receptor that is only found on angiogenic blood vessels, but that tumor cells co-localize with these vessels, possibly increasing the ability of tumor to induce neovascularization and therefore thrive.

Immunodeficient mice bearing components of a human immune system present a novel approach for studying human immune responses. We investigated the number, phenotype, developmental kinetics, and function of developing human immune cells following transfer of CD34(+) hematopoietic stem cell (HSC) preparations originating from second trimester human fetal liver (HFL), umbilical cord blood (UCB), or granulocyte colony-stimulating factor-mobilized adult blood (G-CSF-AB) delivered via intrahepatic injection into sublethally irradiated neonatal NOD-scid/gammac(-/-), Balb/c-Rag1(-/-)gammac(-/-), and C.B-17-scid/bg mice. HFL and UCB HSC provided the greatest number and breadth of developing cells. NOD-scid/gammac(-/-) and Balb/c-Rag1(-/-)gammac(-/-) harbored human B and dendritic cells as well as human platelets in peripheral blood, whereas NOD-scid/gammac(-/-) mice harbored higher levels of human T cells. NOD-scid/gammac(-/-) mice engrafted with HFL CD34(+) HSC demonstrated human immunological competence evidenced by white pulp expansion and increases in total human immunoglobulin following immunization with T-dependent antigens and delayed-type hypersensitivity-infiltrating leukocytes in response to antigenic challenge. In conclusion, we describe an encouraging base system for studying human hematopoietic lineage development and function utilizing human HFL or UCB HSC-engrafted NOD-scid/gammac(-/-) mice that is well suited for future studies toward the development of a fully competent humanized mouse model.

Recently, mass spectrometry has been employed in many studies to provide unbiased, reproducible, and quantitative protein abundance information on a proteome-wide scale. However, how instruments' limited dynamic ranges impact the accuracy of such measurements has remained largely unexplored, especially in the context of complex mixtures. Here, we examined the distribution of peptide signal versus background noise (S/N) and its correlation with quantitative accuracy. With the use of metabolically labeled Jurkat cell lysate, over half of all confidently identified peptides had S/N ratios less than 10 when examined using both hybrid linear ion trap-Fourier transform ion cyclotron resonance and Orbitrap mass spectrometers. Quantification accuracy was also highly correlated with S/N. We developed a mass precision algorithm that significantly reduced measurement variance at low S/N beyond the use of highly accurate mass information alone and expanded it into a new software suite, Vista. We also evaluated the interplay between mass measurement accuracy and S/N; finding a balance between both parameters produced the greatest identification and quantification rates. Finally, we demonstrate that S/N can be a useful surrogate for relative abundance ratios when only a single species is detected.

Checkpoint with forkhead-associated and RING (Chfr) is a ubiquitin ligase (E3) that establishes an antephase or prometaphase checkpoint in response to mitotic stress. Though ubiquitination is essential for checkpoint function, the sites, linkages and ubiquitin conjugating enzyme (E2) specificity are controversial. Here we dissect the function of the two Chfr homologs in S. cerevisiae, Chf1 and Chf2, overexpression of which retard cell cycle at both G(1) and G(2). Using a genetic assay, we establish that Ubc4 is required for Chf2-dependent G(1) cell cycle delay and Chf protein turnover. In contrast, Ubc13/Mms2 is required for G(2) delay and does not contribute to Chf protein turnover. By reconstituting cis and trans-ubiquitination activities of Chf proteins in purified systems and characterizing sites modified and linkages formed by tandem mass spectrometry, we discovered that Ubc13/Mms2- dependent modifications are a distinct subset of those catalyzed by Ubc4. Mutagenesis of Lys residues identified in vitro indicates that site-specific Ubc4-dependent Chf protein autoubiquitination is responsible for Chf protein turnover. Thus, combined genetic and biochemical analyses indicate that Chf proteins have dual E2 specificity accounting for different functions in the cell cycle.

Necroptosis is a cellular mechanism of necrotic cell death induced by apoptotic stimuli in the form of death domain receptor engagement by their respective ligands under conditions where apoptotic execution is prevented. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphological features as unregulated necrotic death. Here we report that necrostatin-1, a previously identified small-molecule inhibitor of necroptosis, is a selective allosteric inhibitor of the death domain receptor-associated adaptor kinase RIP1 in vitro. We show that RIP1 is the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. In addition, we show that two other necrostatins, necrostatin-3 and necrostatin-5, also target the RIP1 kinase step in the necroptosis pathway, but through mechanisms distinct from that of necrostatin-1. Overall, our data establish necrostatins as the first-in-class inhibitors of RIP1 kinase, the key upstream kinase involved in the activation of necroptosis.

The absolute quantification (AQUA) strategy provides a means to determine the precise protein or modified protein levels directly from cells or tissues. The technique is based on two major principles: stable isotope dilution theory and the use of synthetic peptides containing such stable isotopes to exactly mimic native counterparts after proteolysis. These peptides can be synthesized with modifications such as phosphorylation. methylation, and acetylation to allow for the direct, quantitative analysis of posttranslationally modified proteins. In this chapter, we discuss the development of an AQUA method and demonstrate its usefulness in the measurement of endogenous levels of the human protein separase at a functionally relevant phosphorylation site, serine 1126.

International audience; Assembly of heterochromatin at centromeric DNA regions in the fission yeast Schizosaccharomyces pombe involves an intimate interplay between chromatin modifying complexes and components of the RNAi pathway. The RNA-induced transcriptional silencing (RITS) complex, containing Chp1, Ago1, Tas3, and centromeric siRNAs, localizes to centromeric DNA repeats and is required for the assembly and maintenance of heterochromatin. RITS brings together two types of molecular recognition modules: a chromodomain protein, which binds to lysine 9 methylated histone H3 (H3K9), and Argonaute, which binds to specific sequences by siRNA-directed base-pairing interactions. The RNA-directed RNA polymerase complex (RDRC), composed of Rdp1, the Hrr1 helicase, and the Cid12 Poly(A) polymerase family member, synthesizes double-stranded RNA and creates the substrate for Dicer to generate siRNAs. RDRC physically associates with RITS, and both complexes localize to noncoding centromeric RNAs and centromeric DNA repeats, suggesting that recognition of nascent RNA transcripts may be involved in localization of these complexes to specific chromosome regions. In support of this possibility, tethering of the RITS complex to the transcript of the normally euchromatic ura4 (+) gene results in siRNA generation and RNAi- and heterochromatin-dependent silencing of the ura4 (+) gene. Finally, silencing of a subset of endogenous and transgene promoters within heterochromatic DNA domains occurs by RNAi-dependent degradation of nascent transcripts by a mechanism that we have termed co-transcriptional gene silencing (CTGS).

Protein phosphorylation is essential for numerous cellular processes. Large-scale profiling of phosphoproteins continues to enhance the depth and speed at which we understand these processes. The development of effective phosphoprotein and peptide enrichment techniques and improvements to mass spectrometric instrumentation have intensified phosphoproteomic research in recent years, leading to unprecedented achievements. Here, we describe a large-scale phosphorylation analysis of alpha-factor-arrested yeast. Using a multidimensional separation strategy involving preparative SDS-PAGE for prefractionation, in-gel digestion with trypsin, and immobilized metal affinity chromatography (IMAC) enrichment of phosphopeptides, followed by LC-MS/MS analysis employing a hybrid LTQ-Orbitrap mass spectrometer, we were able to catalog a substantial portion of the phosphoproteins present in yeast whole-cell lysate. This analysis yielded the confident identification of 2288 nonredundant phosphorylation sites from 985 proteins. The ambiguity score (Ascore) algorithm was utilized to determine the certainty of site localization for the entire data set. In addition, the size of the data set permitted extraction of known and novel kinase motifs using the Motif-X algorithm. Finally, a large number of members of the pheromone signaling pathway were found as phosphoproteins and are discussed.

Mammalian β-globin loci contain multiple genes that are activated at different developmental stages. Studies have suggested that the transcription of one gene in a locus can influence the expression of the other locus genes. The prevalent model to explain this transcriptional interference is that all potentially active genes compete for locus control region (LCR) activity. To investigate the influence of transcription by the murine embryonic genes on transcription of the other β-like genes, we generated mice with deletions of the promoter regions of Ey and βh1 and measured transcription of the remaining genes. Deletion of the Ey and βh1 promoters increased transcription of βmajor and βminor 2-fold to 3-fold during primitive erythropoiesis. Deletion of Ey did not affect βh1 nor did deletion of βh1 affect Ey, but Ey deletion uniquely activated transcription from βh0, a β-like globin gene immediately downstream of Ey. Protein analysis showed that βh0 encodes a translatable β-like globin protein that can pair with alpha globin. The lack of transcriptional interference between Ey and βh1 and the gene-specific repression of βh0 did not support LCR competition among the embryonic genes and suggested that direct transcriptional interference from Ey suppressed βh0.

The absolute quantification (AQUA) strategy provides a means to determine the precise protein or modified protein levels directly from cells or tissues. The technique is based on two major principles: stable isotope dilution theory and the use of synthetic peptides containing such stable isotopes to exactly mimic native counterparts after proteolysis. These peptides can be synthesized with modifications such as phosphorylation. methylation, and acetylation to allow for the direct, quantitative analysis of posttranslationally modified proteins. In this chapter, we discuss the development of an AQUA method and demonstrate its usefulness in the measurement of endogenous levels of the human protein separase at a functionally relevant phosphorylation site, serine 1126.

Glioblastoma is the most common and aggressive type of primary brain tumor in adults with more than 14,000 new cases diagnosed each year in the US. Although surgical resection, radiation, and cytotoxic therapies are available these current treatment methods are not curative and the median survival of patients remains at 12-15 months. Inhibitor of DNA binding proteins (Id1-Id4) are a family of genetically encoded dominant negative regulators of basic helix-loop-helix (bHLH) transcription factors. Id proteins are widely reported to inhibit differentiation and promote cell cycle transit in neural progenitor cells (NPCs) and have been implicated in the development of glioma. Our laboratory has observed a poor correlation between Id2 mRNA levels and Id2 protein levels in multiple cell types which led us to seek post-translational modifications that effected steady state Id2 protein levels and key Id2 mediated cellular functions. Using mass spectrometry we have identified three phosphorylation sites within the N-terminus of Id2 in proliferating NPCs. To interrogate the importance of Id2 N-terminal phosphorylation, Id2-/- NPCs were modified to express WT Id2 or various Id2 mutants expressing proteins that could not be phosphorylated at the N-terminus. We observed that NPCs expressing these mutants had higher steady state levels of Id2 than NPCs expressing WT Id2. It is known that WT Id2 is rapidly degraded by the proteasome. However, when proliferating NPCs were treated with cyclohexamide, phospho-ablated Id2 molecules exhibited a longer half-life than WT Id2 molecules indicating that loss of N-terminal phosphorylation results in resistance to proteasome-mediated degradation. Moreover, NPCs expressing this degradation-resistant, phospho-ablated Id2 protein proliferate more rapidly than NPCs expressing WT Id2, a finding consistent with the well-characterized function of Id2 in driving proliferation. Seeking to identify molecules whose inhibition might enhance Id2 phosphorylation, we evaluated the activity of multiple phosphatase inhibitors and identified phosphatases that could potentially function to stabilize pro-proliferative Id2 in NPCs. Calyculin A treatment caused a significant loss of Id2 protein suggesting that the PP1, PP2A, PP4 and/or PP6 phosphatases were likely regulators of Id2. To complement these pharmacologic studies we used a genetic approach to decrease PP2A expression and found that Id2 protein levels were decreased. Our findings indicate that inhibition of this phosphatase may provide a novel mechanism to decrease Id2 protein levels by causing rapid degradation of Id2. Citation Format: Jaclyn Sullivan, Matthew Havrda, Brenton Paolella, Arminja Kettenbach, Scott Gerber, Mark A. Israel. Phosphorylation of Id2 at the N-terminus modulates Id2 degradation and mediates cell cycle regulation in neural progenitor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2128. doi:10.1158/1538-7445.AM2015-2128

Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.

The primary goal of proteomics is to gain a better understanding of biological function at the protein expression level. As the field matures, numerous technologies are being developed to aid in the identification, quantification and characterization of protein expression and post-translational modifications on a near-global scale. Stable isotope labeling by amino acids in cell culture is one such technique that has shown broad biological applications. While we have recently shown the application of this technology to a model of metastatic prostate cancer, we now report a substantial improvement in quantitative analysis using a linear ion-trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ FT) and novel quantification software. This resulted in the quantification of nearly 1400 proteins, a greater than 3-fold increase in comparison to our earlier study. This dramatic increase in proteome coverage can be attributed to (1) use of a double-labeling strategy, (2) greater sensitivity, speed and mass accuracy provided by the LTQ FT mass spectrometer, and (3) more robust quantification software. Finally, by using a concatenated target/decoy protein database for our peptide searches, we now report these data in the context of an estimated false-positive rate of one percent.

Mass spectrometers that provide high mass accuracy such as FT-ICR instruments are increasingly used in proteomic studies. Although the importance of accurately determined molecular masses for the identification of biomolecules is generally accepted, its role in the analysis of shotgun proteomic data has not been thoroughly studied. To gain insight into this role, we used a hybrid linear quadrupole ion trap/FT-ICR (LTQ FT) mass spectrometer for LC-MS/MS analysis of a highly complex peptide mixture derived from a fraction of the yeast proteome. We applied three data-dependent MS/MS acquisition methods. The FT-ICR part of the hybrid mass spectrometer was either not exploited, used only for survey MS scans, or also used for acquiring selected ion monitoring scans to optimize mass accuracy. MS/MS data were assigned with the SEQUEST algorithm, and peptide identifications were validated by estimating the number of incorrect assignments using the composite target/decoy database search strategy. We developed a simple mass calibration strategy exploiting polydimethylcyclosiloxane background ions as calibrant ions. This strategy allowed us to substantially improve mass accuracy without reducing the number of MS/MS spectra acquired in an LC-MS/MS run. The benefits of high mass accuracy were greatest for assigning MS/MS spectra with low signal-to-noise ratios and for assigning phosphopeptides. Confident peptide identification rates from these data sets could be doubled by the use of mass accuracy information. It was also shown that improving mass accuracy at a cost to the MS/MS acquisition rate substantially lowered the sensitivity of LC-MS/MS analyses. The use of FT-ICR selected ion monitoring scans to maximize mass accuracy reduced the number of protein identifications by 40%.

Major analytical challenges encountered by shotgun proteome analysis include both the diversity and dynamic range of protein expression. Often new instrumentation can provide breakthroughs in areas where other analytical improvements have not been successful. In the current study, we utilized new instrumentation (LTQ FT) to characterize complex protein samples by shotgun proteomics. Proteomic analyses were performed on murine spleen tissue separated by magnetic beads into distinct CD45 and CD45 cell populations. Using shotgun protein analysis we identified 2,000 proteins per cell group by over 12,000 peptides with mass deviations of less than 4.5 ppm. Datasets obtained by LTQ FT analysis provided a significant increase in the number of proteins identified and greater confidence in those identifications and improved reproducibility in replicate analyses. Because CD45 and not CD45 cells are able to regenerate functional pancreatic islet cells in a mouse model of type I diabetes, protein expression was further compared by a subtractive proteomic approach in search of an exclusive protein expression profile in CD45 cells. Characterization of the proteins exclusively identified in CD45 cells was performed using gene ontology terms via the Javascript GoMiner. The CD45 cell subset readily revealed proteins involved in development, suggesting the persistence of a fetal stem cell in an adult animal. Molecular & Cellular Proteomics 4:1459–1470, 2005. The use of proteomic technologies for global characterization of proteins expressed in cells, tissues, and biological fluids is a key component in furthering our ability to understand biological processes in normal and diseased states. In many proteomic studies, cells or tissues are characterized by profiling and comparing proteins expressed in treated and untreated cells, cancerous versus normal tissues, or various cell populations. A seminal tool in this development has been the application of mass spectrometry technologies to identify proteins and post-translation modifications in complex protein mixtures. The term “shotgun proteomics” is used to de

NASA is developing technologies for nuclear electric propulsion for proposed deep space missions in support of the Exploration initiative under Project Prometheus. Electrical power produced by the combination of a fission-based power source and a Brayton power conversion and distribution system is used by a high specific impulse ion propulsion system to propel the spaceship. The ion propulsion system include the thruster, power processor and propellant feed system. A power processor technology development effort was initiated under Project Prometheus to develop high performance and lightweight power-processing technologies suitable for the application. This effort faces multiple challenges including developing radiation hardened power modules and converters with very high power capability and efficiency to minimize the impact on the power conversion and distribution system as well as the heat rejection system. This paper documents the design and test results of the first version of the beam supply, the design of a second version of the beam supply and the design and test results of the ancillary supplies.

International audience; RNAi-mediated heterochromatin assembly in fission yeast requires the RNA-induced transcriptional silencing (RITS) complex and a putative RNA-directed RNA polymerase (Rdp1). Here we show that Rdp1 is associated with two conserved proteins, Hrr1, an RNA helicase, and Cid12, a member of the polyA polymerase family, in a complex that has RNA-directed RNA polymerase activity (RDRC, RNA-directed RNA polymerase complex). RDRC physically interacts with RITS in a manner that requires the Dicer ribonuclease (Dcr1) and the Clr4 histone methyltransferase. Moreover, both complexes are localized to the nucleus and associate with noncoding centromeric RNAs in a Dcr1-dependent manner. In cells lacking Rdp1, Hrr1, or Cid12, RITS complexes are devoid of siRNAs and fail to localize to centromeric DNA repeats to initiate heterochromatin assembly. These findings reveal a physical and functional link between Rdp1 and RITS and suggest that noncoding RNAs provide a platform for siRNA-dependent localization of RNAi complexes to specific chromosome regions.

BACKGROUND Due to the prevalence of prostate disease, there is a growing interest in the immunobiology of the prostate and its contribution to such pathologies. Further study is needed to fully characterize immune responses within the prostate. METHODS Mouse ventral prostates were removed and analyzed using conventional immunohistochemistry, or by a novel whole mount technique, which allows the visualization of complete structures within the prostate. RESULTS A lymphoid structure was detected within the base of the mouse ventral prostate. Whole mount and cryostat sections revealed an organized arrangement of lymphocytes, along with dendritic cells interspersed throughout the node. It possessed a rich network of lymph node specific high endothelial venules (HEVs) and adjoining lymphatics containing immune cells. CONCLUSIONS Our results demonstrate the presence of a lymphoid structure within the base of the mouse ventral prostate possessing traits characteristic of an organized peripheral lymph node. © 2004 Wiley-Liss, Inc.

The free-piston Stirling power convertor is being considered as an advanced power conversion technology to be used for future NASA deep space missions requiring long life radioisotope power systems. This technology has a conversion efficiency of over 25%, which is significantly higher than the efficiency of the Radioisotope Thermal-electric Generators (RTG) now in use. The NASA Glenn Research Center has long been recognized as a leader in Stirling technology and is responsible for the development of advanced technologies that are intended to significantly improve key characteristics of the Stirling convertor. The advanced technologies identified for development also consider the requirements of potential future missions and the new capabilities that have become available in the associated technical areas. One of the key areas identified for technology development is the engine controller. To support this activity, an advanced controller is being developed for the Stirling power convertor. This controller utilizes active power factor correction electronics and microcontroller-based controls. The object of this paper is to present an overview of the advanced controller concept with modeling, simulation and hardware test data.

NASA has proposed a bold mission to orbit and explore the moons of Jupiter. This mission, known as the Jupiter Icy Moons Orbiter (JIMO), would significantly increase NASA s capability to explore deep space by making use of high power electric propulsion. One electric propulsion option under study for JIMO is an ion propulsion system. An early version of an ion propulsion system was successfully used on NASA's Deep Space 1 mission. One concept for an ion thruster system capable of meeting the current JIMO mission requirement would have individual thrusters that are 16 to 25 kW each and require voltages as high as 8.0 kV. The purpose of this work is to develop power processing schemes for delivering the high voltage power to the spacecraft ion thrusters based upon a three-phase AC distribution system. In addition, a proposed DC-DC converter topology is presented for an ion thruster ancillary supply based upon a DC distribution system. All specifications discussed in this paper are for design convenience and are speculative in nature.

RNA interference (RNAi) is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels. Here, we describe the purification of an RNAi effector complex termed RITS (RNA-induced initiation of transcriptional gene silencing) that is required for heterochromatin assembly in fission yeast. The RITS complex contains Ago1 (the fission yeast Argonaute homolog), Chp1 (a heterochromatin-associated chromodomain protein), and Tas3 (a novel protein). In addition, the complex contains small RNAs that require the Dicer ribonuclease for their production. These small RNAs are homologous to centromeric repeats and are required for the localization of RITS to heterochromatic domains. The results suggest a mechanism for the role of the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci.

We show here that affinity-purified Saccharomyces cerevisiae septin complexes contain stoichiometric amounts of guanine nucleotides, specifically GTP and GDP. Using a 15N-dilution assay read-out by liquid chromatography-tandem mass spectrometry, we determined that the majority of the bound guanine nucleotides do not turn over in vivo during one cell cycle period. In vitro, the isolated S. cerevisiae septin complexes have similar GTP binding and hydrolytic properties to the Drosophila septin complexes (Field, C. M., al-Awar, O., Rosenblatt, J., Wong, M. L., Alberts, B., and Mitchison, T. J. (1996) J. Cell Biol. 133, 605-616). In particular, the GTP turnover of septins is very slow when compared with the GTP turnover for Ras-like GTPases. We conclude that bound GTP and GDP play a structural, rather then regulatory, role for the majority of septins in proliferating cells as GTP does for α-tubulin.

Interleukin 12 (IL-12) is a pleiotropic cytokine with multiple effects on the immune system. The antitumor effects of locally produced IL-12 were examined in 2 tumor model systems. IL-12 expressing EMT6 mammary sarcomas (EMT6/IL-12) grew temporarily and then regressed resulting in mice that were immune to a further challenge of EMT6 cells. Interestingly, the IL-12 expressing K1735 melanomas (K1735/IL-12) maintained a lag phase of nonmeasurable growth for several weeks, followed by tumor outgrowth that was associated with a loss of IL-12 production. Tumor-infiltrating lymphocytes (TILs) isolated from EMT6/IL-12 tumors effectively lysed EMT6 target cells, whereas K1735/IL-12 TILs lacked lytic activity. Both IL-12 expressing tumors, however, grew progressively in nude mice indicating an important role for T cells in each case. Recombinant murine interferon gamma (rmIFN-gamma) inhibited the growth of EMT6 cells, but not K1735 cells in vitro, and strongly induced the expression of the antiangiogenic chemokine interferon-inducible protein 10 (IP-10) by both cell lines. Of interest, only the EMT6 cell line was able to secrete the proangiogenic molecule, vascular endothelial growth factor (VEGF), in response to low oxygen conditions. Fluorescent staining of the vascular endothelium at the tumor injection site provided images depicting early stages of angiogenesis prior to K1735/IL-12 tumor outgrowth. These results indicate that locally produced IL-12 likely mediates the rejection of EMT6 tumors through tumor cell lysis by host immune cells, whereas its antiangiogenic potential may be counterbalanced by the strong induction of VEGF by hypoxic tumor cells. In contrast, IL-12 does not induce protective immunity to K1735 tumors. However, an antiangiogenic mechanism may be responsible for controlling tumor growth.

Electronic modules and power circuits designed for use on many of NASA space missions are required to be efficient, reliable, and capable of operation in harsh environments. Some of these environmental stresses that are encountered in a typical deep space mission include high levels of radiation and extreme temperature. While numerous electronic devices and packages are designed to operate and withstand exposure to ionizing and other types of radiation, little is known about their performance under extreme temperatures, in particular cryogenic environments. In this work, the performance of International Rectifier radiationhardened power MOSFETS was investigated under wide temperature conditions. I. BACKGROUND

DC/DC converters are widely used in power management, conditioning, and control of space power systems. Deep space applications require electronics that withstand cryogenic temperature and meet a stringent radiation tolerance. In this work, the performance of an advanced, radiation-hardened (radhard) commercial DC/DC converter module was investigated at cryogenic temperatures. The converter was investigated in terms of its steady state and dynamic operations. The output voltage regulation, efficiency, terminal current ripple characteristics, and output voltage response to load changes were determined in the temperature range of 20/spl deg/C to - 140/spl deg/C. These parameters were obtained at various load levels and at different input voltages. The experimental procedures along with the results obtained on the investigated converter are presented and discussed.

A need exists for technologies that permit the direct quantification of differences in protein and posttranslationally modified protein expression levels. Here we present a strategy for the absolute quantification (termed AQUA) of proteins and their modification states. Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis. These synthetic peptides can also be prepared with covalent modifications (e.g., phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications. Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and posttranslationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer. In the present work, the AQUA strategy was used to ( i ) quantify low abundance yeast proteins involved in gene silencing, ( ii ) quantitatively determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase protein, and ( iii ) identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay. The methods described here represent focused, alternative approaches for studying the dynamically changing proteome.

A dynamic model for a free‐piston Stirling convertor is being developed at the NASA Glenn Research Center. The model is an end‐to‐end system model that includes the cycle thermodynamics, the dynamics, and electrical aspects of the system. The subsystems of interest are the heat source, the springs, the moving masses, the linear alternator, the controller and the end‐user load. The envisioned use of the model will be in evaluating how changes in a subsystem could affect the operation of the convertor. The model under development will speed the evaluation of improvements to a subsystem and aid in determining areas in which most significant improvements may be found. One of the first uses of the end‐to‐end model will be in the development of controller architectures. Another related area is in evaluating changes to details in the linear alternator.

Invariably, constitutional law courses begin with constitutional powers issues: the origins and scope of judicial review, Congress and the development of national power, the powers of the presidency, the modern administrative state, the states and American federalism, and representation, voting, and electoral politics. In two-semester courses, these issues are addressed in the fall. In one-semes

A 10 W 30 V/5.0 V push-pull DC-DC power converter breadboard, designed by the Jet Propulsion Laboratory (JPL) with a +50/spl deg/C to +5/spl deg/C operating range for the Cassini space probe, was characterized for lower operating temperatures. The breadboard power converter which failed to operate for temperatures below -125/spl deg/C was then modified to operate at temperatures approaching that of liquid nitrogen (LN2). Associated with this low operating temperature range (>-196/spl deg/C) was a variety of performance problems such as a significant change in output voltage, power converter switching instability and failure to restart at temperatures below -154/spl deg/C. An investigation into these problems yielded additional modifications to the power converter which improved low temperature performance even further.

The low temperature (25/spl deg/C to -175/spl deg/C) performance of a 120 W, 100 kHz, 42/12 V phase-shifted zero-voltage-switched full-bridge DC-DC power converter designed with commercially available components is reported. Its efficiency increased from 80.8% at 25/spl deg/C to 81.8% at -175/spl deg/C. The power MOSFET and filter inductor loss decreased whereas the diode rectifier loss increased with decreasing temperatures. Low temperature operation of the open-loop control circuit based on CMOS and BiCMOS ICs is also discussed. The switching frequency of the converter increased with decreasing temperatures with maximum deviation of less than 4% compared to the room temperature operation. The converter successfully restarted at low temperatures without any visible degradation.

Low temperature performance of a 150 W, 50 kHz, 24/48 V boost PWM DC-to-DC power converter is reported. The efficiency of the power converter using a molypermalloy powder (MPP) core based inductor went up from 94% at room temperature (23/spl deg/C) to 95.9% at liquid nitrogen temperature (-196/spl deg/C). A BSCCO based high temperature superconducting (HTS) inductor with a transition temperature of approximately -158/spl deg/C was compared to a MPP core based inductor in terms of the power converter performance at liquid nitrogen temperature. The use of the HTS inductor in the power converter tested yielded no significant performance improvement over the same power converter with the MPP inductor. The experimental results are discussed along with the HTS inductor characteristics.

The L-C resonant decay technique for measuring circuit Q or losses is improved by eliminating the switch from the inductor-capacitor loop. A MOSFET switch is used instead to momentarily connect the resonant circuit to an exciting voltage source, which itself is gated off during the decay transient. Very reproducible low duty cycle data could be taken this way over a dynamic voltage range of at least 10:1. Circuit Q is computed from a polynomial fit to the sequence of the decaying voltage maxima. This method was applied to measure the losses at 60 kHz in inductors having loose powder cores of moly permalloy (MPP) and a Mn-Zn power ferrite. After the copper and capacitor losses are separated, the resulting specific core loss is shown to be roughly as expected for the MPP powder, but anomalously high for the ferrite powder. Possible causes are mentioned.

Low-temperature (77 K) operation of a 42/28 V, 175 W, 50 kHz PWM buck DC/DC power converter designed with commercially available components is reported. Overall, the power converter losses decreased at 77 K compared to room temperature operation. A full-load efficiency of 97% was recorded at liquid-nitrogen temperature, compared to 95.8% at room temperature. Power MOSFET operation improved significantly whereas the output rectifier operation deteriorated at low-temperature. The performance of the output filter inductor and capacitor did not change significantly at 77 K compared to room temperature performance. It is possible to achieve high-density and high efficiency power conversion at low-temperatures due to improved electronic, electrical and thermal properties of materials. >