Science.gov

Sample records for abundance protein depletion

  1. Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study.

    PubMed

    Lee, Hye Min; Gupta, Ravi; Kim, Sun Hyung; Wang, Yiming; Rakwal, Randeep; Agrawal, Ganesh Kumar; Kim, Sun Tae

    2015-05-01

    High-abundance proteins (HAPs) hamper in-depth proteome study necessitating development of a HAPs depletion method. Here, we report a novel ethanol precipitation method (EPM) for HAPs depletion from total tuber proteins. Ethanol showed a dose-dependent effect on depletion of sporamin from sweet potato and patatin from potato tubers, respectively. The 50% ethanol was an optimal concentration. 2DE analysis of EPM-prepared sweet potato proteins also revealed enrichment of storage proteins (SPs) in ethanol supernatant (ES) resulting in detection of new low-abundance proteins in ethanol pellet (EP), compared to total fraction. The ES fraction showed even higher trypsin inhibitor activity than total proteins, further showing the efficacy of EPM in enrichment of sporamin in ES fraction. Application of this method was demonstrated for comparative proteomics of two sweet potato cultivars (Hwang-geum and Ho-bac) and purification of SP (sporamin) in its native form, as examples. Comparative proteomics identified many cultivar specific protein spots and selected spots were confidently assigned for their protein identity using MALDI-TOF-TOF analysis. Overall, the EPM is simple, reproducible, and economical for depletion of SPs and is suitable for downstream proteomics study. This study opens a door for its potential application to other tuber crops or fruits rich in carbohydrates. PMID:25689267

  2. Comparison of Depletion Strategies for the Enrichment of Low-Abundance Proteins in Urine

    PubMed Central

    Filip, Szymon; Vougas, Konstantinos; Zoidakis, Jerome; Latosinska, Agnieszka; Mullen, William; Spasovski, Goce; Mischak, Harald; Vlahou, Antonia; Jankowski, Joachim

    2015-01-01

    Proteome analysis of complex biological samples for biomarker identification remains challenging, among others due to the extended range of protein concentrations. High-abundance proteins like albumin or IgG of plasma and urine, may interfere with the detection of potential disease biomarkers. Currently, several options are available for the depletion of abundant proteins in plasma. However, the applicability of these methods in urine has not been thoroughly investigated. In this study, we compared different, commercially available immunodepletion and ion-exchange based approaches on urine samples from both healthy subjects and CKD patients, for their reproducibility and efficiency in protein depletion. A starting urine volume of 500 μL was used to simulate conditions of a multi-institutional biomarker discovery study. All depletion approaches showed satisfactory reproducibility (n=5) in protein identification as well as protein abundance. Comparison of the depletion efficiency between the unfractionated and fractionated samples and the different depletion strategies, showed efficient depletion in all cases, with the exception of the ion-exchange kit. The depletion efficiency was found slightly higher in normal than in CKD samples and normal samples yielded more protein identifications than CKD samples when using both initial as well as corresponding depleted fractions. Along these lines, decrease in the amount of albumin and other targets as applicable, following depletion, was observed. Nevertheless, these depletion strategies did not yield a higher number of identifications in neither the urine from normal nor CKD patients. Collectively, when analyzing urine in the context of CKD biomarker identification, no added value of depletion strategies can be observed and analysis of unfractionated starting urine appears to be preferable. PMID:26208298

  3. Depletion of cells and abundant proteins from biological samples by enhanced dielectrophoresis✩

    PubMed Central

    Gupta, C.; Provine, J.; Davis, R.W.; Howe, R.T.

    2016-01-01

    Platforms that are sensitive and specific enough to assay low-abundance protein biomarkers, in a high throughput multiplex format, within a complex biological fluid specimen, are necessary to enable protein biomarker based diagnostics for diseases such as cancer. The signal from an assay for a low-abundance protein biomarker in a biological fluid sample like blood is typically buried in a background that arises from the presence of blood cells and from high-abundance proteins that make up 90% of the assayed protein mass. We present an automated on-chip platform for the depletion of cells and highly abundant serum proteins in blood. Our platform consists of two components, the first of which is a microfluidic mixer that mixes beads containing antibodies against the highly abundant proteins in the whole blood. This complex mixture (consisting of beads, cells, and serum proteins) is then injected into the second component of our microfluidic platform, which comprises a filter trench to capture all the cells and the beads. The size-based trapping of the cells and beads into the filter trench is significantly enhanced by leveraging additional negative dielectrophoretic forces to push the micron sized particles (cells and beads which have captured the highly abundant proteins) down into the trench, allowing the serum proteins of lower abundance to flow through. In general, dielectrophoresis using bare electrodes is incapable of producing forces beyond the low piconewton range that tend to be insufficient for separation applications. However, by using electrodes passivated with atomic layer deposition, we demonstrate the application of enhanced negative DEP electrodes together with size-based flltration induced by the filter trench, to deplete 100% of the micron sized particles in the mixture. PMID:26924893

  4. Evaluation of two high-abundance protein depletion kits and optimization of downstream isoelectric focusing.

    PubMed

    Qiu, Fanghua; Hou, Tieying; Huang, Dehong; Xue, Zhifeng; Liang, Dongyan; Li, Qiuming; Lin, Weimiao

    2015-11-01

    Disease biomarkers for diagnostic and prognostic purposes are most likely within an extremely low concentration range and are thus masked by the presence of high‑abundance proteins. Therefore, removing high‑abundance proteins is the main challenge for identifying disease biomarkers. In addition, the solution obtained from high‑abundance protein depletion kits contains a rich array of compounds, which interfere with isoelectric focusing (IEF). In the present study, the effect of two commercial kits was evaluated and the downstream IEF protocol was optimized. High‑resolution results could be obtained according to the following conditions: The ProteoPrep Blue Albumin and IgG Depletion kit depleted albumin and IgG; immobilized pH gradient strips (typically 18 cm) were rehydrated with sample buffer containing 250 µg serum proteins at 30 v for 6 h, 60 v for 6 h, 200 v for 2 h, 500 v for 2 h, 1,000 v for 2 h, 5,000 v for 2 h, 10,000 v for 2 h and then focusing at 10,000 v up to 110 k vhs. In addition, the protein spots identified by matrix‑assisted laser desorption ionization time‑of‑flight mass spectrometry demonstrated that all proteins had a low abundance. The present study not only provides a definite and effective method for removing high‑abundance proteins, but also provides a proper protocol (protocol C) for downstream IEF. The present study includes a comprehensive investigation of serum proteomics, which paves the way for serum protein research. PMID:26460178

  5. Depletion of the highly abundant protein albumin from human plasma using the Gradiflow.

    PubMed

    Rothemund, Deborah L; Locke, Vicki L; Liew, Audrey; Thomas, Theresa M; Wasinger, Valerie; Rylatt, Dennis B

    2003-03-01

    Analysis of complex protein samples by two-dimensional electrophoresis (2-DE) is often more difficult in the presence of a few predominant proteins. In plasma, proteins such as albumin mask proteins of lower abundance, as well as significantly limiting the amount of protein that can be loaded onto the immobilized pH gradient strip. In this paper the Gradiflow, a preparative electrophoresis system, has been used to deplete human plasma of the highly abundant protein albumin under native and denatured conditions. A three step protocol incorporating a charge separation to collect proteins with an isoelectric point greater than albumin and two size separations to isolate proteins larger and smaller than albumin, was used. When the albumin depleted fractions were analysed on pH 3-10 2-DE gels, proteins that were masked by albumin were revealed and proteins not seen in the unfractionated plasma sample were visualised. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analysis confirmed the identification of the protein that lies beneath albumin to be C4B-binding protein alpha chain. The liquid fractions from the Gradiflow separations were also analysed by liquid chromatography-tandem mass spectrometry to confirm the proteins were separated according to their size and charge mobility in an electric field. PMID:12627381

  6. Depletion of abundant plant RuBisCO protein using the protamine sulfate precipitation method.

    PubMed

    Kim, Yu Ji; Lee, Hye Min; Wang, Yiming; Wu, Jingni; Kim, Sang Gon; Kang, Kyu Young; Park, Ki Hun; Kim, Yong Chul; Choi, In Soo; Agrawal, Ganesh Kumar; Rakwal, Randeep; Kim, Sun Tae

    2013-07-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant plant leaf protein, hampering deep analysis of the leaf proteome. Here, we describe a novel protamine sulfate precipitation (PSP) method for the depletion of RuBisCO. For this purpose, soybean leaf total proteins were extracted using Tris-Mg/NP-40 extraction buffer. Obtained clear supernatant was subjected to the PSP method, followed by 13% SDS-PAGE analysis of total, PS-supernatant and -precipitation derived protein samples. In a dose-dependent experiment, 0.1% w/v PS was found to be sufficient for precipitating RuBisCO large and small subunits (LSU and SSU). Western blot analysis confirmed no detection of RuBisCO LSU in the PS-supernatant proteins. Application of this method to Arabidopsis, rice, and maize leaf proteins revealed results similar to soybean. Furthermore, 2DE analyses of PS-treated soybean leaf displayed enriched protein profile for the protein sample derived from the PS-supernatant than total proteins. Some enriched 2D spots were subjected to MALDI-TOF-TOF analysis and were successfully assigned for their protein identity. Hence, the PSP method is: (i) simple, fast, economical, and reproducible for RuBisCO precipitation from the plant leaf sample; (ii) applicable to both dicot and monocot plants; and (iii) suitable for downstream proteomics analysis. PMID:23576416

  7. A rapid method for depletion of Rubisco from soybean (Glycine max) leaf for proteomic analysis of lower abundance proteins.

    PubMed

    Krishnan, Hari B; Natarajan, Savithiry S

    2009-12-01

    2-DE analysis of complex plant proteomes has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the low abundance proteins within leaf tissue is difficult when it is comprised of 30-50% of the CO(2) fixation enzyme Rubisco. Resolution can be improved through depletion of Rubisco using fractionation techniques based upon different physiological or biochemical principles. We have developed a fast and simple fractionation technique using 10 mM Ca(2+) and 10 mM phytate to precipitate Rubisco from soybean leaf soluble protein extract. This method is not only rapid, but also inexpensive, and capable of removing 85% of the extremely abundant Rubisco enzyme from soybean leaf soluble protein extract. This method allowed for roughly 230 previously inconspicuous protein spots in soybean leaf to be more easily detectable (3-fold increase in vol%) using fluorescent detection and allowed 28 phosphorylated proteins previously undetected, to be isolated and identified by MALDI-TOF-MS. PMID:19766275

  8. Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: Analysis of human plasma and cerebrospinal fluid

    SciTech Connect

    Hyung, Seok Won; Piehowski, Paul D.; Moore, Ronald J.; Orton, Daniel J.; Schepmoes, Athena A.; Clauss, Therese R.; Chu, Rosalie K.; Fillmore, Thomas L.; Brewer, Heather M.; Liu, Tao; Zhao, Rui; Smith, Richard D.

    2014-09-06

    Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (<20 µL) due to low yields stemming from losses caused by nonspecific binding to the column matrix. Additionally, the cost of the depletion media can be prohibitive for larger scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large scale studies. We characterized the performance of a 346 µL column volume micro-scale depletion system, using four different flow rates to determine the most effective depletion conditions for ~6 μL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10 mL depletion column served as the control. Results showed depletion efficiency of the micro-scale column increased as flow rate decreased, and that our micro-depletion was reproducible. We found, in an initial application, a 600 µL sample of human cerebral spinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.

  9. Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: Analysis of human plasma and cerebrospinal fluid

    DOE PAGESBeta

    Hyung, Seok Won; Piehowski, Paul D.; Moore, Ronald J.; Orton, Daniel J.; Schepmoes, Athena A.; Clauss, Therese R.; Chu, Rosalie K.; Fillmore, Thomas L.; Brewer, Heather M.; Liu, Tao; et al

    2014-09-06

    Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (<20 µL) due to low yields stemming from losses caused by nonspecific binding to the column matrix. Additionally, the cost of the depletion media can be prohibitive for larger scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizingmore » sample recovery when samples are limited, as well as for reducing the expense of large scale studies. We characterized the performance of a 346 µL column volume micro-scale depletion system, using four different flow rates to determine the most effective depletion conditions for ~6 μL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10 mL depletion column served as the control. Results showed depletion efficiency of the micro-scale column increased as flow rate decreased, and that our micro-depletion was reproducible. We found, in an initial application, a 600 µL sample of human cerebral spinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.« less

  10. Microscale Depletion of High Abundance Proteins in Human Biofluids using IgY14 Immunoaffinity Resin. Analysis of Human Plasma and Cerebrospinal Fluid

    SciTech Connect

    Hyung, Seok Won; Piehowski, Paul D.; Moore, Ronald J.; Orton, Daniel J.; Schepmoes, Athena A.; Clauss, Therese RW; Chu, Rosalie K.; Fillmore, Thomas L.; Brewer, Heather M.; Liu, Tao; Zhao, Rui; Smith, Richard D.

    2014-09-06

    Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (<20 µL) due to low yields stemming from losses caused by nonspecific binding to the column matrix. Additionally, the cost of the depletion media can be prohibitive for larger scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large scale studies. We characterized the performance of a 346 µL column volume micro-scale depletion system, using four different flow rates to determine the most effective depletion conditions for ~6 μL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10 mL depletion column served as the control. Results showed depletion efficiency of the micro-scale column increased as flow rate decreased, and that our micro-depletion was reproducible. In an initial application, a 600 µL sample of human cerebral spinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.

  11. Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: analysis of human plasma and cerebrospinal fluid

    PubMed Central

    Hyung, Seok-Won; Piehowski, Paul D.; Moore, Ronald J.; Orton, Daniel J.; Schepmoes, Athena A.; Clauss, Therese R.; Chu, Rosalie K.; Fillmore, Thomas L.; Brewer, Heather; Liu, Tao; Zhao, Rui; Smith, Richard D.

    2014-01-01

    Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (<20 μL) due to low yields stemming from losses caused by nonspecific binding to the column matrix and concentration of large eluent volumes. Additionally, the cost of the depletion media can be prohibitive for larger-scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large-scale studies. We characterized the performance of a 346 μL column volume microscale depletion system, using four different flow rates to determine the most effective depletion conditions for ~6-μL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10-mL depletion column served as the control. Results showed depletion efficiency of the microscale column increased as flow rate decreased, and that our microdepletion was reproducible. In an initial application, a 600-μL sample of human cerebrospinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited. PMID:25192788

  12. Combining subproteome enrichment and Rubisco depletion enables identification of low abundance proteins differentially regulated during plant defense.

    PubMed

    Widjaja, Ivy; Naumann, Kai; Roth, Udo; Wolf, Noreen; Mackey, David; Dangl, Jeffery L; Scheel, Dierk; Lee, Justin

    2009-01-01

    Transgenic Arabidopsis conditionally expressing the bacterial avrRpm1 type III effector under the control of a dexamethasone-responsive promoter were used for proteomics studies. This model system permits study of an individual effector without interference from additional bacterial components. Coupling of different prefractionation approaches to high resolution 2-DE facilitated the discovery of low abundance proteins - enabling the identification of proteins that have escaped detection in similar experiments. A total of 34 differentially regulated protein spots were identified. Four of these (a remorin, a protein phosphatase 2C (PP2C), an RNA-binding protein, and a C2-domain-containing protein) are potentially early signaling components in the interaction between AvrRpm1 and the cognate disease resistance gene product, resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). For the remorin and RNA-binding protein, involvement of PTM and post-transcriptional regulation are implicated, respectively. PMID:19053141

  13. Late embryogenesis abundant proteins

    PubMed Central

    Olvera-Carrillo, Yadira; Reyes, José Luis

    2011-01-01

    Late Embryogenesis Abundant (LEA) proteins accumulate at the onset of seed desiccation and in response to water deficit in vegetative plant tissues. The typical LEA proteins are highly hydrophilic and intrinsically unstructured. They have been classified in different families, each one showing distinctive conserved motifs. In this manuscript we present and discuss some of the recent findings regarding their role in plant adaptation to water deficit, as well as those concerning to their possible function, and how it can be related to their intrinsic structural flexibility. PMID:21447997

  14. Interstellar abundance and depletion studies along Galactic sightlines

    NASA Astrophysics Data System (ADS)

    Haris, U.; Murthy, Jayant; Sofia, Ulysses

    2016-07-01

    The Far Ultraviolet Spectroscopic Explorer (FUSE) and Hubble Space Telescope (HST) has enhanced our understanding many aspects of interstellar medium of our galaxy. The wavelength coverage of FUSE and HST is of great astrophysical importance. We use FUSE and HST data for interstellar abundances studies of some important atomic species, such as sulphur and silicon. We report the newly derived column densities by measuring the equivalent widths of several ultraviolet absorption lines. Comparisons of observed depletions and grain properties with existing dust models will be discussed.

  15. Structurally Resolved Abundances and Depletions in the Rho OPH Cloud

    NASA Astrophysics Data System (ADS)

    Seab, C.

    1995-07-01

    The mechanism that determines the pattern of depletion ofelements in the interstellar medium has been a problem for along time. It is clear that some of the most refractoryelements such as Si, Fe, and Mg, are heavily depleted onto theinterstellar grains. On the other hand, some elements such asS and Zn are normally either undepleted or very lightlydepleted. The difference between the two cases is notunderstood. We propose to address this question with adetailed study of the depletion patterns in the Rho Ophiuchicloud. This study is strongly based on a combination of thecapabilities of two modern instruments: the GHRS for high-resolution UV data, and the Ultra High Resolution Facility(UHRF) of the AAT. This instrument has been used to obtain NaI line profiles in the Rho Oph cloud with a resolution ofR=1,000,000. The combination of these two types of data willbe used to resolve the velocity structure of the elementdepletions in the cloud.

  16. Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications.

    PubMed

    Gu, W; Crawford, E D; O'Donovan, B D; Wilson, M R; Chow, E D; Retallack, H; DeRisi, J L

    2016-01-01

    Next-generation sequencing has generated a need for a broadly applicable method to remove unwanted high-abundance species prior to sequencing. We introduce DASH (Depletion of Abundant Sequences by Hybridization). Sequencing libraries are 'DASHed' with recombinant Cas9 protein complexed with a library of guide RNAs targeting unwanted species for cleavage, thus preventing them from consuming sequencing space. We demonstrate a more than 99 % reduction of mitochondrial rRNA in HeLa cells, and enrichment of pathogen sequences in patient samples. We also demonstrate an application of DASH in cancer. This simple method can be adapted for any sample type and increases sequencing yield without additional cost. PMID:26944702

  17. Predicting the dynamics of protein abundance.

    PubMed

    Mehdi, Ahmed M; Patrick, Ralph; Bailey, Timothy L; Bodén, Mikael

    2014-05-01

    Protein synthesis is finely regulated across all organisms, from bacteria to humans, and its integrity underpins many important processes. Emerging evidence suggests that the dynamic range of protein abundance is greater than that observed at the transcript level. Technological breakthroughs now mean that sequencing-based measurement of mRNA levels is routine, but protocols for measuring protein abundance remain both complex and expensive. This paper introduces a Bayesian network that integrates transcriptomic and proteomic data to predict protein abundance and to model the effects of its determinants. We aim to use this model to follow a molecular response over time, from condition-specific data, in order to understand adaptation during processes such as the cell cycle. With microarray data now available for many conditions, the general utility of a protein abundance predictor is broad. Whereas most quantitative proteomics studies have focused on higher organisms, we developed a predictive model of protein abundance for both Saccharomyces cerevisiae and Schizosaccharomyces pombe to explore the latitude at the protein level. Our predictor primarily relies on mRNA level, mRNA-protein interaction, mRNA folding energy and half-life, and tRNA adaptation. The combination of key features, allowing for the low certainty and uneven coverage of experimental observations, gives comparatively minor but robust prediction accuracy. The model substantially improved the analysis of protein regulation during the cell cycle: predicted protein abundance identified twice as many cell-cycle-associated proteins as experimental mRNA levels. Predicted protein abundance was more dynamic than observed mRNA expression, agreeing with experimental protein abundance from a human cell line. We illustrate how the same model can be used to predict the folding energy of mRNA when protein abundance is available, lending credence to the emerging view that mRNA folding affects translation efficiency

  18. Predicting the Dynamics of Protein Abundance

    PubMed Central

    Mehdi, Ahmed M.; Patrick, Ralph; Bailey, Timothy L.; Bodén, Mikael

    2014-01-01

    Protein synthesis is finely regulated across all organisms, from bacteria to humans, and its integrity underpins many important processes. Emerging evidence suggests that the dynamic range of protein abundance is greater than that observed at the transcript level. Technological breakthroughs now mean that sequencing-based measurement of mRNA levels is routine, but protocols for measuring protein abundance remain both complex and expensive. This paper introduces a Bayesian network that integrates transcriptomic and proteomic data to predict protein abundance and to model the effects of its determinants. We aim to use this model to follow a molecular response over time, from condition-specific data, in order to understand adaptation during processes such as the cell cycle. With microarray data now available for many conditions, the general utility of a protein abundance predictor is broad. Whereas most quantitative proteomics studies have focused on higher organisms, we developed a predictive model of protein abundance for both Saccharomyces cerevisiae and Schizosaccharomyces pombe to explore the latitude at the protein level. Our predictor primarily relies on mRNA level, mRNA–protein interaction, mRNA folding energy and half-life, and tRNA adaptation. The combination of key features, allowing for the low certainty and uneven coverage of experimental observations, gives comparatively minor but robust prediction accuracy. The model substantially improved the analysis of protein regulation during the cell cycle: predicted protein abundance identified twice as many cell-cycle-associated proteins as experimental mRNA levels. Predicted protein abundance was more dynamic than observed mRNA expression, agreeing with experimental protein abundance from a human cell line. We illustrate how the same model can be used to predict the folding energy of mRNA when protein abundance is available, lending credence to the emerging view that mRNA folding affects translation

  19. On protein abundance distributions in complex mixtures

    PubMed Central

    2013-01-01

    Mass spectrometry, an analytical technique that measures the mass-to-charge ratio of ionized atoms or molecules, dates back more than 100 years, and has both qualitative and quantitative uses for determining chemical and structural information. Quantitative proteomic mass spectrometry on biological samples focuses on identifying the proteins present in the samples, and establishing the relative abundances of those proteins. Such protein inventories create the opportunity to discover novel biomarkers and disease targets. We have previously introduced a normalized, label-free method for quantification of protein abundances under a shotgun proteomics platform (Griffin et al., 2010). The introduction of this method for quantifying and comparing protein levels leads naturally to the issue of modeling protein abundances in individual samples. We here report that protein abundance levels from two recent proteomics experiments conducted by the authors can be adequately represented by Sichel distributions. Mathematically, Sichel distributions are mixtures of Poisson distributions with a rather complex mixing distribution, and have been previously and successfully applied to linguistics and species abundance data. The Sichel model can provide a direct measure of the heterogeneity of protein abundances, and can reveal protein abundance differences that simpler models fail to show. PMID:23360617

  20. SoyProLow: A protein database enriched in low abundant soybean proteins

    PubMed Central

    Tavakolan, Mona; Alkharouf, Nadim W; Matthews, Benjamin F; Natarajan, Savithiry S

    2014-01-01

    Soybeans are an important legume crop that contain 2 major storage proteins, β-conglycinin and glycinin, which account about 70- 80% of total seed proteins. These abundant proteins hinder the isolation and characterization of several low abundant proteins in soybean seeds. Several protein extraction methodologies were developed in our laboratory to decrease these abundant storage proteins in seed extracts and to also decrease the amount of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), which is normally very abundant in leaf extracts. One of the extraction methodologies used 40% isopropanol and was more effective in depleting soybean storage proteins and enhancing low abundant seed proteins than similar methods using 10-80% isopropanol. Extractions performed with 40% isopropanol decreased the amount of storage proteins and revealed 107 low abundant proteins when using the combined approaches of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Mass Spectrometry (MS). The separation of proteins was achieved by iso-electric focusing (IEF) and 2D-PAGE. The proteins were analyzed with MS techniques to provide amino acid sequence. The proteins were identified by comparing their amino acid sequences with those in different databases including NCBI-non redundant, UniprotKB and MSDB databases. In this investigation, previously published results on low abundant soybean seed proteins were used to create an online database (SoyProLow) to provide a data repository that can be used as a reference to identify and characterize low abundance proteins. This database is freely accessible to individuals using similar techniques and can be for the subsequent genetic manipulation to produce value added soybean traits. An intuitive user interface based on dynamic HTML enables users to browse the network and the profiles of the low abundant proteins. Availability http://bioinformatics.towson.edu/Soybean_low_abundance_proteins_2D_Gel_DB/Gel1.aspx PMID:25352730

  1. Precise Li abundances in metal-poor stars: depletion in the Spite plateau

    NASA Astrophysics Data System (ADS)

    Meléndez, J.; Casagrande, L.; Ramírez, I.; Asplund, M.

    2010-03-01

    We present Li abundances for 73 stars in the metallicity range -3.5 < [Fe/H] < -1.0 using improved IRFM temperatures (Casagrande et al. 2009) with precise E(B-V) values obtained mostly from interstellar NaI D lines, and high-quality equivalent widths (σEW ~ 3%). At all metallicities we uncover a fine-structure in the Li abundances of Spite plateau stars, which we trace to Li depletion that depends on both metallicity and mass. Models including atomic diffusion and turbulent mixing seem to reproduce the observed Li depletion assuming a primordial Li abundance ALi = 2.64 dex (MARCS models) or 2.72 (Kurucz overshooting models), in good agreement with current predictions (ALi = 2.72) from standard BBN.

  2. Click chemistry: a new facile and efficient strategy for the preparation of Fe3O4 nanoparticles covalently functionalized with IDA-Cu and their application in the depletion of abundant protein in blood samples

    NASA Astrophysics Data System (ADS)

    Jian, Guiqin; Liu, Yuxing; He, Xiwen; Chen, Langxing; Zhang, Yukui

    2012-09-01

    In this study, we report a novel method to synthesize core-shell structured Fe3O4 nanoparticles (NPs) covalently functionalized with iminodiacetic acid (IDA) via click chemistry between the azide and alkyne groups and charged with Cu2+. Firstly, the Fe3O4@SiO2 NPs were obtained using tetraethoxysilane (TEOS) to form a silica shell on the surface of the Fe3O4 core. The azide group-modified Fe3O4@SiO2 NPs were obtained by a sol-gel process using 3-azidopropyltriethoxysilane (AzPTES) as the silane agent. Fe3O4@SiO2-N3 was directly reacted with N-propargyl iminodiacetic via click chemistry, in the presence of a Cu(I) catalyst, to acquire the IDA-modified Fe3O4 NPs. Finally, through the addition of Cu2+, the Fe3O4@SiO2-IDA-Cu NP product was obtained. The morphology, structure and composition of the NPs were characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The resulting NPs showed a strong magnetic response to an externally applied magnetic field, a high adsorption capacity and excellent specificity towards hemoglobin (Hb). In addition, the Fe3O4@SiO2-IDA-Cu NPs can be used for the selective removal of abundant Hb protein in bovine and human blood samples.

  3. Click chemistry: a new facile and efficient strategy for the preparation of Fe3O4 nanoparticles covalently functionalized with IDA-Cu and their application in the depletion of abundant protein in blood samples.

    PubMed

    Jian, Guiqin; Liu, Yuxing; He, Xiwen; Chen, Langxing; Zhang, Yukui

    2012-10-21

    In this study, we report a novel method to synthesize core-shell structured Fe(3)O(4) nanoparticles (NPs) covalently functionalized with iminodiacetic acid (IDA) via click chemistry between the azide and alkyne groups and charged with Cu(2+). Firstly, the Fe(3)O(4)@SiO(2) NPs were obtained using tetraethoxysilane (TEOS) to form a silica shell on the surface of the Fe(3)O(4) core. The azide group-modified Fe(3)O(4)@SiO(2) NPs were obtained by a sol-gel process using 3-azidopropyltriethoxysilane (AzPTES) as the silane agent. Fe(3)O(4)@SiO(2)-N(3) was directly reacted with N-propargyl iminodiacetic via click chemistry, in the presence of a Cu(I) catalyst, to acquire the IDA-modified Fe(3)O(4) NPs. Finally, through the addition of Cu(2+), the Fe(3)O(4)@SiO(2)-IDA-Cu NP product was obtained. The morphology, structure and composition of the NPs were characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The resulting NPs showed a strong magnetic response to an externally applied magnetic field, a high adsorption capacity and excellent specificity towards hemoglobin (Hb). In addition, the Fe(3)O(4)@SiO(2)-IDA-Cu NPs can be used for the selective removal of abundant Hb protein in bovine and human blood samples. PMID:22941423

  4. Decline in the tropospheric abundance of halogen from halocarbons: Implications for stratospheric ozone depletion

    SciTech Connect

    Montzka, S.A.; Butler, J.H.; Myers, R.C.

    1996-05-31

    Analyses of air sampled from remote locations across the globe reveal that tropospheric chlorine attributable to anthropogenic halocarbons peaked near the beginning of 1994 and was decreasing at a rate of 25 {+-} parts per trillion per year by mid-1995. Although bromine from halons was still increasing in mid-1995, the summed abundance of these halogens in the troposphere is decreasing. To assess the effect of this trend on stratospheric ozone, estimates of the future stratospheric abundance of ozone-depleting gases were made for mid-latitude and polar regions on the basis of these tropospheric measurements. These results suggest that the amount of reactive chlorine and bromine will reach a maximum in the stratosphere between 1997 and 1999 and will decline thereafter if limits outlined in the adjusted and amended Montreal Protocol on Substances That Deplete the Ozone Layer are not exceeded in future years. 30 refs., 4 figs., 1 tab.

  5. C/O abundance ratios, iron depletions, and infrared dust features in galactic planetary nebulae

    SciTech Connect

    Delgado-Inglada, Gloria; Rodríguez, Mónica E-mail: mrodri@inaoep.mx

    2014-04-01

    We study the dust present in 56 Galactic planetary nebulae (PNe) through their iron depletion factors, their C/O abundance ratios (in 51 objects), and the dust features that appear in their infrared spectra (for 33 objects). Our sample objects have deep optical spectra of good quality, and most of them also have ultraviolet observations. We use these observations to derive the iron abundances and the C/O abundance ratios in a homogeneous way for all the objects. We compile detections of infrared dust features from the literature and we analyze the available Spitzer/IRS spectra. Most of the PNe have C/O ratios below one and show crystalline silicates in their infrared spectra. The PNe with silicates have C/O <1, with the exception of Cn 1-5. Most of the PNe with dust features related to C-rich environments (SiC or the 30 μm feature usually associated to MgS) have C/O ≳ 0.8. Polycyclic aromatic hydrocarbons are detected over the full range of C/O values, including 6 objects that also show silicates. Iron abundances are low in all the objects, implying that more than 90% of their iron atoms are deposited into dust grains. The range of iron depletions in the sample covers about two orders of magnitude, and we find that the highest depletion factors are found in C-rich objects with SiC or the 30 μm feature in their infrared spectra, whereas some of the O-rich objects with silicates show the lowest depletion factors.

  6. C/O Abundance Ratios, Iron Depletions, and Infrared Dust Features in Galactic Planetary Nebulae

    NASA Astrophysics Data System (ADS)

    Delgado-Inglada, Gloria; Rodríguez, Mónica

    2014-04-01

    We study the dust present in 56 Galactic planetary nebulae (PNe) through their iron depletion factors, their C/O abundance ratios (in 51 objects), and the dust features that appear in their infrared spectra (for 33 objects). Our sample objects have deep optical spectra of good quality, and most of them also have ultraviolet observations. We use these observations to derive the iron abundances and the C/O abundance ratios in a homogeneous way for all the objects. We compile detections of infrared dust features from the literature and we analyze the available Spitzer/IRS spectra. Most of the PNe have C/O ratios below one and show crystalline silicates in their infrared spectra. The PNe with silicates have C/O <1, with the exception of Cn 1-5. Most of the PNe with dust features related to C-rich environments (SiC or the 30 μm feature usually associated to MgS) have C/O >~ 0.8. Polycyclic aromatic hydrocarbons are detected over the full range of C/O values, including 6 objects that also show silicates. Iron abundances are low in all the objects, implying that more than 90% of their iron atoms are deposited into dust grains. The range of iron depletions in the sample covers about two orders of magnitude, and we find that the highest depletion factors are found in C-rich objects with SiC or the 30 μm feature in their infrared spectra, whereas some of the O-rich objects with silicates show the lowest depletion factors.

  7. The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans.

    PubMed

    Zhang, Liangyu; Ward, Jordan D; Cheng, Ze; Dernburg, Abby F

    2015-12-15

    Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation of biological regulatory mechanisms. Whereas powerful techniques for protein expression have been developed in Caenorhabditis elegans, existing tools for conditional disruption of protein function are far more limited. To address this, we have adapted the auxin-inducible degradation (AID) system discovered in plants to enable conditional protein depletion in C. elegans. We report that expression of a modified Arabidopsis TIR1 F-box protein mediates robust auxin-dependent depletion of degron-tagged targets. We document the effectiveness of this system for depletion of nuclear and cytoplasmic proteins in diverse somatic and germline tissues throughout development. Target proteins were depleted in as little as 20-30 min, and their expression could be re-established upon auxin removal. We have engineered strains expressing TIR1 under the control of various promoter and 3' UTR sequences to drive tissue-specific or temporally regulated expression. The degron tag can be efficiently introduced by CRISPR/Cas9-based genome editing. We have harnessed this system to explore the roles of dynamically expressed nuclear hormone receptors in molting, and to analyze meiosis-specific roles for proteins required for germ line proliferation. Together, our results demonstrate that the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism. PMID:26552885

  8. The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans

    PubMed Central

    Zhang, Liangyu; Ward, Jordan D.; Cheng, Ze; Dernburg, Abby F.

    2015-01-01

    Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation of biological regulatory mechanisms. Whereas powerful techniques for protein expression have been developed in Caenorhabditis elegans, existing tools for conditional disruption of protein function are far more limited. To address this, we have adapted the auxin-inducible degradation (AID) system discovered in plants to enable conditional protein depletion in C. elegans. We report that expression of a modified Arabidopsis TIR1 F-box protein mediates robust auxin-dependent depletion of degron-tagged targets. We document the effectiveness of this system for depletion of nuclear and cytoplasmic proteins in diverse somatic and germline tissues throughout development. Target proteins were depleted in as little as 20-30 min, and their expression could be re-established upon auxin removal. We have engineered strains expressing TIR1 under the control of various promoter and 3′ UTR sequences to drive tissue-specific or temporally regulated expression. The degron tag can be efficiently introduced by CRISPR/Cas9-based genome editing. We have harnessed this system to explore the roles of dynamically expressed nuclear hormone receptors in molting, and to analyze meiosis-specific roles for proteins required for germ line proliferation. Together, our results demonstrate that the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism. PMID:26552885

  9. Late Embryogenesis Abundant (LEA) proteins in legumes

    PubMed Central

    Battaglia, Marina; Covarrubias, Alejandra A.

    2013-01-01

    Plants are exposed to different external conditions that affect growth, development, and productivity. Water deficit is one of these adverse conditions caused by drought, salinity, and extreme temperatures. Plants have developed different responses to prevent, ameliorate or repair the damage inflicted by these stressful environments. One of these responses is the activation of a set of genes encoding a group of hydrophilic proteins that typically accumulate to high levels during seed dehydration, at the last stage of embryogenesis, hence named Late Embryogenesis Abundant (LEA) proteins. LEA proteins also accumulate in response to water limitation in vegetative tissues, and have been classified in seven groups based on their amino acid sequence similarity and on the presence of distinctive conserved motifs. These proteins are widely distributed in the plant kingdom, from ferns to angiosperms, suggesting a relevant role in the plant response to this unfavorable environmental condition. In this review, we analyzed the LEA proteins from those legumes whose complete genomes have been sequenced such as Phaseolus vulgaris, Glycine max, Medicago truncatula, Lotus japonicus, Cajanus cajan, and Cicer arietinum. Considering their distinctive motifs, LEA proteins from the different groups were identified, and their sequence analysis allowed the recognition of novel legume specific motifs. Moreover, we compile their transcript accumulation patterns based on publicly available data. In spite of the limited information on these proteins in legumes, the analysis and data compiled here confirm the high correlation between their accumulation and water deficit, reinforcing their functional relevance under this detrimental conditions. PMID:23805145

  10. Late Embryogenesis Abundant (LEA) proteins in legumes.

    PubMed

    Battaglia, Marina; Covarrubias, Alejandra A

    2013-01-01

    Plants are exposed to different external conditions that affect growth, development, and productivity. Water deficit is one of these adverse conditions caused by drought, salinity, and extreme temperatures. Plants have developed different responses to prevent, ameliorate or repair the damage inflicted by these stressful environments. One of these responses is the activation of a set of genes encoding a group of hydrophilic proteins that typically accumulate to high levels during seed dehydration, at the last stage of embryogenesis, hence named Late Embryogenesis Abundant (LEA) proteins. LEA proteins also accumulate in response to water limitation in vegetative tissues, and have been classified in seven groups based on their amino acid sequence similarity and on the presence of distinctive conserved motifs. These proteins are widely distributed in the plant kingdom, from ferns to angiosperms, suggesting a relevant role in the plant response to this unfavorable environmental condition. In this review, we analyzed the LEA proteins from those legumes whose complete genomes have been sequenced such as Phaseolus vulgaris, Glycine max, Medicago truncatula, Lotus japonicus, Cajanus cajan, and Cicer arietinum. Considering their distinctive motifs, LEA proteins from the different groups were identified, and their sequence analysis allowed the recognition of novel legume specific motifs. Moreover, we compile their transcript accumulation patterns based on publicly available data. In spite of the limited information on these proteins in legumes, the analysis and data compiled here confirm the high correlation between their accumulation and water deficit, reinforcing their functional relevance under this detrimental conditions. PMID:23805145

  11. Mouse liver protein sulfhydryl depletion after acetaminophen exposure.

    PubMed

    Yang, Xi; Greenhaw, James; Shi, Qiang; Roberts, Dean W; Hinson, Jack A; Muskhelishvili, Levan; Davis, Kelly; Salminen, William F

    2013-01-01

    Acetaminophen (APAP)-induced liver injury is the leading cause of acute liver failure in many countries. This study determined the extent of liver protein sulfhydryl depletion not only in whole liver homogenate but also in the zonal pattern of sulfhydryl depletion within the liver lobule. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase levels, liver necrosis, and glutathione depletion in a dose-dependent manner. Free protein sulfhydryls were measured in liver protein homogenates by labeling with maleimide linked to a near infrared fluorescent dye followed by SDS-polyacrylamide gel electrophoresis. Global protein sulfhydryl levels were decreased significantly (48.4%) starting at 1 hour after the APAP dose and maintained at this reduced level through 24 hours. To visualize the specific hepatocytes that had reduced protein sulfhydryl levels, frozen liver sections were labeled with maleimide linked to horseradish peroxidase. The centrilobular areas exhibited dramatic decreases in free protein sulfhydryls while the periportal regions were essentially spared. These protein sulfhydryl-depleted regions correlated with areas exhibiting histopathologic injury and APAP binding to protein. The majority of protein sulfhydryl depletion was due to reversible oxidation since the global- and lobule-specific effects were essentially reversed when the samples were reduced with tris(2-carboxyethy)phosphine before maleimide labeling. These temporal and zonal pattern changes in protein sulfhydryl oxidation shed new light on the importance that changes in protein redox status might play in the pathogenesis of APAP hepatotoxicity. PMID:23093024

  12. Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity.

    PubMed

    Okada, Kazushi; Fukui, Masayuki; Zhu, Bao-Ting

    2016-08-26

    Glutathione depletion is a distinct cause underlying many forms of pathogenesis associated with oxidative stress and cytotoxicity. Earlier studies showed that glutamate-induced glutathione depletion in immortalized murine HT22 hippocampal neuronal cells leads to accumulation of reactive oxygen species (ROS) and ultimately cell death, but the precise mechanism underlying these processes is not clear. Here we show that during the induction of glutathione depletion, nitric oxide (NO) accumulation precedes ROS accumulation. While neuronal NO synthase (nNOS) in untreated HT22 cells exists mostly as a monomer, glutathione depletion results in increased formation of the dimer nNOS, accompanied by increases in the catalytic activity. We identified that nNOS dimerization is catalyzed by protein disulfide isomerase (PDI). Inhibition of PDI's isomerase activity effectively abrogates glutathione depletion-induced conversion of monomer nNOS into dimer nNOS, accumulation of NO and ROS, and cytotoxicity. Furthermore, we found that PDI is present in untreated cells in an inactive S-nitrosylated form, which becomes activated following glutathione depletion via S-denitrosylation. These results reveal a novel role for PDI in mediating glutathione depletion-induced oxidative cytotoxicity, as well as its role as a valuable therapeutic target for protection against oxidative cytotoxicity. PMID:27317486

  13. NEBULAR WATER DEPLETION AS THE CAUSE OF JUPITER'S LOW OXYGEN ABUNDANCE

    SciTech Connect

    Mousis, Olivier; Madhusudhan, Nikku; Johnson, Torrence V.

    2012-05-20

    Motivated by recent spectroscopic observations suggesting that atmospheres of some extrasolar giant planets are carbon-rich, i.e., carbon/oxygen ratio (C/O) {>=} 1, we find that the whole set of compositional data for Jupiter is consistent with the hypothesis that it should be a carbon-rich giant planet. We show that the formation of Jupiter in the cold outer part of an oxygen-depleted disk (C/O {approx} 1) reproduces the measured Jovian elemental abundances at least as well as the hitherto canonical model of Jupiter formed in a disk of solar composition (C/O 0.54). The resulting O abundance in Jupiter's envelope is then moderately enriched by a factor of {approx}2 Multiplication-Sign solar (instead of {approx}7 Multiplication-Sign solar) and is found to be consistent with values predicted by thermochemical models of the atmosphere. That Jupiter formed in a disk with C/O {approx} 1 implies that water ice was heterogeneously distributed over several AU beyond the snow line in the primordial nebula and that the fraction of water contained in icy planetesimals was a strong function of their formation location and time. The Jovian oxygen abundance to be measured by NASA's Juno mission en route to Jupiter will provide a direct and strict test of our predictions.

  14. Gas-phase CO depletion and N2H+ abundances in starless cores

    NASA Astrophysics Data System (ADS)

    Lippok, N.; Launhardt, R.; Semenov, D.; Stutz, A. M.; Balog, Z.; Henning, Th.; Krause, O.; Linz, H.; Nielbock, M.; Pavlyuchenkov, Ya. N.; Schmalzl, M.; Schmiedeke, A.; Bieging, J. H.

    2013-12-01

    Context. In the dense and cold interiors of starless molecular cloud cores, a number of chemical processes allow for the formation of complex molecules and the deposition of ice layers on dust grains. Dust density and temperature maps of starless cores derived from Herschel continuum observations constrain the physical structure of the cloud cores better than ever before. We use these to model the temporal chemical evolution of starless cores. Aims: We derive molecular abundance profiles for a sample of starless cores. We then analyze these using chemical modeling based on dust temperature and hydrogen density maps derived from Herschel continuum observations. Methods: We observed the 12CO (2-1), 13CO (2-1), C18O (2-1) and N2H+ (1-0) transitions towards seven isolated, nearby low-mass starless molecular cloud cores. Using far infrared (FIR) and submillimeter (submm) dust emission maps from the Herschel key program Earliest Phases of Star formation (EPoS) and by applying a ray-tracing technique, we derived the physical structure (density, dust temperature) of these cores. Based on these results we applied time-dependent chemical modeling of the molecular abundances. We modeled the molecular emission profiles with a line-radiative transfer code and compared them to the observed emission profiles. Results: CO is frozen onto the grains in the center of all cores in our sample. The level of CO depletion increases with hydrogen density and ranges from 46% up to more than 95% in the core centers of the three cores with the highest hydrogen density. The average hydrogen density at which 50% of CO is frozen onto the grains is 1.1 ± 0.4 × 105 cm-3. At about this density, the cores typically have the highest relative abundance of N2H+. The cores with higher central densities show depletion of N2H+ at levels of 13% to 55%. The chemical ages for the individual species are on average (2 ± 1) × 105 yr for 13CO, (6 ± 3) × 104 yr for C18O, and (9 ± 2) × 104 yr for N2H

  15. RNase J depletion leads to massive changes in mRNA abundance in Helicobacter pylori.

    PubMed

    Redko, Yulia; Galtier, Eloïse; Arnion, Hélène; Darfeuille, Fabien; Sismeiro, Odile; Coppée, Jean-Yves; Médigue, Claudine; Weiman, Marion; Cruveiller, Stéphane; De Reuse, Hilde

    2016-02-01

    Degradation of RNA as an intermediate message between genes and corresponding proteins is important for rapid attenuation of gene expression and maintenance of cellular homeostasis. This process is controlled by ribonucleases that have different target specificities. In the bacterial pathogen Helicobacter pylori, an exo- and endoribonuclease RNase J is essential for growth. To explore the role of RNase J in H. pylori, we identified its putative targets at a global scale using next generation RNA sequencing. We found that strong depletion for RNase J led to a massive increase in the steady-state levels of non-rRNAs. mRNAs and RNAs antisense to open reading frames were most affected with over 80% increased more than 2-fold. Non-coding RNAs expressed in the intergenic regions were much less affected by RNase J depletion. Northern blotting of selected messenger and non-coding RNAs validated these results. Globally, our data suggest that RNase J of H. pylori is a major RNase involved in degradation of most cellular RNAs. PMID:26726773

  16. Dietary zinc depletion and repletion affects plasma proteins: an analysis of the plasma proteome

    PubMed Central

    Wickwire, Kathie; Ho, Emily; Chung, Carolyn S.; King, Janet

    2014-01-01

    Zinc (Zn) deficiency is a problem worldwide. Current methods for assessing Zn status are limited to measuring plasma or serum Zn within populations suspected of deficiency. Despite the high prevalence of Zn deficiency in the human population there are no methods currently available for sensitively assessing Zn status among individuals. The purpose of this research was to utilize a proteomic approach using two-dimensional gel electrophoresis (2DE) and mass spectrometry to identify protein biomarkers that were sensitive to changes in dietary Zn levels in humans. Proteomic analysis was performed in human plasma samples (n = 6) obtained from healthy adult male subjects that completed a dietary Zn depletion/repletion protocol, current dietary zinc intake has a greater effect on fractional zinc absorption than does longer term zinc consumption in healthy adult men. Chung et al. (Am J Clin Nutr 87 (5):1224–1229, 2008). After a 13 day Zn acclimatization period where subjects consumed a Zn-adequate diet, the male subjects consumed a marginal Zn-depleted diet for 42 days followed by consumption of a Zn-repleted diet for 28 days. The samples at baseline, end of depletion and end of repletion were pre-fractionated through immuno-affinity columns to remove 14 highly abundant proteins, and each fraction separated by 2DE. Following staining by colloidal Coomassie blue and densitometric analysis, three proteins were identified by mass spectrometry as affected by changes in dietary Zn. Fibrin β and chain E, fragment double D were observed in the plasma protein fraction that remained bound to the immuno-affinity column. An unnamed protein that was related to immunoglobulins was observed in the immunode-pleted plasma fraction. Fibrin β increased two-fold following the Zn depletion period and decreased to baseline values following the Zn repletion period; this protein may serve as a viable biomarker for Zn status in the future. PMID:23255060

  17. Abundance, composition, and distribution of crustacean zooplankton in relation to hypolimnetic oxygen depletion in west-central Lake Erie

    USGS Publications Warehouse

    Heberger, Roy F.; Reynolds, James B.

    1977-01-01

    Samples of crustacean zooplankton were collected monthly in west-central Lake Erie in April and June to October 1968, and in July and August 1970, before and during periods of hypolimnetic dissolved oxygen (DO) depletion. The water column at offshore stations was thermally stratified from June through September 1968, and the hypolimnion contained no DO in mid-August of 1968 or 1970. Composition, abundance, and vertical distribution of crustacean zooplankton changed coincidentally with oxygen depletion. From July to early August, zooplankton abundance dropped 79% in 1968 and 50% in 1970. The declines were attributed largely to a sharp decrease in abundance of planktonic Cyclops bicuspidatus thomasi. Zooplankton composition shifted from mainly cyclopoid copepods in July to mainly cladocerans and copepod nauplii in middle to late August. We believe that mortality of adults and dormancy of copepodites in response to anoxia was the probable reason for the late summer decline in planktonic C. b. thomasi.

  18. Improvements in proteomic metrics of low abundance proteins through proteome equalization using ProteoMiner prior to MudPIT

    PubMed Central

    Fonslow, Bryan R.; Carvalho, Paulo C.; Academia, Katrina; Freeby, Steve; Xu, Tao; Nakorchevsky, Aleksey; Paulus, Aran; Yates, John R.

    2011-01-01

    Ideally shotgun proteomics would facilitate the identification of an entire proteome with 100% protein sequence coverage. In reality, the large dynamic range and complexity of cellular proteomes results in oversampling of abundant proteins, while peptides from low abundance proteins are undersampled or remain undetected. We tested the proteome equalization technology, ProteoMiner, in conjunction with Multidimensional Protein Identification Technology (MudPIT) to determine how the equalization of protein dynamic range could improve shotgun proteomics methods for the analysis of cellular proteomes. Our results suggest low abundance protein identifications were improved by two mechanisms: (1) depletion of high abundance proteins freed ion trap sampling space usually occupied by high abundance peptides and (2) enrichment of low abundance proteins increased the probability of sampling their corresponding more abundant peptides. Both mechanisms also contributed to dramatic increases in the quantity of peptides identified and the quality of MS/MS spectra acquired due to increases in precursor intensity of peptides from low abundance proteins. From our large data set of identified proteins, we categorized the dominant physicochemical factors which facilitate proteome equalization with a hexapeptide library. These results illustrate that equalization of the dynamic range of the cellular proteome is a promising methodology to improve low abundance protein identification confidence, reproducibility, and sequence coverage in shotgun proteomics experiments, opening a new avenue of research for improving proteome coverage. PMID:21702434

  19. Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

    SciTech Connect

    Shi, Tujin; Sun, Xuefei; Gao, Yuqian; Fillmore, Thomas L.; Schepmoes, Athena A.; Zhao, Rui; He, Jintang; Moore, Ronald J.; Kagan, Jacob; Rodland, Karin D.; Liu, Tao; Liu, Alvin Y.; Smith, Richard D.; Tang, Keqi; Camp, David G.; Qian, Weijun

    2013-07-05

    We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50-100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion and the multiplexing potential of this technique. Limits of quantification (LOQs) at low ng/mL levels with a median CV of ~12% were achieved for proteins spiked into human female serum using as little as 2 µL serum. PRISM-SRM provided up to ~1000-fold improvement in the LOQ when compared to conventional SRM measurements. Multiplexing capability of PRISM-SRM was also evaluated by two sets of serum samples with 6 and 21 target peptides spiked at the low attomole/µL levels. The results from SRM measurements for pooled or post-concatenated samples were comparable to those obtained from individual peptide fractions in terms of signal-to-noise ratios and SRM peak area ratios of light to heavy peptides. PRISM-SRM was applied to measure several ng/mL-level endogenous plasma proteins, including prostate-specific antigen, in clinical patient sera where correlation coefficients > 0.99 were observed between the results from PRISM-SRM and ELISA assays. Our results demonstrate that PRISM-SRM can be successfully used for quantification of low-abundance endogenous proteins in highly complex samples. Moderate throughput (50 samples/week) can be achieved by applying the post-concatenation or fraction multiplexing strategies. We anticipate broad applications for targeted PRISM

  20. Protein rotational motion in solution measured by polarized fluorescence depletion.

    PubMed Central

    Yoshida, T M; Barisas, B G

    1986-01-01

    A microscope-based system is described for directly measuring protein rotational motion in viscous environments such as cell membranes by polarized fluorescence depletion (PFD). Proteins labeled with fluorophores having a high quantum yield for triplet formation, such as eosin isothiocyanate (EITC), are examined anaerobically in a fluorescence microscope. An acousto-optic modulator generates a several-microsecond pulse of linearly polarized light which produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. When the sample is then probed with light polarized parallel to the excitation pulse, fluorescence recovers over 0-1,000 microseconds as the sum of two exponentials. One exponential corresponds to triplet decay and the other to the rotational relaxation. An exciting pulse perpendicular to the probe beam is then applied. Fluorescence recovery following this pulse is the difference of the same two exponentials. Equations for fluorescence recovery kinetics to be expected in various experimentally significant cases are derived. Least-squares analysis using these equations then permits the triplet lifetime and rotational correlation time to be determined directly from PFD data. Instrumentation for PFD measurements is discussed that permits photobleaching recovery measurements of lateral diffusion coefficients using the same microscope system. With this apparatus, both rotational and translational diffusion coefficients (Dr, Dt) were measured for EITC-labeled bovine serum albumin in glycerol solutions. Values obtained for Dr and Dt are discussed in light of both the PFD models and the experimental system. PMID:3730506

  1. New observations of interstellar abundances and depletions of boron, vanadium, chromium, and cobalt

    NASA Technical Reports Server (NTRS)

    Snow, T. P., Jr.; Weiler, W. J.; Oegerle, W. R.

    1979-01-01

    New observations of interstellar lines of boron, vanadium, chromium, and cobalt in the spectra of Zeta Oph and Xi Per have been obtained with the Copernicus satellite. Chromium has been detected for the first time toward a reddened star, and cobalt has been seen for the first time in any interstellar line of sight. New limits have been obtained for boron and vanadium. These new data, along with limits on scandium and other species from the literature, have been compared with models for the depletion process. No fully conclusive test of depletion models is yet possible, but the new data on boron appear to favor the hypothesis that the depletions are dominated by accretion of gas-phase particles onto grains, rather than being due to grain condensation under pressure equilibrium. The impact of these new data on the study of grain surface properties is described.

  2. Estimating relative abundances of proteins from shotgun proteomics data

    PubMed Central

    2012-01-01

    Background Spectral counting methods provide an easy means of identifying proteins with differing abundances between complex mixtures using shotgun proteomics data. The crux spectral-counts command, implemented as part of the Crux software toolkit, implements four previously reported spectral counting methods, the spectral index (SIN), the exponentially modified protein abundance index (emPAI), the normalized spectral abundance factor (NSAF), and the distributed normalized spectral abundance factor (dNSAF). Results We compared the reproducibility and the linearity relative to each protein’s abundance of the four spectral counting metrics. Our analysis suggests that NSAF yields the most reproducible counts across technical and biological replicates, and both SIN and NSAF achieve the best linearity. Conclusions With the crux spectral-counts command, Crux provides open-source modular methods to analyze mass spectrometry data for identifying and now quantifying peptides and proteins. The C++ source code, compiled binaries, spectra and sequence databases are available at http://noble.gs.washington.edu/proj/crux-spectral-counts. PMID:23164367

  3. Consequences of defective vitamin A transportation on mitochondrial membrane integrity during protein depletion.

    PubMed

    Olowookere, J O

    1986-01-01

    The relationships between the structural integrity and functionality of rat liver mitochondrial membranes, and different levels of dietary protein and vitamin A transportation during protein depletion in animals have been investigated. Although the vitamin A content of the protein-depleted diet was 1680 +/- 35 IU/kg diet, and that of the control diet was 1,650 +/- 30 IU/kg diet, the vitamin A content of the liver of depleted rats was reduced to 16.7% of controls. The hepatic mitochondria of rats fed a protein-depleted diet showed excessive passive swelling (about 3-fold of controls) in isotonic solutions. Whereas a seemingly inverse relationship existed between the vitamin A content of the liver and the osmotic behaviour of hepatic mitochondria of rats fed a protein-depleted diet, there is a direct relationship between their hepatic mitochondrial vitamin A and the respiratory control ratio. The implications of these observations are discussed. PMID:3717896

  4. Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

    SciTech Connect

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B.; Bag, Jnanankur

    2011-05-13

    Highlights: {yields} Depletion of cellular PABP level arrests mRNA translation in HeLa cells. {yields} PABP knock down leads to apoptotic cell death. {yields} PABP depletion does not affect transcription. {yields} PABP depletion does not lead to nuclear accumulation of mRNA. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

  5. Total protein or high-abundance protein: Which offers the best loading control for Western blotting?

    PubMed

    Thacker, Jonathan S; Yeung, Derrick H; Staines, W Richard; Mielke, John G

    2016-03-01

    Western blotting routinely involves a control for variability in the amount of protein across immunoblot lanes. Normalizing a target signal to one found for an abundantly expressed protein is widely regarded as a reliable loading control; however, this approach is being increasingly questioned. As a result, we compared blotting for two high-abundance proteins (actin and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) and two total protein membrane staining methods (Ponceau and Coomassie Brilliant Blue) to determine the best control for loading variability. We found that Ponceau staining optimally balanced accuracy and precision, and we suggest that this approach be considered as an alternative to normalizing with a high-abundance protein. PMID:26706797

  6. Tumor cell migration and invasion are enhanced by depletion of Rap1 GTPase-activating protein (Rap1GAP).

    PubMed

    Tsygankova, Oxana M; Wang, Hongbin; Meinkoth, Judy L

    2013-08-23

    The functional significance of the widespread down-regulation of Rap1 GTPase-activating protein (Rap1GAP), a negative regulator of Rap activity, in human tumors is unknown. Here we show that human colon cancer cells depleted of Rap1GAP are endowed with more aggressive migratory and invasive properties. Silencing Rap1GAP enhanced the migration of confluent and single cells. In the latter, migration distance, velocity, and directionality were increased. Enhanced migration was a consequence of increased endogenous Rap activity as silencing Rap expression selectively abolished the migration of Rap1GAP-depleted cells. ROCK-mediated cell contractility was suppressed in Rap1GAP-depleted cells, which exhibited a spindle-shaped morphology and abundant membrane protrusions. Tumor cells can switch between Rho/ROCK-mediated contractility-based migration and Rac1-mediated mesenchymal motility. Strikingly, the migration of Rap1GAP-depleted, but not control cells required Rac1 activity, suggesting that loss of Rap1GAP alters migratory mechanisms. Inhibition of Rac1 activity restored membrane blebbing and increased ROCK activity in Rap1GAP-depleted cells, suggesting that Rac1 contributes to the suppression of contractility. Collectively, these findings identify Rap1GAP as a critical regulator of aggressive tumor cell behavior and suggest that the level of Rap1GAP expression influences the migratory mechanisms that are operative in tumor cells. PMID:23864657

  7. Antibody arrays for determination of relative protein abundances.

    PubMed

    Chaga, Grigoriy S

    2008-01-01

    As a large number of genome-sequencing projects reached completion, the attention of the scientific community is turning toward understanding the structure-functions of gene translation products-the proteins as well as the complete complement of proteins-the proteome. One goal of proteomics is to correlate changes in protein abundance with biological processes and disease states. To help accelerate this avenue of proteomics, a significant effort has been devoted to the development of multiplexed methods for protein analyses. We have developed an Antibody Microarray, a chip-based technology for multiparallel determination of relative abundance of hundreds of proteins. The Antibody Microarray is composed of hundreds of distinct monoclonal antibodies printed at high density on a glass slide. It utilizes a novel experimental setup and data analysis algorithm, which enables scientists to assay hundreds of cytosolic, nuclear, and membrane-bound proteins with a single experiment. Examples of biological samples that are analyzed on the Antibody Microarray include tissue samples, cell cultures, and body fluids. PMID:18370316

  8. Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions, LC-MS/MS, and multivariate analysis

    PubMed Central

    Madsen, James A; Farutin, Victor; Carbeau, Theresa; Wudyka, Steve; Yin, Yan; Smith, Stephen; Anderson, James; Capila, Ishan

    2015-01-01

    Host cell protein (HCP) impurities are generated by the host organism during the production of therapeutic recombinant proteins, and are difficult to remove completely. Though commonly present in small quantities, if levels are not controlled, HCPs can potentially reduce drug efficacy and cause adverse patient reactions. A high resolution approach for thorough HCP characterization of therapeutic monoclonal antibodies is presented herein. In this method, antibody samples are first depleted via affinity enrichment (e.g., Protein A, Protein L) using milligram quantities of material. The HCP-containing flow-through is then enzymatically digested, analyzed using nano-UPLC-MS/MS, and proteins are identified through database searching. Nearly 700 HCPs were identified from samples with very low total HCP levels (< 1 ppm to ∼10 ppm) using this method. Quantitation of individual HCPs was performed using normalized spectral counting as the number of peptide spectrum matches (PSMs) per protein is proportional to protein abundance. Multivariate analysis tools were utilized to assess similarities between HCP profiles by: 1) quantifying overlaps between HCP identities; and 2) comparing correlations between individual protein abundances as calculated by spectral counts. Clustering analysis using these measures of dissimilarity between HCP profiles enabled high resolution differentiation of commercial grade monoclonal antibody samples generated from different cell lines, cell culture, and purification processes. PMID:26291024

  9. Abundant protein phosphorylation potentially regulates Arabidopsis anther development

    PubMed Central

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-01-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana. However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4–7 and 8–12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  10. Abundant protein phosphorylation potentially regulates Arabidopsis anther development.

    PubMed

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-09-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4-7 and 8-12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  11. Topology of Protein Interaction Network Shapes Protein Abundances and Strengths of Their Functional and Nonspecific Interactions

    SciTech Connect

    Maslov, S.; Heo, M.; Shakhnovich, E.

    2011-03-08

    How do living cells achieve sufficient abundances of functional protein complexes while minimizing promiscuous nonfunctional interactions? Here we study this problem using a first-principle model of the cell whose phenotypic traits are directly determined from its genome through biophysical properties of protein structures and binding interactions in a crowded cellular environment. The model cell includes three independent prototypical pathways, whose topologies of protein-protein interaction (PPI) subnetworks are different, but whose contributions to the cell fitness are equal. Model cells evolve through genotypic mutations and phenotypic protein copy number variations. We found a strong relationship between evolved physical-chemical properties of protein interactions and their abundances due to a 'frustration' effect: Strengthening of functional interactions brings about hydrophobic interfaces, which make proteins prone to promiscuous binding. The balancing act is achieved by lowering concentrations of hub proteins while raising solubilities and abundances of functional monomers. On the basis of these principles we generated and analyzed a possible realization of the proteome-wide PPI network in yeast. In this simulation we found that high-throughput affinity capture-mass spectroscopy experiments can detect functional interactions with high fidelity only for high-abundance proteins while missing most interactions for low-abundance proteins.

  12. Cellular retinol-binding protein and retinoic acid-binding protein in rat testes: effect of retinol depletion.

    PubMed

    Ong, D E; Tsai, C H; Chytil, F

    1976-02-01

    Testes of rats contain two cellular binding proteins of interest in vitamin A metabolism. One protein binds retinoic acid with high specificity; the other binds retinol with high specificity. When the cellular retinol-binding protein was partially purified from rat testes, it exhibited fluorescence excitation and emission spectra similar to that of all-trans-retinol in hexane. Exposure of this preparation to UV light destroyed this fluorescence but spectra identical to the original were obtained after addition of retinol. Hexane extracts of the binding protein had fluorescence spectra identical to all-trans-retinol, suggesting that this compound is bound to the protein in vivo. Extracts of testes from retinol depleted rats were submitted to gel filtration but failed to show a retinol-like fluorescence at the elution position of retinol binding protein. This fluorescence was observed in the preparations from pair fed control animals. However, after addition of all-trans-retinol to the extracts from the depleted rats, fluorescence at that elution position was observed. This indicates that in testes of retinol depleted rats the cellular retinol binding protein is present but without bound retinol, in contrast to the non-depleted rats where 30-43% of the binding protein had bound retinol. The amounts of cellular retinol binding protein and retinoic acid binding protein in testes, as determined by sucrose gradient centrifugation, were found to be similar for retinol depleted and pair fed control rats. PMID:942996

  13. Determination of optimal protein quantity required to identify abundant and less abundant soybean seed proteins by 2D-PAGE and MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Optimizing the amounts of proteins required to separate and characterize both abundant and less abundant proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is critical for conducting proteomic research. In this study, we tested five different levels of soybean seed proteins (7...

  14. Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants.

    PubMed

    Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Mendes-Silva, Leonardo; Pinho Melo, Eduardo; Harding, Heather P; Ron, David

    2014-01-01

    Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1(CtoS) purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER.DOI: http://dx.doi.org/10.7554/eLife.03421.001. PMID:25073928

  15. Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants

    PubMed Central

    Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Mendes-Silva, Leonardo; Pinho Melo, Eduardo; Harding, Heather P; Ron, David

    2014-01-01

    Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER. DOI: http://dx.doi.org/10.7554/eLife.03421.001 PMID:25073928

  16. Practical Immunoaffinity-Enrichment LC-MS for Measuring Protein Kinetics of Low-Abundance Proteins

    PubMed Central

    Lassman, Michael E.; McAvoy, Thomas; Lee, Anita Y.H.; Chappell, Derek; Wong, Oitak; Zhou, Haihong; Reyes-Soffer, Gissette; Ginsberg, Henry N.; Millar, John S.; Rader, Daniel J.; Gutstein, David E.; Laterza, Omar

    2016-01-01

    BACKGROUND For a more complete understanding of pharmacodynamic, metabolic, and pathophysiologic effects, protein kinetics, such as production rate and fractional catabolic rate, can offer substantially more information than protein concentration alone. Kinetic experiments with stable isotope tracers typically require laborious sample preparation and are most often used for studying abundant proteins. Here we describe a practical methodology for measuring isotope enrichment into low-abundance proteins that uses an automated procedure and immunoaffinity enrichment (IA) with LC-MS. Low-abundance plasma proteins cholesteryl ester transfer protein (CETP) and proprotein convertase subtilisin/kexin type 9 (PCSK9) were studied as examples. METHODS Human participants (n = 39) were infused with [2H3]leucine, and blood samples were collected at multiple time points. Sample preparation and analysis were automated and multiplexed to increase throughput. Proteins were concentrated from plasma by use of IA and digested with trypsin to yield proteotypic peptides that were analyzed by microflow chromatography-mass spectrometry to measure isotope enrichment. RESULTS The IA procedure was optimized to provide the greatest signal intensity. Use of a gel-free method increased throughput while increasing the signal. The intra- and interassay CVs were <15% at all isotope enrichment levels studied. More than 1400 samples were analyzed in <3 weeks without the need for instrument stoppages or user interventions. CONCLUSIONS The use of automated gel-free methods to multiplex the measurement of isotope enrichment was applied to the low-abundance proteins CETP and PCSK9. PMID:24751376

  17. Arginine Depletion by Arginine Deiminase Does Not Affect Whole Protein Metabolism or Muscle Fractional Protein Synthesis Rate in Mice

    PubMed Central

    Marini, Juan C.; Didelija, Inka Cajo

    2015-01-01

    Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depletion can potentially exacerbate the progressive loss of body weight, and especially lean body mass, in cancer patients we determined the effect of arginine depletion by pegylated arginine deiminase (ADI-PEG 20) on whole body protein synthesis and fractional protein synthesis rate in multiple tissues of mice. ADI-PEG 20 successfully depleted circulating arginine (<1 μmol/L), and increased citrulline concentration more than tenfold. Body weight and body composition, however, were not affected by ADI-PEG 20. Despite the depletion of arginine, whole body protein synthesis and breakdown were maintained in the ADI-PEG 20 treated mice. The fractional protein synthesis rate of muscle was also not affected by arginine depletion. Most tissues (liver, kidney, spleen, heart, lungs, stomach, small and large intestine, pancreas) were able to maintain their fractional protein synthesis rate; however, the fractional protein synthesis rate of brain, thymus and testicles was reduced due to the ADI-PEG 20 treatment. Furthermore, these results were confirmed by the incorporation of ureido [14C]citrulline, which indicate the local conversion into arginine, into protein. In conclusion, the intracellular recycling pathway of citrulline is able to provide enough arginine to maintain protein synthesis rate and prevent the loss of lean body mass and body weight. PMID:25775142

  18. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.

    PubMed

    Natsume, Toyoaki; Kiyomitsu, Tomomi; Saga, Yumiko; Kanemaki, Masato T

    2016-04-01

    Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function. PMID:27052166

  19. Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance1[W

    PubMed Central

    Nikolovski, Nino; Shliaha, Pavel V.; Gatto, Laurent; Dupree, Paul; Lilley, Kathryn S.

    2014-01-01

    The proteomic composition of the Arabidopsis (Arabidopsis thaliana) Golgi apparatus is currently reasonably well documented; however, little is known about the relative abundances between different proteins within this compartment. Accurate quantitative information of Golgi resident proteins is of great importance: it facilitates a better understanding of the biochemical processes that take place within this organelle, especially those of different polysaccharide synthesis pathways. Golgi resident proteins are challenging to quantify because the abundance of this organelle is relatively low within the cell. In this study, an organelle fractionation approach targeting the Golgi apparatus was combined with a label-free quantitative mass spectrometry (data-independent acquisition method using ion mobility separation known as LC-IMS-MSE [or HDMSE]) to simultaneously localize proteins to the Golgi apparatus and assess their relative quantity. In total, 102 Golgi-localized proteins were quantified. These data show that organelle fractionation in conjunction with label-free quantitative mass spectrometry is a powerful and relatively simple tool to access protein organelle localization and their relative abundances. The findings presented open a unique view on the organization of the plant Golgi apparatus, leading toward unique hypotheses centered on the biochemical processes of this organelle. PMID:25122472

  20. Experimental evidence for the stability of the depletion zone around a growing protein crystal under microgravity.

    PubMed

    Otálora, F; Novella, M L; Gavira, J A; Thomas, B R; García Ruiz, J M

    2001-03-01

    Experimental evidence is presented for the first time for the development and time evolution of concentration-depletion zones around protein crystals growing in microgravity and gelled on-ground experiments. Crystal motion and buoyancy-driven fluid movements as a result of residual accelerations and g-jitters are demonstrated to have an adverse effect on the stability of these depletion zones, provoking the breakdown of their radial symmetry. These findings may explain some of the controversial results reported on the quality of single crystals grown under microgravity in previous space missions. PMID:11223518

  1. An efficient extraction method to enhance analysis of low abundant proteins from soybean seed.

    PubMed

    Natarajan, Savithiry S; Krishnan, Hari B; Lakshman, Sukla; Garrett, Wesley M

    2009-11-15

    Large amounts of the major storage proteins, beta-conglycinin and glycinin, in soybean (Glycine max) seeds hinder the isolation and characterization of less abundant seed proteins. We investigated whether isopropanol extraction could facilitate resolution of the low abundant proteins, different from the main storage protein fractions, in one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 1D-PAGE of proteins extracted by different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%) of isopropanol showed that greater than 30% isopropanol was suitable for preferential enrichment of low abundant proteins. Analysis of 2D-PAGE showed that proteins which were less abundant or absent by the conventional extraction procedure were clearly seen in the 40% isopropanol extracts. Increasing isopropanol concentration above 40% resulted in a decrease in the number of less abundant protein spots. We have identified a total of 107 protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrophotometry (MALDI-TOF-MS) and liquid chromatography-mass spectrometry (LC-MS/MS). Our results suggest that extraction of soybean seed powder with 40% isopropanol enriches lower abundance proteins and is a suitable method for 2D-PAGE separation and identification. This methodology could potentially allow the extraction and characterization of low abundant proteins of other legume seeds containing highly abundant storage proteins. PMID:19651100

  2. An efficient extraction method to enhance analysis of low abundant proteins from soybean seed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Large amounts of the major seed storage proteins, such as ß-conglycinin and glycinin, in soybean (Glycine max) seeds hinder the isolation and characterization of less abundant seed proteins. We investigated whether isopropanol extraction could facilitate resolution of the less abundant proteins fro...

  3. EXAMINATION OF THE MASS-DEPENDENT Li DEPLETION HYPOTHESIS BY THE Li ABUNDANCES OF THE VERY METAL-POOR DOUBLE-LINED SPECTROSCOPIC BINARY G166-45

    SciTech Connect

    Aoki, Wako; Ito, Hiroko; Tajitsu, Akito

    2012-05-20

    The Li abundances of the two components of the very metal-poor ([Fe/H] -2.5) double-lined spectroscopic binary G166-45 (BD+26 Degree-Sign 2606) are determined separately based on high-resolution spectra obtained with the Subaru Telescope High Dispersion Spectrograph and its image slicer. From the photometric colors and the mass ratio, the effective temperatures of the primary and secondary components are estimated to be 6350 {+-} 100 K and 5830 {+-} 170 K, respectively. The Li abundance of the primary (A(Li) = 2.23) agrees well with the Spite plateau value, while that of the secondary is slightly lower (A(Li) = 2.11). Such a discrepancy of the Li abundances between the two components is previously found in the extremely metal-poor, double-lined spectroscopic binary CS 22876-032; however, the discrepancy in G166-45 is much smaller. The results agree with the trends found for Li abundance as a function of effective temperature (and of stellar mass) of main-sequence stars with -3.0 < [Fe/H] < -2.0, suggesting that the depletion of Li at T{sub eff} {approx} 5800 K is not particularly large in this metallicity range. The significant Li depletion found in CS 22876-032B is a phenomenon only found in the lowest metallicity range ([Fe/H] < -3).

  4. Mass spectrometry in cancer biomarker research: a case for immunodepletion of abundant blood-derived proteins from clinical tissue specimens

    PubMed Central

    Prieto, DaRue A; Johann, Donald J; Wei, Bih-Rong; Ye, Xiaoying; Chan, King C; Nissley, Dwight V; Simpson, R Mark; Citrin, Deborah E; Mackall, Crystal L; Linehan, W Marston; Blonder, Josip

    2014-01-01

    The discovery of clinically relevant cancer biomarkers using mass spectrometry (MS)-based proteomics has proven difficult, primarily because of the enormous dynamic range of blood-derived protein concentrations and the fact that the 22 most abundant blood-derived proteins constitute approximately 99% of the total plasma protein mass. Immunodepletion of clinical body fluid specimens (e.g., serum/plasma) for the removal of highly abundant proteins is a reasonable and reproducible solution. Often overlooked, clinical tissue specimens also contain a formidable amount of highly abundant blood-derived proteins present in tissue-embedded networks of blood/lymph capillaries and interstitial fluid. Hence, the dynamic range impediment to biomarker discovery remains a formidable obstacle, regardless of clinical sample type (solid tissue and/or body fluid). Thus, we optimized and applied simultaneous immunodepletion of blood-derived proteins from solid tissue and peripheral blood, using clear cell renal cell carcinoma as a model disease. Integrative analysis of data from this approach and genomic data obtained from the same type of tumor revealed concordant key pathways and protein targets germane to clear cell renal cell carcinoma. This includes the activation of the lipogenic pathway characterized by increased expression of adipophilin (PLIN2) along with 'cadherin switching', a phenomenon indicative of transcriptional reprogramming linked to renal epithelial dedifferentiation. We also applied immunodepletion of abundant blood-derived proteins to various tissue types (e.g., adipose tissue and breast tissue) showing unambiguously that the removal of abundant blood-derived proteins represents a powerful tool for the reproducible profiling of tissue proteomes. Herein, we show that the removal of abundant blood-derived proteins from solid tissue specimens is of equal importance to depletion of body fluids and recommend its routine use in the context of biological discovery and

  5. Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depl...

  6. Expect the Unexpected Enrichment of "Hidden Proteome" of Seeds and Tubers by Depletion of Storage Proteins.

    PubMed

    Gupta, Ravi; Min, Cheol W; Wang, Yiming; Kim, Yong C; Agrawal, Ganesh K; Rakwal, Randeep; Kim, Sun T

    2016-01-01

    Dynamic resolution of seed and tuber protein samples is highly limited due to the presence of high-abundance storage proteins (SPs). These proteins inevitably obscure the low-abundance proteins (LAPs) impeding their identification and characterization. To facilitate the detection of LAPs, several methods have been developed during the past decade, enriching the proteome with extreme proteins. Most of these methods, if not all, are based on the specific removal of SPs which ultimately magnify the proteome coverage. In this mini-review, we summarize the available methods that have been developed over the years for the enrichment of LAPs either from seeds or tubers. Incorporation of these methods during the protein extraction step will be helpful in understanding the seed/tuber biology in greater detail. PMID:27313590

  7. Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control[W

    PubMed Central

    Ramundo, Silvia; Casero, David; Mühlhaus, Timo; Hemme, Dorothea; Sommer, Frederik; Crèvecoeur, Michèle; Rahire, Michèle; Schroda, Michael; Rusch, Jannette; Goodenough, Ursula; Pellegrini, Matteo; Perez-Perez, Maria Esther; Crespo, José Luis; Schaad, Olivier; Civic, Natacha; Rochaix, Jean David

    2014-01-01

    Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria. PMID:24879428

  8. Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control.

    PubMed

    Ramundo, Silvia; Casero, David; Mühlhaus, Timo; Hemme, Dorothea; Sommer, Frederik; Crèvecoeur, Michèle; Rahire, Michèle; Schroda, Michael; Rusch, Jannette; Goodenough, Ursula; Pellegrini, Matteo; Perez-Perez, Maria Esther; Crespo, José Luis; Schaad, Olivier; Civic, Natacha; Rochaix, Jean David

    2014-05-30

    Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria. PMID:24879428

  9. Regular Patterns for Proteome-Wide Distribution of Protein Abundance across Species

    PubMed Central

    Jiang, Ying; Ying, Wantao; Wu, Songfeng; Zhu, Yunping; Liu, Siqi; Yang, Pengyuan; Qian, Xiaohong; He, Fuchu

    2012-01-01

    A proteome of the bio-entity, including cell, tissue, organ, and organism, consists of proteins of diverse abundance. The principle that determines the abundance of different proteins in a proteome is of fundamental significance for an understanding of the building blocks of the bio-entity. Here, we report three regular patterns in the proteome-wide distribution of protein abundance across species such as human, mouse, fly, worm, yeast, and bacteria: in most cases, protein abundance is positively correlated with the protein's origination time or sequence conservation during evolution; it is negatively correlated with the protein's domain number and positively correlated with domain coverage in protein structure, and the correlations became stronger during the course of evolution; protein abundance can be further stratified by the function of the protein, whereby proteins that act on material conversion and transportation (mass category) are more abundant than those that act on information modulation (information category). Thus, protein abundance is intrinsically related to the protein's inherent characters of evolution, structure, and function. PMID:22427835

  10. Rationalization of Membrane Protein Crystallization with Polyethylene Glycol Using a Simple Depletion Model

    PubMed Central

    Tanaka, Shinpei; Ataka, Mitsuo; Onuma, Kazuo; Kubota, Tomomi

    2003-01-01

    Based on the importance of crystallizing membrane proteins in a rational way, cytochrome bc1 complex (BC1) was crystallized using polyethylene glycol (PEG) as a sole crystallization agent. Interaction between protein-detergent complexes of BC1 was estimated by dynamic light scattering, and was compared with the numerical calculation using the Derjaguin-Landau-Verwey-Overbeek potential plus a depletion potential, without considering specific surface properties of the protein-detergent complexes. The experiments and calculation were found to be consistent and we obtained a relation between PEG molecular weight M and the range of depletion zone δ as δ ∼ M0.48±0.02. The stability of liquid phase of BC1 solutions was controlled by a ratio of (the range of depletion zone)/(the radius of a BC1 particle), which was consistent with recent theoretical predictions. The crystallization was most successful under a condition where the stability of the liquid phase changed from stable to unstable. The PEG molecular weight that fulfilled this condition coincided with the one used empirically to crystallize BC1 in the past by a number of groups. These results are compared to the fact that membrane proteins were often successfully crystallized close to the detergent cloud point. PMID:12719259

  11. Glutathione depletion and acute exercise increase O-GlcNAc protein modification in rat skeletal muscle.

    PubMed

    Peternelj, Tina Tinkara; Marsh, Susan A; Strobel, Natalie A; Matsumoto, Aya; Briskey, David; Dalbo, Vincent J; Tucker, Patrick S; Coombes, Jeff S

    2015-02-01

    Post-translational modification of intracellular proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) profoundly affects protein structure, function, and metabolism. Although many skeletal muscle proteins are O-GlcNAcylated, the modification has not been extensively studied in this tissue, especially in the context of exercise. This study investigated the effects of glutathione depletion and acute exercise on O-GlcNAc protein modification in rat skeletal muscle. Diethyl maleate (DEM) was used to deplete intracellular glutathione and rats were subjected to a treadmill run. White gastrocnemius and soleus muscles were analyzed for glutathione status, O-GlcNAc and O-GlcNAc transferase (OGT) protein levels, and mRNA expression of OGT, O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase. DEM and exercise both reduced intracellular glutathione and increased O-GlcNAc. DEM upregulated OGT protein expression. The effects of the interventions were significant 4 h after exercise (P < 0.05). The changes in the mRNA levels of O-GlcNAc enzymes were different in the two muscles, potentially resulting from different rates of oxidative stress and metabolic demands between the muscle types. These findings indicate that oxidative environment promotes O-GlcNAcylation in skeletal muscle and suggest an interrelationship between cellular redox state and O-GlcNAc protein modification. This could represent one mechanism underlying cellular adaptation to oxidative stress and health benefits of exercise. PMID:25416863

  12. Proteomics-Based Identification of Differentially Abundant Proteins from Human Keratinocytes Exposed to Arsenic Trioxide

    PubMed Central

    Udensi, Udensi K; Tackett, Alan J; Byrum, Stephanie; Avaritt, Nathan L; Sengupta, Deepanwita; Moreland, Linley W; Tchounwou, Paul B; Isokpehi, Raphael D

    2014-01-01

    Introduction Arsenic is a widely distributed environmental toxicant that can cause multi-tissue pathologies. Proteomic assays allow for the identification of biological processes modulated by arsenic in diverse tissue types. Method The altered abundance of proteins from HaCaT human keratinocyte cell line exposed to arsenic was quantified using a label-free LC-MS/MS mass spectrometry workflow. Selected proteomics results were validated using western blot and RT-PCR. A functional annotation analytics strategy that included visual analytical integration of heterogeneous data sets was developed to elucidate functional categories. The annotations integrated were mainly tissue localization, biological process and gene family. Result The abundance of 173 proteins was altered in keratinocytes exposed to arsenic; in which 96 proteins had increased abundance while 77 proteins had decreased abundance. These proteins were also classified into 69 Gene Ontology biological process terms. The increased abundance of transferrin receptor protein (TFRC) was validated and also annotated to participate in response to hypoxia. A total of 33 proteins (11 increased abundance and 22 decreased abundance) were associated with 18 metabolic process terms. The Glutamate--cysteine ligase catalytic subunit (GCLC), the only protein annotated with the term sulfur amino acid metabolism process, had increased abundance while succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial precursor (SDHB), a tumor suppressor, had decreased abundance. Conclusion A list of 173 differentially abundant proteins in response to arsenic trioxide was grouped using three major functional annotations covering tissue localization, biological process and protein families. A possible explanation for hyperpigmentation pathologies observed in arsenic toxicity is that arsenic exposure leads to increased iron uptake in the normally hypoxic human skin. The proteins mapped to metabolic process terms and

  13. A Simple Procedure for Depletion of Storage Proteins From Soybean (Glycine max) Seeds to Aid Proteome Analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two-dimensional electrophoretic analysis of plant proteomes containing thousands of proteins, has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the low abundance proteins within seeds is difficult when 60 – 80% is storage proteins. Resolution can ...

  14. HIGH PRECISION ABUNDANCES OF THE OLD SOLAR TWIN HIP 102152: INSIGHTS ON Li DEPLETION FROM THE OLDEST SUN

    SciTech Connect

    Monroe, TalaWanda R.; Melendez, Jorge; Tucci Maia, Marcelo; Freitas, Fabricio C.; Yong, David; Asplund, Martin; Alves-Brito, Alan; Casagrande, Luca; Bergemann, Maria; Bedell, Megan; Bean, Jacob; Lind, Karin; Castro, Matthieu; Do Nascimento, Jose-Dias; Bazot, Michael

    2013-09-10

    We present the first detailed chemical abundance analysis of the old 8.2 Gyr solar twin, HIP 102152. We derive differential abundances of 21 elements relative to the Sun with precisions as high as 0.004 dex ({approx}<1%), using ultra high-resolution (R = 110,000), high S/N UVES spectra obtained on the 8.2 m Very Large Telescope. Our determined metallicity of HIP 102152 is [Fe/H] = -0.013 {+-} 0.004. The atmospheric parameters of the star were determined to be 54 K cooler than the Sun, 0.09 dex lower in surface gravity, and a microturbulence identical to our derived solar value. Elemental abundance ratios examined versus dust condensation temperature reveal a solar abundance pattern for this star, in contrast to most solar twins. The abundance pattern of HIP 102152 appears to be the most similar to solar of any known solar twin. Abundances of the younger, 2.9 Gyr solar twin, 18 Sco, were also determined from UVES spectra to serve as a comparison for HIP 102152. The solar chemical pattern of HIP 102152 makes it a potential candidate to host terrestrial planets, which is reinforced by the lack of giant planets in its terrestrial planet region. The following non-local thermodynamic equilibrium Li abundances were obtained for HIP 102152, 18 Sco, and the Sun: log {epsilon} (Li) = 0.48 {+-} 0.07, 1.62 {+-} 0.02, and 1.07 {+-} 0.02, respectively. The Li abundance of HIP 102152 is the lowest reported to date for a solar twin, and allows us to consider an emerging, tightly constrained Li-age trend for solar twin stars.

  15. High Precision Abundances of the Old Solar Twin HIP 102152: Insights on Li Depletion from the Oldest Sun

    NASA Astrophysics Data System (ADS)

    Monroe, TalaWanda R.; Meléndez, Jorge; Ramírez, Iván; Yong, David; Bergemann, Maria; Asplund, Martin; Bedell, Megan; Tucci Maia, Marcelo; Bean, Jacob; Lind, Karin; Alves-Brito, Alan; Casagrande, Luca; Castro, Matthieu; do Nascimento, José-Dias; Bazot, Michael; Freitas, Fabrício C.

    2013-09-01

    We present the first detailed chemical abundance analysis of the old 8.2 Gyr solar twin, HIP 102152. We derive differential abundances of 21 elements relative to the Sun with precisions as high as 0.004 dex (lsim1%), using ultra high-resolution (R = 110,000), high S/N UVES spectra obtained on the 8.2 m Very Large Telescope. Our determined metallicity of HIP 102152 is [Fe/H] = -0.013 ± 0.004. The atmospheric parameters of the star were determined to be 54 K cooler than the Sun, 0.09 dex lower in surface gravity, and a microturbulence identical to our derived solar value. Elemental abundance ratios examined versus dust condensation temperature reveal a solar abundance pattern for this star, in contrast to most solar twins. The abundance pattern of HIP 102152 appears to be the most similar to solar of any known solar twin. Abundances of the younger, 2.9 Gyr solar twin, 18 Sco, were also determined from UVES spectra to serve as a comparison for HIP 102152. The solar chemical pattern of HIP 102152 makes it a potential candidate to host terrestrial planets, which is reinforced by the lack of giant planets in its terrestrial planet region. The following non-local thermodynamic equilibrium Li abundances were obtained for HIP 102152, 18 Sco, and the Sun: log epsilon (Li) = 0.48 ± 0.07, 1.62 ± 0.02, and 1.07 ± 0.02, respectively. The Li abundance of HIP 102152 is the lowest reported to date for a solar twin, and allows us to consider an emerging, tightly constrained Li-age trend for solar twin stars. Based on observations obtained at the European Southern Observatory (observing programs 083.D-0871 and 188.C-0265).

  16. Increasing the immune activity of exosomes: the effect of miRNA-depleted exosome proteins on activating dendritic cell/cytokine-induced killer cells against pancreatic cancer* #

    PubMed Central

    Que, Ri-sheng; Lin, Cheng; Ding, Guo-ping; Wu, Zheng-rong; Cao, Li-ping

    2016-01-01

    Background: Tumor-derived exosomes were considered to be potential candidates for tumor vaccines because they are abundant in immune-regulating proteins, whereas tumor exosomal miRNAs may induce immune tolerance, thereby having an opposite immune function. Objective: This study was designed to separate exosomal protein and depleted exosomal microRNAs (miRNAs), increasing the immune activity of exosomes for activating dendritic cell/cytokine-induced killer cells (DC/CIKs) against pancreatic cancer (PC). Methods: PC-derived exosomes (PEs) were extracted from cultured PANC-1 cell supernatants and then ruptured; this was followed by ultrafiltered exosome lysates (UELs). DCs were stimulated with lipopolysaccharide (LPS), PE, and UEL, followed by co-culture with CIKs. The anti-tumor effects of DC/CIKs against PC were evaluated by proliferation and killing rates, tumor necrosis factor-α (TNF-α) and perforin secretion. Exosomal miRNAs were depleted after lysis and ultrafiltration, while 128 proteins were retained, including several immune-activating proteins. Results: UEL-stimulated DC/CIKs showed a higher killing rate than LPS- and PE-stimulated DC/CIKs. Conclusions: miRNA-depleted exosome proteins may be promising agonists for specifically activating DC/CIKs against PC. PMID:27143262

  17. Mitotic catastrophe and cell death induced by depletion of centrosomal proteins

    PubMed Central

    Kimura, M; Yoshioka, T; Saio, M; Banno, Y; Nagaoka, H; Okano, Y

    2013-01-01

    Mitotic catastrophe, which refers to cell death or its prologue triggered by aberrant mitosis, can be induced by a heterogeneous group of stimuli, including chromosome damage or perturbation of the mitotic apparatus. We investigated the mechanism of mitotic catastrophe and cell death induced by depletion of centrosomal proteins that perturbs microtubule organization. We transfected cells harboring wild-type or mutated p53 with siRNAs targeting Aurora A, ninein, TOG, TACC3, γ-tubulin, or pericentriolar material-1, and monitored the effects on cell death. Knockdown of Aurora A, ninein, TOG, and TACC3 led to cell death, regardless of p53 status. Knockdown of Aurora A, ninein, and TOG, led to aberrant spindle formation and subsequent cell death, which was accompanied by several features of apoptosis, including nuclear condensation and Annexin V binding in HeLa cells. During this process, cleavage of poly(ADP-ribose) polymerase-1, caspase-3, and caspase-9 was detected, but cleavage of caspase-8 was not. Cell death, monitored by time-lapse imaging, occurred during both interphase and M phase. In cells depleted of a centrosomal protein (Aurora A, ninein, or TOG), the rate of cell death was higher if the cells were cotransfected with siRNA against BubR1 or Mad2 than if they were transfected with siRNA against Bub1 or a control siRNA. These results suggest that metaphase arrest is necessary for the mitotic catastrophe and cell death caused by depletion of centrosomal proteins. Knockdown of centrosomal proteins led to increased phosphorylation of Chk2. Enhanced p-Chk2 localization was also observed at the centrosome in cells arrested in M phase, as well as in the nuclei of dying cells. Cotransfection of siRNAs against Chk2, in combination with depletion of a centrosomal protein, decreased the amount of cell death. Thus, Chk2 activity is indispensable for apoptosis after mitotic catastrophe induced by depletion of centrosomal proteins that perturbs microtubule organization

  18. Impact of Protein Stability, Cellular Localization, and Abundance on Proteomic Detection of Tumor-Derived Proteins in Plasma

    PubMed Central

    Faca, Vitor M.; Zhang, Wenxuan; Zhang, Qing; Jain, Anjali; Hanash, Sam; Agus, David B.; McIntosh, Martin W.; Mallick, Parag

    2011-01-01

    Tumor-derived, circulating proteins are potentially useful as biomarkers for detection of cancer, for monitoring of disease progression, regression and recurrence, and for assessment of therapeutic response. Here we interrogated how a protein's stability, cellular localization, and abundance affect its observability in blood by mass-spectrometry-based proteomics techniques. We performed proteomic profiling on tumors and plasma from two different xenograft mouse models. A statistical analysis of this data revealed protein properties indicative of the detection level in plasma. Though 20% of the proteins identified in plasma were tumor-derived, only 5% of the proteins observed in the tumor tissue were found in plasma. Both intracellular and extracellular tumor proteins were observed in plasma; however, after normalizing for tumor abundance, extracellular proteins were seven times more likely to be detected. Although proteins that were more abundant in the tumor were also more likely to be observed in plasma, the relationship was nonlinear: Doubling the spectral count increased detection rate by only 50%. Many secreted proteins, even those with relatively low spectral count, were observed in plasma, but few low abundance intracellular proteins were observed. Proteins predicted to be stable by dipeptide composition were significantly more likely to be identified in plasma than less stable proteins. The number of tryptic peptides in a protein was not significantly related to the chance of a protein being observed in plasma. Quantitative comparison of large versus small tumors revealed that the abundance of proteins in plasma as measured by spectral count was associated with the tumor size, but the relationship was not one-to-one; a 3-fold decrease in tumor size resulted in a 16-fold decrease in protein abundance in plasma. This study provides quantitative support for a tumor-derived marker prioritization strategy that favors secreted and stable proteins over all but the

  19. Impact of protein stability, cellular localization, and abundance on proteomic detection of tumor-derived proteins in plasma.

    PubMed

    Fang, Qiaojun; Kani, Kian; Faca, Vitor M; Zhang, Wenxuan; Zhang, Qing; Jain, Anjali; Hanash, Sam; Agus, David B; McIntosh, Martin W; Mallick, Parag

    2011-01-01

    Tumor-derived, circulating proteins are potentially useful as biomarkers for detection of cancer, for monitoring of disease progression, regression and recurrence, and for assessment of therapeutic response. Here we interrogated how a protein's stability, cellular localization, and abundance affect its observability in blood by mass-spectrometry-based proteomics techniques. We performed proteomic profiling on tumors and plasma from two different xenograft mouse models. A statistical analysis of this data revealed protein properties indicative of the detection level in plasma. Though 20% of the proteins identified in plasma were tumor-derived, only 5% of the proteins observed in the tumor tissue were found in plasma. Both intracellular and extracellular tumor proteins were observed in plasma; however, after normalizing for tumor abundance, extracellular proteins were seven times more likely to be detected. Although proteins that were more abundant in the tumor were also more likely to be observed in plasma, the relationship was nonlinear: Doubling the spectral count increased detection rate by only 50%. Many secreted proteins, even those with relatively low spectral count, were observed in plasma, but few low abundance intracellular proteins were observed. Proteins predicted to be stable by dipeptide composition were significantly more likely to be identified in plasma than less stable proteins. The number of tryptic peptides in a protein was not significantly related to the chance of a protein being observed in plasma. Quantitative comparison of large versus small tumors revealed that the abundance of proteins in plasma as measured by spectral count was associated with the tumor size, but the relationship was not one-to-one; a 3-fold decrease in tumor size resulted in a 16-fold decrease in protein abundance in plasma. This study provides quantitative support for a tumor-derived marker prioritization strategy that favors secreted and stable proteins over all but the

  20. Sensing Small Changes in Protein Abundance: Stimulation of Caco-2 Cells by Human Whey Proteins.

    PubMed

    Cundiff, Judy K; McConnell, Elizabeth J; Lohe, Kimberly J; Maria, Sarah D; McMahon, Robert J; Zhang, Qiang

    2016-01-01

    Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FCs) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein, we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5-40% changes in protein abundance, with the variance controls of ± 5% FC for around 95% of TMT ratios when sampling 9-12 biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development. PMID:26586228

  1. Selective Membrane Redistribution and Depletion of Gαq-Protein by Pasteurella multocida Toxin.

    PubMed

    Clemons, Nathan C; Luo, Shuhong; Ho, Mengfei; Wilson, Brenda A

    2016-01-01

    Pasteurella multocida toxin (PMT), the major virulence factor responsible for zoonotic atrophic rhinitis, is a protein deamidase that activates the alpha subunit of heterotrimeric G proteins. Initial activation of G alpha-q-coupled phospholipase C-beta-1 signaling by PMT is followed by uncoupling of G alpha-q-dependent signaling, causing downregulation of downstream calcium and mitogenic signaling pathways. Here, we show that PMT decreases endogenous and exogenously expressed G alpha-q protein content in host cell plasma membranes and in detergent resistant membrane (DRM) fractions. This membrane depletion of G alpha-q protein was dependent upon the catalytic activity of PMT. Results indicate that PMT-modified G alpha-q redistributes within the host cell membrane from the DRM fraction into the soluble membrane and cytosolic fractions. In contrast, PMT had no affect on G alpha-s or G beta protein levels, which are not substrate targets of PMT. PMT also had no affect on G alpha-11 levels, even though G alpha-11 can serve as a substrate for deamidation by PMT, suggesting that membrane depletion of PMT-modified G-alpha-q has specificity. PMID:27490568

  2. Selective Membrane Redistribution and Depletion of Gαq-Protein by Pasteurella multocida Toxin

    PubMed Central

    Clemons, Nathan C.; Luo, Shuhong; Ho, Mengfei; Wilson, Brenda A.

    2016-01-01

    Pasteurella multocida toxin (PMT), the major virulence factor responsible for zoonotic atrophic rhinitis, is a protein deamidase that activates the alpha subunit of heterotrimeric G proteins. Initial activation of G alpha-q-coupled phospholipase C-beta-1 signaling by PMT is followed by uncoupling of G alpha-q-dependent signaling, causing downregulation of downstream calcium and mitogenic signaling pathways. Here, we show that PMT decreases endogenous and exogenously expressed G alpha-q protein content in host cell plasma membranes and in detergent resistant membrane (DRM) fractions. This membrane depletion of G alpha-q protein was dependent upon the catalytic activity of PMT. Results indicate that PMT-modified G alpha-q redistributes within the host cell membrane from the DRM fraction into the soluble membrane and cytosolic fractions. In contrast, PMT had no affect on G alpha-s or G beta protein levels, which are not substrate targets of PMT. PMT also had no affect on G alpha-11 levels, even though G alpha-11 can serve as a substrate for deamidation by PMT, suggesting that membrane depletion of PMT-modified G-alpha-q has specificity. PMID:27490568

  3. KRIT1 Protein Depletion Modifies Endothelial Cell Behavior via Increased Vascular Endothelial Growth Factor (VEGF) Signaling*

    PubMed Central

    DiStefano, Peter V.; Kuebel, Julia M.; Sarelius, Ingrid H.; Glading, Angela J.

    2014-01-01

    Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), PDCD10, and CCM2) are critical regulators of endothelial cell-cell contact and vascular homeostasis. Here we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1-depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (VEGFR2) activation and subsequent alteration of cytoskeletal organization, migration, and barrier function and to in vivo endothelial permeability in KRIT1-deficient animals. VEGFR2 activation also increases β-catenin phosphorylation but is only partially responsible for KRIT1 depletion-dependent disruption of cell-cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1-deficient cells and microvessel permeability in Krit1+/− mice; however, VEGF signaling is likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1. PMID:25320085

  4. Computer simulations and theoretical aspects of the depletion interaction in protein-oligomer mixtures

    NASA Astrophysics Data System (ADS)

    Bončina, M.; Reščič, J.; Kalyuzhnyi, Yu. V.; Vlachy, V.

    2007-07-01

    The depletion interaction between proteins caused by addition of either uncharged or partially charged oligomers was studied using the canonical Monte Carlo simulation technique and the integral equation theory. A protein molecule was modeled in two different ways: either as (i) a hard sphere of diameter 30.0Å with net charge 0, or +5, or (ii) as a hard sphere with discrete charges (depending on the pH of solution) of diameter 45.4Å. The oligomers were pictured as tangentially jointed, uncharged, or partially charged, hard spheres. The ions of a simple electrolyte present in solution were represented by charged hard spheres distributed in the dielectric continuum. In this study we were particularly interested in changes of the protein-protein pair-distribution function, caused by addition of the oligomer component. In agreement with previous studies we found that addition of a nonadsorbing oligomer reduces the phase stability of solution, which is reflected in the shape of the protein-protein pair-distribution function. The value of this function in protein-protein contact increases with increasing oligomer concentration, and is larger for charged oligomers. The range of the depletion interaction and its strength also depend on the length (number of monomer units) of the oligomer chain. The integral equation theory, based on the Wertheim Ornstein-Zernike approach applied in this study, was found to be in fair agreement with Monte Carlo results only for very short oligomers. The computer simulations for a model mimicking the lysozyme molecule (ii) are in qualitative agreement with small-angle neutron experiments for lysozyme-dextran mixtures.

  5. Chloroplast isolation and affinity chromatography for enrichment of low-abundant proteins in complex proteomes.

    PubMed

    Bayer, Roman G; Stael, Simon; Teige, Markus

    2015-01-01

    Detailed knowledge of the proteome is crucial to advance the biological sciences. Low-abundant proteins are of particular interest to many biologists as they include, for example those proteins involved in signal transduction. Recent technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS). However, the dynamic range in protein abundance still forms a fundamental problem that limits the detection of low-abundant proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of other proteins at an abundance several-fold higher in order of magnitude. Therefore, specific enrichment strategies are required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundant proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles. PMID:25820724

  6. A New 6Li Detection in a Halo Subgiant, and Constraints for the Depletion of the Big Bang 7Li Abundance

    NASA Astrophysics Data System (ADS)

    Deliyannis, C. P.; Ryan, S. G.

    2000-05-01

    We present measurements of the 6Li/7Li isotope ratio in ten metal-poor stars derived from very high resolution (100,000) and S/N (300-800/pixel) McDonald 2.7-meter coude spectra, including two possible 6Li detections. We present specific new evidence that we have indeed detected the 6Li absorption feature, and not a convective asymmetry of the 7Li feature. One of our detections argues in favor of a protostellar (and not a surface-spallated) origin for this 6Li. We find that 6Li has either not evolved strongly with metallicity, in contrast to what is observed for Be and B, or else concurrent 6Li production is matched by stellar depletion. While such fine-tuning seems unlikely, no models can explain the origin of 6Li without such depletion. In the context of the observed 9Be/7Li depletion correlation and its slow-mixing explanation, taking our data at face value implies that the Big Bang 7Li abundance is no more than 0.2-0.3 dex higher than the values observed in the halo Li plateau.

  7. Identification of Differentially Abundant Proteins of Edwardsiella ictaluri during Iron Restriction

    PubMed Central

    Dumpala, Pradeep R.; Peterson, Brian C.; Lawrence, Mark L.; Karsi, Attila

    2015-01-01

    Edwardsiella ictaluri is a Gram-negative facultative anaerobe intracellular bacterium that causes enteric septicemia in channel catfish. Iron is an essential inorganic nutrient of bacteria and is crucial for bacterial invasion. Reduced availability of iron by the host may cause significant stress for bacterial pathogens and is considered a signal that leads to significant alteration in virulence gene expression. However, the precise effect of iron-restriction on E. ictaluri protein abundance is unknown. The purpose of this study was to identify differentially abundant proteins of E. ictaluri during in vitro iron-restricted conditions. We applied two-dimensional difference in gel electrophoresis (2D-DIGE) for determining differentially abundant proteins and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS) for protein identification. Gene ontology and pathway-based functional modeling of differentially abundant proteins was also conducted. A total of 50 unique differentially abundant proteins at a minimum of 2-fold (p ≤ 0.05) difference in abundance due to iron-restriction were detected. The numbers of up- and down-regulated proteins were 37 and 13, respectively. We noted several proteins, including EsrB, LamB, MalM, MalE, FdaA, and TonB-dependent heme/hemoglobin receptor family proteins responded to iron restriction in E. ictaluri. PMID:26168192

  8. Natural variability in abundance of prevalent soybean proteins.

    PubMed

    Natarajan, Savithiry S

    2010-12-01

    Soybean is an inexpensive source of protein for humans and animals. Genetic modifications (GMO) to soybean have become inevitable on two fronts, both quality and yield will need to improve to meet increasing global demand. To ensure the safety of the crop for consumers it is important to determine the natural variation in seed protein constituents as well as any unintended changes that may occur in the GMO as a result of genetic modification. Understanding the natural variation of seed proteins in wild and cultivated soybeans that have been used in conventional soybean breeding programs is critical for determining unintended protein expression in GMO soybeans. In recent years, proteomic technologies have been used as an effective analytical tool for examining modifications of protein profiles. We have standardized and applied these technologies to determine and quantify the spectrum of proteins present in soybean seed. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and liquid chromatography mass spectrometry (LC-MS) for the separation, quantification, and identification of different classes of soybean seed proteins. We have observed significant variations in different classes of proteins, including storage, allergen and anti-nutritional protein profiles, between non-GMO cultivated and wild soybean varieties. This information is useful for scientists and regulatory agencies to determine whether the unintended expression of proteins found in transgenic soybean is within the range of natural variation. PMID:20709130

  9. Mantle in the Manihiki Plateau source with ultra-depleted incompatible element abundances but FOZO-like isotopic signature

    NASA Astrophysics Data System (ADS)

    Golowin, R.; Hoernle, K.; Portnyagin, M.; Hauff, F.; Gurenko, A.; Garbe-Schoenberg, C. D.; Werner, R.

    2014-12-01

    The ~120Ma Manihiki Plateau basement consists of high-Ti tholeiitic basalts with EM-I type isotopic signatures, similar to the Singgalo basalts at Ontong Java, and low-Ti tholeiitic basalts with FOZO (Kwaimbaita/Kroenke) to HIMU-type isotopic compositions, similar to late stage volcanism on Hikurangi and Manihiki Plateaus (Hoernle et al. 2010; Timm et al. 2011). The low-Ti basalts have affinities to boninites and have been interpreted to be derived from residual mantle wedge mantle (Ingle et al. 2007). New major, volatile and trace element and radiogenic isotope data have been generated from fresh low-Ti glass samples recovered during R/V Sonne cruises SO193 and SO225. The low-Ti samples have distinctly lower Ti/V ratios compared to lavas from Ontong Java Plateau (Kwaimbaita-Kroenke and Singgalo), but similar to boninitic rocks. Glasses and melt inclusions in olivine have low volatile contents (0.12-0.25 wt% H2O). Olivine chemistry points to derivation from peridotite source. Therefore we interpret the low-Ti lavas to have formed through melting of dry and depleted peridotite at high temperatures, consistent with Timm et al (2011). The low-Ti group is characterized by U-shaped trace element patterns. The glass samples form linear mixing arrays on radiogenic isotope diagrams, pointing to the involvement of two components: 1) a component ultra-depleted in highly incompatible elements (UDC) but with intermediate Pb, Sr and Nd isotopic compositions, being similar to Kwaimbaita/Kroenke lavas from Ontong Java, and 2) an enriched component with HIMU-type incompatible element and isotopic characteristics, similar to late-stage volcanism on Manihiki, Hikurangi and Ontong Java (e.g. Hoernle et al. 2010). The ultra-depleted, FOZO-like mantle component could represent second stage melting of FOZO type mantle or re-melting of young recycled oceanic lithosphere within the plume head. Enrichment with HIMU type melts is required to explain the enrichment in the most incompatible

  10. Automated SDS Depletion for Mass Spectrometry of Intact Membrane Proteins though Transmembrane Electrophoresis.

    PubMed

    Kachuk, Carolyn; Faulkner, Melissa; Liu, Fang; Doucette, Alan A

    2016-08-01

    Membrane proteins are underrepresented in proteome analysis platforms because of their hydrophobic character, contributing to decreased solubility. Sodium dodecyl sulfate is a favored denaturant in proteomic workflows, facilitating cell lysis and protein dissolution; however, SDS impedes MS detection and therefore must be removed prior to analysis. Although strategies exist for SDS removal, they provide low recovery, purity, or reproducibility. Here we present a simple automated device, termed transmembrane electrophoresis (TME), incorporating the principles of membrane filtration, but with an applied electric current to ensure near-complete (99.9%) removal of the surfactant, including protein-bound SDS. Intact proteins are recovered in solution phase in high yield (90-100%) within 1 h of operation. The strategy is applied to protein standards and proteome mixtures, including an enriched membrane fraction from E. coli, resulting in quality MS spectra free of SDS adducts. The TME platform is applicable to both bottom-up MS/MS as well as LC-ESI-MS analysis of intact proteins. SDS-depleted fractions reveal a similar number of protein identifications (285) compared wit a non-SDS control (280), being highly correlated in terms of protein spectral counts. This fully automated approach to SDS removal presents a viable tool for proteome sample processing ahead of MS analysis. Data are available via ProteomeXchange, identifier PXD003941. PMID:27376408

  11. Genomic analysis of membrane protein families: abundance and conserved motifs

    PubMed Central

    Liu, Yang; Engelman, Donald M; Gerstein, Mark

    2002-01-01

    Background Polytopic membrane proteins can be related to each other on the basis of the number of transmembrane helices and sequence similarities. Building on the Pfam classification of protein domain families, and using transmembrane-helix prediction and sequence-similarity searching, we identified a total of 526 well-characterized membrane protein families in 26 recently sequenced genomes. To this we added a clustering of a number of predicted but unclassified membrane proteins, resulting in a total of 637 membrane protein families. Results Analysis of the occurrence and composition of these families revealed several interesting trends. The number of assigned membrane protein domains has an approximately linear relationship to the total number of open reading frames (ORFs) in 26 genomes studied. Caenorhabditis elegans is an apparent outlier, because of its high representation of seven-span transmembrane (7-TM) chemoreceptor families. In all genomes, including that of C. elegans, the number of distinct membrane protein families has a logarithmic relation to the number of ORFs. Glycine, proline, and tyrosine locations tend to be conserved in transmembrane regions within families, whereas isoleucine, valine, and methionine locations are relatively mutable. Analysis of motifs in putative transmembrane helices reveals that GxxxG and GxxxxxxG (which can be written GG4 and GG7, respectively; see Materials and methods) are among the most prevalent. This was noted in earlier studies; we now find these motifs are particularly well conserved in families, however, especially those corresponding to transporters, symporters, and channels. Conclusions We carried out a genome-wide analysis on patterns of the classified polytopic membrane protein families and analyzed the distribution of conserved amino acids and motifs in the transmembrane helix regions in these families. PMID:12372142

  12. Transcriptional abundance is not the single force driving the evolution of bacterial proteins

    PubMed Central

    2013-01-01

    Background Despite rapid progress in understanding the mechanisms that shape the evolution of proteins, the relative importance of various factors remain to be elucidated. In this study, we have assessed the effects of 16 different biological features on the evolutionary rates (ERs) of protein-coding sequences in bacterial genomes. Results Our analysis of 18 bacterial species revealed new correlations between ERs and constraining factors. Previous studies have suggested that transcriptional abundance overwhelmingly constrains the evolution of yeast protein sequences. This transcriptional abundance leads to selection against misfolding or misinteractions. In this study we found that there was no single factor in determining the evolution of bacterial proteins. Not only transcriptional abundance (codon adaptation index and expression level), but also protein-protein associations (PPAs), essentiality (ESS), subcellular localization of cytoplasmic membrane (SLM), transmembrane helices (TMH) and hydropathicity score (HS) independently and significantly affected the ERs of bacterial proteins. In some species, PPA and ESS demonstrate higher correlations with ER than transcriptional abundance. Conclusions Different forces drive the evolution of protein sequences in yeast and bacteria. In bacteria, the constraints are involved in avoiding a build-up of toxic molecules caused by misfolding/misinteraction (transcriptional abundance), while retaining important functions (ESS, PPA) and maintaining the cell membrane (SLM, TMH and HS). Each of these independently contributes to the variation in protein evolution. PMID:23914835

  13. Inhibition of protein kinase C affects on mode of synaptic vesicle exocytosis due to cholesterol depletion.

    PubMed

    Petrov, Alexey M; Zakyrjanova, Guzalija F; Yakovleva, Anastasia A; Zefirov, Andrei L

    2015-01-01

    Previous studies demonstrated that depletion of membrane cholesterol by 10mM methyl-beta-cyclodextrin (MCD) results in increased spontaneous exocytosis at both peripheral and central synapses. Here, we investigated the role of protein kinase C in the enhancement of spontaneous exocytosis at frog motor nerve terminals after cholesterol depletion using electrophysiological and optical methods. Inhibition of the protein kinase C by myristoylated peptide and chelerythrine chloride prevented MCD-induced increases in FM1-43 unloading, whereas the frequency of spontaneous postsynaptic events remained enhanced. The increase in FM1-43 unloading still could be observed if sulforhodamine 101 (the water soluble FM1-43 quencher that can pass through the fusion pore) was added to the extracellular solution. This suggests a possibility that exocytosis of synaptic vesicles under these conditions could occur through the kiss-and-run mechanism with the formation of a transient fusion pore. Inhibition of phospholipase C did not lead to similar change in MCD-induced exocytosis. PMID:25446113

  14. Inhibition of protein kinase C affects on mode of synaptic vesicle exocytosis due to cholesterol depletion

    SciTech Connect

    Petrov, Alexey M. Zakyrjanova, Guzalija F. Yakovleva, Anastasia A. Zefirov, Andrei L.

    2015-01-02

    Highlights: • We examine the involvement of PKC in MCD induced synaptic vesicle exocytosis. • PKC inhibitor does not decrease the effect MCD on MEPP frequency. • PKC inhibitor prevents MCD induced FM1-43 unloading. • PKC activation may switch MCD induced exocytosis from kiss-and-run to a full mode. • Inhibition of phospholipase C does not lead to similar change in exocytosis. - Abstract: Previous studies demonstrated that depletion of membrane cholesterol by 10 mM methyl-beta-cyclodextrin (MCD) results in increased spontaneous exocytosis at both peripheral and central synapses. Here, we investigated the role of protein kinase C in the enhancement of spontaneous exocytosis at frog motor nerve terminals after cholesterol depletion using electrophysiological and optical methods. Inhibition of the protein kinase C by myristoylated peptide and chelerythrine chloride prevented MCD-induced increases in FM1-43 unloading, whereas the frequency of spontaneous postsynaptic events remained enhanced. The increase in FM1-43 unloading still could be observed if sulforhodamine 101 (the water soluble FM1-43 quencher that can pass through the fusion pore) was added to the extracellular solution. This suggests a possibility that exocytosis of synaptic vesicles under these conditions could occur through the kiss-and-run mechanism with the formation of a transient fusion pore. Inhibition of phospholipase C did not lead to similar change in MCD-induced exocytosis.

  15. Two Novel Heat-Soluble Protein Families Abundantly Expressed in an Anhydrobiotic Tardigrade

    PubMed Central

    Yamaguchi, Ayami; Tanaka, Sae; Yamaguchi, Shiho; Kuwahara, Hirokazu; Takamura, Chizuko; Imajoh-Ohmi, Shinobu; Horikawa, Daiki D.; Toyoda, Atsushi; Katayama, Toshiaki; Arakawa, Kazuharu; Fujiyama, Asao; Kubo, Takeo; Kunieda, Takekazu

    2012-01-01

    Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals. PMID:22937162

  16. Protein abundance changes of Zygosaccharomyces rouxii in different sugar concentrations.

    PubMed

    Guo, Hong; Niu, Chen; Liu, Bin; Wei, JianPing; Wang, HuXuan; Yuan, YaHong; Yue, TianLi

    2016-09-16

    Zygosaccharomyces rouxii is a yeast which can cause spoilage in the concentrated juice industries. It exhibits resistance to high sugar concentrations but genome- and proteome-wide studies on Z. rouxii in response to high sugar concentrations have been poorly investigated. Herein, by using a 2-D electrophoresis based workflow, the proteome of a wild strain of Z. rouxii under different sugar concentrations has been analyzed. Proteins were extracted, quantified, and subjected to 2-DE analysis in the pH range 4-7. Differences in growth (lag phase), protein content (13.97-19.23mg/g cell dry weight) and number of resolved spots (196-296) were found between sugar concentrations. ANOVA test showed that 168 spots were different, and 47 spots, corresponding to 40 unique gene products have been identified. These protein species are involved in carbohydrate and energy metabolism, amino acid metabolism, response to stimulus, protein transport and vesicle organization, cell morphogenesis regulation, transcription and translation, nucleotide metabolism, amino-sugar nucleotide-sugar pathways, oxidoreductases balancing, and ribosome biogenesis. The present study provides important information about how Z. rouxii acts to cope with high sugar concentration at molecular levels, which might enhance our global understanding of Z. rouxii's high sugar-tolerance trait. PMID:27322723

  17. Heterogeneous distribution of H2O in the Martian interior: Implications for the abundance of H2O in depleted and enriched mantle sources

    NASA Astrophysics Data System (ADS)

    McCubbin, Francis M.; Boyce, Jeremy W.; Srinivasan, Poorna; Santos, Alison R.; Elardo, Stephen M.; Filiberto, Justin; Steele, Andrew; Shearer, Charles K.

    2016-04-01

    We conducted a petrologic study of apatite within 12 Martian meteorites, including 11 shergottites and one basaltic regolith breccia. These data were combined with previously published data to gain a better understanding of the abundance and distribution of volatiles in the Martian interior. Apatites in individual Martian meteorites span a wide range of compositions, indicating they did not form by equilibrium crystallization. In fact, the intrasample variation in apatite is best described by either fractional crystallization or crustal contamination with a Cl-rich crustal component. We determined that most Martian meteorites investigated here have been affected by crustal contamination and hence cannot be used to estimate volatile abundances of the Martian mantle. Using the subset of samples that did not exhibit crustal contamination, we determined that the enriched shergottite source has 36-73 ppm H2O and the depleted source has 14-23 ppm H2O. This result is consistent with other observed geochemical differences between enriched and depleted shergottites and supports the idea that there are at least two geochemically distinct reservoirs in the Martian mantle. We also estimated the H2O, Cl, and F content of the Martian crust using known crust-mantle distributions for incompatible lithophile elements. We determined that the bulk Martian crust has ~1410 ppm H2O, 450 ppm Cl, and 106 ppm F, and Cl and H2O are preferentially distributed toward the Martian surface. The estimate of crustal H2O results in a global equivalent surface layer (GEL) of ~229 m, which can account for at least some of the surface features on Mars attributed to flowing water and may be sufficient to support the past presence of a shallow sea on Mars' surface.

  18. Depletion of BBS Protein LZTFL1 Affects Growth and Causes Retinal Degeneration in Mice.

    PubMed

    Jiang, Jiangsong; Promchan, Kanyarat; Jiang, Hong; Awasthi, Parirokh; Marshall, Heather; Harned, Adam; Natarajan, Ven

    2016-06-20

    Bardet-Biedl syndrome (BBS) is a heterogeneous disease characterized by deficiencies in various organs that are caused by defects in genes involved in the genesis, structural maintenance, and protein trafficking of cilia. Leucine zipper transcription factor-like 1 (LZTFL1) has been identified as a BBS protein (BBS17), because patients with mutations in this gene exhibit the common BBS phenotypes. In this study, we generated a knockout mouse model to investigate the effects of LZTFL1 depletion. Lztfl1 knockout mice were born with low birth weight, reached similar weight to those of wild-type mice at 10 weeks of age, and later gained more weight than their wild-type counterparts. LZTFL1 was localized to the primary cilium of kidney cells, and the absence of LZTFL1 increased the ciliary localization of BBS9. Moreover, in the retinas of Lztfl1 knockout mice, the photoreceptor outer segment was shortened, the distal axoneme of photoreceptor connecting cilium was significantly enlarged, and rhodopsin was targeted to the outer nuclear layer. TUNEL assay showed that many of these abnormal photoreceptor cells in Lztfl1 knockout mice underwent apoptosis. Interestingly, the absence of LZTFL1 caused an abnormal increase of the adaptor protein complex 1 (AP1) in some photoreceptor cells. Based on these data, we conclude that LZTFL1 is a cilium protein and regulates animal weight and photoreceptor connecting cilium function probably by controlling microtubule assembly and protein trafficking in cilia. PMID:27312011

  19. Protein carbonylation and aggregation precede neuronal apoptosis induced by partial glutathione depletion

    PubMed Central

    Dasgupta, Anushka; Zheng, Jianzheng; Bizzozero, Oscar A.

    2012-01-01

    While the build-up of oxidized proteins within cells is believed to be toxic, there is currently no evidence linking protein carbonylation and cell death. In the present study, we show that incubation of nPC12 (neuron-like PC12) cells with 50 μM DEM (diethyl maleate) leads to a partial and transient depletion of glutathione (GSH). Concomitant with GSH disappearance there is increased accumulation of PCOs (protein carbonyls) and cell death (both by necrosis and apoptosis). Immunocytochemical studies also revealed a temporal/spatial relationship between carbonylation and cellular apoptosis. In addition, the extent of all three, PCO accumulation, protein aggregation and cell death, augments if oxidized proteins are not removed by proteasomal degradation. Furthermore, the effectiveness of the carbonyl scavengers hydralazine, histidine hydrazide and methoxylamine at preventing cell death identifies PCOs as the toxic species. Experiments using well-characterized apoptosis inhibitors place protein carbonylation downstream of the mitochondrial transition pore opening and upstream of caspase activation. While the study focused mostly on nPC12 cells, experiments in primary neuronal cultures yielded the same results. The findings are also not restricted to DEM-induced cell death, since a similar relationship between carbonylation and apoptosis was found in staurosporine- and buthionine sulfoximine-treated nPC12 cells. In sum, the above results show for the first time a causal relationship between carbonylation, protein aggregation and apoptosis of neurons undergoing oxidative damage. To the best of our knowledge, this is the first study to place direct (oxidative) protein carbonylation within the apoptotic pathway. PMID:22376187

  20. Assessment of the natural variation of low abundant metabolic proteins in soybean seeds using proteomics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, we investigated the distribution of the low abundant proteins that are involved in soybean seed development in four wild and twelve cultivated soybean genotypes. We found proteomic variation of these proteins within and...

  1. Measurement of Cysteine Dioxygenase Activity and Protein Abundance

    PubMed Central

    Stipanuk, Martha H.; Dominy, John E.; Ueki, Iori; Hirschberger, Lawrence L.

    2009-01-01

    Cysteine dioxygenase is an iron (Fe2+)-dependent thiol dioxygenase that uses molecular oxygen to oxidize the sulfhydryl group of cysteine to generate 3-sulfinoalanine (commonly called cysteinesulfinic acid). Cysteine dioxygenase activity is routinely assayed by measuring cysteinesulfinate formation from substrate L-cysteine at pH 6.1 in the presence of ferrous ions to saturate the enzyme with metal cofactor, a copper chelator to diminish substrate oxidation, and hydroxylamine to inhibit pyridoxal 5′-phosphate-dependent degradation of product. The amount of cysteine dioxygenase may be measured by immunoblotting. Upon SDS-PAGE, cysteine dioxygenase can be separated into two major bands, with the upper band representing the 23-kDa protein and the lower band representing the mature enzyme that has undergone formation of an internal thioether cross link in the active site. Formation of this cross link is dependent upon the catalytic turnover of substrate and produces an enzyme with a higher catalytic efficiency and catalytic half-life. PMID:19885389

  2. The effect of colostrum intake on blood plasma proteome profile in newborn lambs: low abundance proteins

    PubMed Central

    2014-01-01

    Background Colostrum intake by newborn lambs plays a fundamental role in the perinatal period, ensuring lamb survival. In this study, blood plasma samples from two groups of newborn lambs (Colostrum group and Delayed Colostrum group) at 2 and 14 h after birth were treated to reduce the content of high abundance proteins and analyzed using Two-Dimensional Differential in Gel Electrophoresis and MALDI MS/MS for protein identification in order to investigate low abundance proteins with immune function in newborn lambs. Results The results showed that four proteins were increased in the blood plasma of lambs due to colostrum intake. These proteins have not been previously described as increased in blood plasma of newborn ruminants by colostrum intake. Moreover, these proteins have been described as having an immune function in other species, some of which were previously identified in colostrum and milk. Conclusions In conclusion, colostrum intake modified the low abundance proteome profile of blood plasma from newborn lambs, increasing the concentration of apolipoprotein A-IV, plasminogen, serum amyloid A and fibrinogen, demonstrating that colostrum is essential, not only for the provision of immunoglobulins, but also because of increases in several low abundance proteins with immune function. PMID:24708841

  3. CD19xCD3 DART protein mediates human B-cell depletion in vivo in humanized BLT mice

    PubMed Central

    Tsai, Perry; Thayer, William O; Liu, Liqin; Silvestri, Guido; Nordstrom, Jeffrey L; Garcia, J Victor

    2016-01-01

    Novel therapeutic strategies are needed for the treatment of hematologic malignancies; and bispecific antibody-derived molecules, such as dual-affinity re-targeting (DART) proteins, are being developed to redirect T cells to kill target cells expressing tumor or viral antigens. Here we present our findings of specific and systemic human B-cell depletion by a CD19xCD3 DART protein in humanized BLT mice. Administration of the CD19xCD3 DART protein resulted in a dramatic sustained depletion of human CD19+ B cells from the peripheral blood, as well as a dramatic systemic reduction of human CD19+ B-cell levels in all tissues (bone marrow, spleen, liver, lung) analyzed. When human CD8+ T cells were depleted from the mice, no significant B-cell depletion was observed in response to CD19xCD3 DART protein treatment, confirming that human CD8+ T cells are the primary effector cells in this in vivo model. These studies validate the use of BLT humanized mice for the in vivo evaluation and preclinical development of bispecific molecules that redirect human T cells to selectively deplete target cells. PMID:27119115

  4. Choice of dietary protein of vegetarians and omnivores is reflected in their hair protein 13C and 15N abundance.

    PubMed

    Petzke, Klaus J; Boeing, Heiner; Metges, Cornelia C

    2005-01-01

    Stable isotopic (15N, 13C) composition of tissues depends on isotopic pattern of food sources. We investigated whether the isotopic compositions of human hair protein and amino acids reflect the habitual dietary protein intake. Hair samples were analyzed from 100 omnivores (selected randomly out of the 1987-1988 German nutrition survey VERA), and from 15 ovo-lacto-vegetarians (OLV), and from 6 vegans recruited separately. Hair bulk and amino acid specific isotopic compositions were analyzed by isotope-ratio mass spectrometry (EA/IRMS and GC/C/IRMS, respectively) and the results were correlated with data of the 7 day dietary records. Hair bulk 15N and 13C abundances clearly reflect the particular eating habits. Vegans can be distinguished from OLV and both are significantly distinct from omnivores in both 15N and 13C abundances. 15N and 13C abundances rose with a higher proportion of animal to total protein intake (PAPI). Individual proportions of animal protein consumption (IPAP) were calculated using isotopic abundances and a linear regression model using animal protein consumption data of vegans (PAPI = 0) and omnivores (mean PAPI = 0.639). IPAP values positively correlated with the intake of protein, meat, meat products, and animal protein. Distinct patterns for hair amino acid specific 15N and 13C abundances were measured but with lower resolution between food preference groups compared with bulk values. In conclusion, hair 13C and 15N values both reflected the extent of animal protein consumption. Bulk isotopic abundance of hair can be tested for future use in the validation of dietary assessment methods. PMID:15880664

  5. Genetics of single-cell protein abundance variation in large yeast populations.

    PubMed

    Albert, Frank W; Treusch, Sebastian; Shockley, Arthur H; Bloom, Joshua S; Kruglyak, Leonid

    2014-02-27

    Variation among individuals arises in part from differences in DNA sequences, but the genetic basis for variation in most traits, including common diseases, remains only partly understood. Many DNA variants influence phenotypes by altering the expression level of one or several genes. The effects of such variants can be detected as expression quantitative trait loci (eQTL). Traditional eQTL mapping requires large-scale genotype and gene expression data for each individual in the study sample, which limits sample sizes to hundreds of individuals in both humans and model organisms and reduces statistical power. Consequently, many eQTL are probably missed, especially those with smaller effects. Furthermore, most studies use messenger RNA rather than protein abundance as the measure of gene expression. Studies that have used mass-spectrometry proteomics reported unexpected differences between eQTL and protein QTL (pQTL) for the same genes, but these studies have been even more limited in scope. Here we introduce a powerful method for identifying genetic loci that influence protein expression in the yeast Saccharomyces cerevisiae. We measure single-cell protein abundance through the use of green fluorescent protein tags in very large populations of genetically variable cells, and use pooled sequencing to compare allele frequencies across the genome in thousands of individuals with high versus low protein abundance. We applied this method to 160 genes and detected many more loci per gene than previous studies. We also observed closer correspondence between loci that influence protein abundance and loci that influence mRNA abundance of a given gene. Most loci that we detected were clustered in 'hotspots' that influence multiple proteins, and some hotspots were found to influence more than half of the proteins that we examined. The variants that underlie these hotspots have profound effects on the gene regulatory network and provide insights into genetic variation in cell

  6. Genetics of single-cell protein abundance variation in large yeast populations

    NASA Astrophysics Data System (ADS)

    Albert, Frank W.; Treusch, Sebastian; Shockley, Arthur H.; Bloom, Joshua S.; Kruglyak, Leonid

    2014-02-01

    Variation among individuals arises in part from differences in DNA sequences, but the genetic basis for variation in most traits, including common diseases, remains only partly understood. Many DNA variants influence phenotypes by altering the expression level of one or several genes. The effects of such variants can be detected as expression quantitative trait loci (eQTL). Traditional eQTL mapping requires large-scale genotype and gene expression data for each individual in the study sample, which limits sample sizes to hundreds of individuals in both humans and model organisms and reduces statistical power. Consequently, many eQTL are probably missed, especially those with smaller effects. Furthermore, most studies use messenger RNA rather than protein abundance as the measure of gene expression. Studies that have used mass-spectrometry proteomics reported unexpected differences between eQTL and protein QTL (pQTL) for the same genes, but these studies have been even more limited in scope. Here we introduce a powerful method for identifying genetic loci that influence protein expression in the yeast Saccharomyces cerevisiae. We measure single-cell protein abundance through the use of green fluorescent protein tags in very large populations of genetically variable cells, and use pooled sequencing to compare allele frequencies across the genome in thousands of individuals with high versus low protein abundance. We applied this method to 160 genes and detected many more loci per gene than previous studies. We also observed closer correspondence between loci that influence protein abundance and loci that influence mRNA abundance of a given gene. Most loci that we detected were clustered in `hotspots' that influence multiple proteins, and some hotspots were found to influence more than half of the proteins that we examined. The variants that underlie these hotspots have profound effects on the gene regulatory network and provide insights into genetic variation in cell

  7. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

    NASA Astrophysics Data System (ADS)

    He, Yi-Ming; Ma, Bin-Guang

    2016-05-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

  8. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

    PubMed Central

    He, Yi-Ming; Ma, Bin-Guang

    2016-01-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions. PMID:27220911

  9. Seasonal changes in mollusc abundance in a tropical intertidal ecosystem, Banc d'Arguin (Mauritania): Testing the ‘depletion by shorebirds' hypothesis

    NASA Astrophysics Data System (ADS)

    Ahmedou Salem, Mohamed Vall; van der Geest, Matthijs; Piersma, Theunis; Saoud, Younès; van Gils, Jan A.

    2014-01-01

    build upon a recently published optimal diet model in which the most abundant molluscivore shorebird, the red knot (Calidris canutus), could choose between Loripes and Dosinia. Observed changes in densities of these two bivalves closely match depletion trajectories predicted by the model. We conclude that molluscivore shorebirds are able to deplete their food stocks in the course of their ‘winter' in a tropical intertidal area.

  10. Natural Genetic Variation Influences Protein Abundances in C. elegans Developmental Signalling Pathways

    PubMed Central

    Singh, Kapil Dev; Roschitzki, Bernd; Snoek, L. Basten; Grossmann, Jonas; Zheng, Xue; Elvin, Mark; Kamkina, Polina; Schrimpf, Sabine P.; Poulin, Gino B.; Kammenga, Jan E.; Hengartner, Michael O.

    2016-01-01

    Complex traits, including common disease-related traits, are affected by many different genes that function in multiple pathways and networks. The apoptosis, MAPK, Notch, and Wnt signalling pathways play important roles in development and disease progression. At the moment we have a poor understanding of how allelic variation affects gene expression in these pathways at the level of translation. Here we report the effect of natural genetic variation on transcript and protein abundance involved in developmental signalling pathways in Caenorhabditis elegans. We used selected reaction monitoring to analyse proteins from the abovementioned four pathways in a set of recombinant inbred lines (RILs) generated from the wild-type strains N2 (Bristol) and CB4856 (Hawaii) to enable quantitative trait locus (QTL) mapping. About half of the cases from the 44 genes tested showed a statistically significant change in protein abundance between various strains, most of these were however very weak (below 1.3-fold change). We detected a distant QTL on the left arm of chromosome II that affected protein abundance of the phosphatidylserine receptor protein PSR-1, and two separate QTLs that influenced embryonic and ionizing radiation-induced apoptosis on chromosome IV. Our results demonstrate that natural variation in C. elegans is sufficient to cause significant changes in signalling pathways both at the gene expression (transcript and protein abundance) and phenotypic levels. PMID:26985669

  11. Visualization and Dissemination of Multidimensional Proteomics Data Comparing Protein Abundance During Caenorhabditis elegans Development

    NASA Astrophysics Data System (ADS)

    Riffle, Michael; Merrihew, Gennifer E.; Jaschob, Daniel; Sharma, Vagisha; Davis, Trisha N.; Noble, William S.; MacCoss, Michael J.

    2015-11-01

    Regulation of protein abundance is a critical aspect of cellular function, organism development, and aging. Alternative splicing may give rise to multiple possible proteoforms of gene products where the abundance of each proteoform is independently regulated. Understanding how the abundances of these distinct gene products change is essential to understanding the underlying mechanisms of many biological processes. Bottom-up proteomics mass spectrometry techniques may be used to estimate protein abundance indirectly by sequencing and quantifying peptides that are later mapped to proteins based on sequence. However, quantifying the abundance of distinct gene products is routinely confounded by peptides that map to multiple possible proteoforms. In this work, we describe a technique that may be used to help mitigate the effects of confounding ambiguous peptides and multiple proteoforms when quantifying proteins. We have applied this technique to visualize the distribution of distinct gene products for the whole proteome across 11 developmental stages of the model organism Caenorhabditis elegans. The result is a large multidimensional dataset for which web-based tools were developed for visualizing how translated gene products change during development and identifying possible proteoforms. The underlying instrument raw files and tandem mass spectra may also be downloaded. The data resource is freely available on the web at http://www.yeastrc.org/wormpes/.

  12. Visualization and dissemination of multidimensional proteomics data comparing protein abundance during Caenorhabditis elegans development.

    PubMed

    Riffle, Michael; Merrihew, Gennifer E; Jaschob, Daniel; Sharma, Vagisha; Davis, Trisha N; Noble, William S; MacCoss, Michael J

    2015-11-01

    Regulation of protein abundance is a critical aspect of cellular function, organism development, and aging. Alternative splicing may give rise to multiple possible proteoforms of gene products where the abundance of each proteoform is independently regulated. Understanding how the abundances of these distinct gene products change is essential to understanding the underlying mechanisms of many biological processes. Bottom-up proteomics mass spectrometry techniques may be used to estimate protein abundance indirectly by sequencing and quantifying peptides that are later mapped to proteins based on sequence. However, quantifying the abundance of distinct gene products is routinely confounded by peptides that map to multiple possible proteoforms. In this work, we describe a technique that may be used to help mitigate the effects of confounding ambiguous peptides and multiple proteoforms when quantifying proteins. We have applied this technique to visualize the distribution of distinct gene products for the whole proteome across 11 developmental stages of the model organism Caenorhabditis elegans. The result is a large multidimensional dataset for which web-based tools were developed for visualizing how translated gene products change during development and identifying possible proteoforms. The underlying instrument raw files and tandem mass spectra may also be downloaded. The data resource is freely available on the web at http://www.yeastrc.org/wormpes/ . Graphical Abstract ᅟ. PMID:26133526

  13. Determination of relative protein abundance by internally normalized ratio algorithm with antibody arrays.

    PubMed

    Andersson, Oskar; Kozlowski, Mark; Garachtchenko, Tatiana; Nikoloff, Corina; Lew, Nancy; Litman, David J; Chaga, Grigoriy

    2005-01-01

    In this paper, we report an experimental setup and mathematical algorithm for determination of relative protein abundance from directly labeled native protein samples applied to an array of antibodies. The application of the proposed experimental system compensates internally at each array element for a number of deficiencies in array experiments such as differential labeling efficiency in dual color assay systems, differential solubility of protein molecules in dual color assay systems, and differential affinity of capture reagents toward proteins labeled with two different fluorescent dyes. This system offers full compensation for variable amounts of capture reagents on separate array structures, as well as limited compensation for nonspecific interactions between capture reagents and analytes. The proposed experimental strategy enables the use of a large number of capture reagents to develop a true multiplex analysis system that will yield complete relative protein abundance information in two biological systems. PMID:15952723

  14. Mapping protein abundance patterns in the brain using voxelation combined with liquid chromatography and mass spectrometry

    SciTech Connect

    Petyuk, Vladislav A.; Qian, Weijun; Smith, Richard D.; Smith, Desmond J.

    2010-02-01

    Voxelation creates expression atlases by high-throughput analysis of spatially registered cubes or voxels harvested from the brain. The modality independence of voxelation allows a variety of bioanalytical techniques to be used to map abundance. Protein expression patterns in the brain can be obtained using liquid chromatography (LC) combined with mass spectrometry (MS). Here we describe the methodology of voxelation as it pertains particularly to LC-MS proteomic analysis: sample preparation, instrumental set up and analysis, peptide identification and protein relative abundance quantitation. We also briefly describe some of the advantages, limitations and insights into the brain that can be obtained using combined proteomic and transcriptomic maps

  15. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway.

    PubMed

    Shi, Tujin; Niepel, Mario; McDermott, Jason E; Gao, Yuqian; Nicora, Carrie D; Chrisler, William B; Markillie, Lye M; Petyuk, Vladislav A; Smith, Richard D; Rodland, Karin D; Sorger, Peter K; Qian, Wei-Jun; Wiley, H Steven

    2016-01-01

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components-16 core proteins and 10 feedback regulators-of the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling. PMID:27405981

  16. Mining the plasma proteome for disease applications across seven logs of protein abundance.

    PubMed

    Zhang, Q; Faca, V; Hanash, S

    2011-01-01

    The current state of proteomics technologies has sufficiently advanced to allow in-depth quantitative analysis of the plasma proteome and development of a related knowledge base. Here we review approaches that have been applied to increase depth of analysis by mass spectrometry given the substantial complexity of plasma and the vast dynamic range of protein abundance. Fractionation strategies resulting in reduced complexity of individual fractions followed by mass spectrometry analysis of digests from individual fractions has allowed well in excess of 1000 proteins to be identified and quantified with high confidence that span more than seven logs of protein abundance. Such depth of analysis has contributed to elucidation of plasma proteome variation in health and of protein changes associated with disease states. PMID:21062094

  17. DEPLETION OF CELLULAR PROTEIN THIOLS AS AN INDICATOR OF ARYLATION IN ISOLATED TROUT HEPATOCYTES EXPOSED TO 1,4-BENZOQUINONE

    EPA Science Inventory

    A method for the measurement of protein thiols (PrSH), un-reacted as well as oxidized, i.e. dithiothreitol recoverable, was adapted for the determination of PrSH depletion in isolated rainbow trout hepatocytes exposed to an arylating agent, 1,4-benzoquinone (BQ). Toxicant analysi...

  18. Hsp90 is involved in the regulation of cytosolic precursor protein abundance in tomato.

    PubMed

    Tillmann, Bodo; Röth, Sascha; Bublak, Daniela; Sommer, Manuel; Stelzer, Ernst H K; Scharf, Klaus-Dieter; Schleiff, Enrico

    2015-02-01

    Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 (Hsp70) and 90 (Hsp90) assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins with respect to the cytosolic chaperone-dependent regulation. Some preproteins such as pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins such as pSSU is more strongly dependent on Hsp70. The E3 ligase, C-terminus of Hsp70-interacting protein (Chip), appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable with the cytosolic unfolded protein response. PMID:25619681

  19. Data set for transcriptional response to depletion of the Shoc2 scaffolding protein

    PubMed Central

    Rouchka, Eric C.; Jeoung, Myoungkun; Jang, Eun Ryoung; Liu, Jinpeng; Wang, Chi; Li, Xiaohong; Galperin, Emilia

    2016-01-01

    The Suppressor of Clear, Caenorhabditis elegans Homolog (SHOC2) is a scaffold protein that positively modulates activity of the RAS/ERK1/2 MAP kinase signaling cascade. We set out to understand the ERK1/2 pathway transcriptional response transduced through the SHOC2 scaffolding module. This data article describes raw gene expression within triplicates of kidney fibroblast-like Cos1 cell line expressing non-targeting shRNA (Cos-NT) and triplicates of Cos1 cells depleted of SHOC2 using shRNA (Cos-LV1) upon activation of ERK1/2 pathway by the Epidermal Growth Factor Receptor (EGFR). The data referred here is available in NCBI׳s Gene Expression Omnibus (GEO), accession GEO: GSE67063 as well as NCBI׳s Sequence Read Archive (SRA), accession SRA: SRP056324. A complete analysis of the results can be found in “Shoc2-tranduced ERK1/2 motility signals – Novel insights from functional genomics”(Jeoung et al., 2016) [1]. PMID:27077079

  20. Data set for transcriptional response to depletion of the Shoc2 scaffolding protein.

    PubMed

    Rouchka, Eric C; Jeoung, Myoungkun; Jang, Eun Ryoung; Liu, Jinpeng; Wang, Chi; Li, Xiaohong; Galperin, Emilia

    2016-06-01

    The Suppressor of Clear, Caenorhabditis elegans Homolog (SHOC2) is a scaffold protein that positively modulates activity of the RAS/ERK1/2 MAP kinase signaling cascade. We set out to understand the ERK1/2 pathway transcriptional response transduced through the SHOC2 scaffolding module. This data article describes raw gene expression within triplicates of kidney fibroblast-like Cos1 cell line expressing non-targeting shRNA (Cos-NT) and triplicates of Cos1 cells depleted of SHOC2 using shRNA (Cos-LV1) upon activation of ERK1/2 pathway by the Epidermal Growth Factor Receptor (EGFR). The data referred here is available in NCBI׳s Gene Expression Omnibus (GEO), accession GEO: GSE67063 as well as NCBI׳s Sequence Read Archive (SRA), accession SRA: SRP056324. A complete analysis of the results can be found in "Shoc2-tranduced ERK1/2 motility signals - Novel insights from functional genomics"(Jeoung et al., 2016) [1]. PMID:27077079

  1. LDL Receptor-related Protein 1 Regulates the Abundance of Diverse Cell-signaling Proteins in the Plasma Membrane Proteome

    PubMed Central

    Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F.; Gonias, Steven L.

    2010-01-01

    LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, which are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 co-immunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not co-immunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome. PMID:20919742

  2. Hsp90 is involved in the regulation of cytosolic precursor protein abundance in tomato.

    PubMed

    Tillmann, Bodo; Röth, Sascha; Bublak, Daniela; Sommer, Manuel; Stelzer, Ernst H K; Scharf, Klaus-Dieter; Schleiff, Enrico

    2014-10-20

    Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the heat stress protein 70 and 90 (Hsp70 or Hsp90) families assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins in respect to the cytosolic chaperone dependent regulation. Some preproteins like pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins like pSSU is more strongly dependent on Hsp70. The E3 ligase Chip appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable to the cytosolic unfolded protein response. PMID:25336566

  3. Using antibody arrays to measure protein abundance and glycosylation: considerations for optimal performance.

    PubMed

    Haab, Brian B; Partyka, Katie; Cao, Zheng

    2013-01-01

    Antibody arrays provide a valuable method for obtaining multiple protein measurements from small volumes of biological samples. Antibody arrays can be designed to target not only core protein abundances (relative or absolute abundances, depending on the availability of standards for calibration), but also posttranslational modifications, provided antibodies or other affinity reagents are available to detect them. Glycosylation is a common modification that has important and diverse functions in both normal and disease biology. Significant progress has been made in developing methods for measuring glycan levels on multiple specific proteins using antibody arrays and glycan-binding reagents. This unit describes practical approaches for developing, optimizing, and using antibody array assays to determine both protein abundance and glycosylation state. Low-volume arrays can be used to reduce sample consumption, and a new way to improve the binding strength of particular glycan-binding reagents through multimerization is discussed. These methods can be useful for a wide range of biological studies in which glycosylation may change and/or affect protein function. PMID:24510592

  4. Identification of Proteins of Altered Abundance in Oil Palm Infected with Ganoderma boninense

    PubMed Central

    Al-Obaidi, Jameel R.; Mohd-Yusuf, Yusmin; Razali, Nurhanani; Jayapalan, Jaime Jacqueline; Tey, Chin-Chong; Md-Noh, Normahnani; Junit, Sarni Mat; Othman, Rofina Yasmin; Hashim, Onn Haji

    2014-01-01

    Basal stem rot is a common disease that affects oil palm, causing loss of yield and finally killing the trees. The disease, caused by fungus Ganoderma boninense, devastates thousands of hectares of oil palm plantings in Southeast Asia every year. In the present study, root proteins of healthy oil palm seedlings, and those infected with G. boninense, were analyzed by 2-dimensional gel electrophoresis (2-DE). When the 2-DE profiles were analyzed for proteins, which exhibit consistent significant change of abundance upon infection with G. boninense, 21 passed our screening criteria. Subsequent analyses by mass spectrometry and database search identified caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, enolase, fructokinase, cysteine synthase, malate dehydrogenase, and ATP synthase as among proteins of which abundances were markedly altered. PMID:24663087

  5. Expect the Unexpected Enrichment of “Hidden Proteome” of Seeds and Tubers by Depletion of Storage Proteins

    PubMed Central

    Gupta, Ravi; Min, Cheol W.; Wang, Yiming; Kim, Yong C.; Agrawal, Ganesh K.; Rakwal, Randeep; Kim, Sun T.

    2016-01-01

    Dynamic resolution of seed and tuber protein samples is highly limited due to the presence of high-abundance storage proteins (SPs). These proteins inevitably obscure the low-abundance proteins (LAPs) impeding their identification and characterization. To facilitate the detection of LAPs, several methods have been developed during the past decade, enriching the proteome with extreme proteins. Most of these methods, if not all, are based on the specific removal of SPs which ultimately magnify the proteome coverage. In this mini-review, we summarize the available methods that have been developed over the years for the enrichment of LAPs either from seeds or tubers. Incorporation of these methods during the protein extraction step will be helpful in understanding the seed/tuber biology in greater detail. PMID:27313590

  6. Hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins.

    PubMed

    Magni, Ruben; Espina, Benjamin H; Liotta, Lance A; Luchini, Alessandra; Espina, Virginia

    2014-01-01

    Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers. PMID:25145492

  7. Protein and lipid refeeding changes protein metabolism and colonic but not small intestinal morphology in protein-depleted rats.

    PubMed

    Qu, Z; Ling, P R; Tahan, S R; Sierra, P; Onderdonk, A B; Bistrian, B R

    1996-04-01

    In this study, we fed rats a 2% casein AIN 76 diet for 2 wk to produce protein malnutrition. We determined in these animals the effects of different concentrations of dietary protein refeeding (2% and 20% casein) on recovery and gut mucosal repletion and the potential role of type of dietary fat in the regulation of protein metabolism and mucosal growth by providing conventional long-chain triglyceride (LCT), a structured lipid composed of long-, medium- and short-chain fatty acids (SC/SL), or a physical mixture of the same components present in the structured lipid given as individual pure triglycerides (SC/PM) along with adequate amounts of protein and energy. The results confirmed that protein malnutrition can be reversed rapidly by protein refeeding, as indicated by an increase in body weight, positive nitrogen balance, liver growth and elevations in plasma concentrations of insulin-like growth factor-1, leucine and albumin. In the colon, crypt cell number, crypt depth and number of crypt cells in the rapidly proliferating fraction of the colon were greater in rats fed the higher protein diet. However, the general architecture of small intestinal mucosa, including duodenum, jejunum and ileum, was not affected by protein malnutrition. Although the number of colonic cells was similar with fat refeeding, there were significantly fewer displaying the proliferating cell nuclear antigen in the colonic epithelium when rats were fed SC/PM compared with SC/SL. Therefore, changes in colonic mucosal proliferation were only seen with repletion by adequate protein and by SC/SL feeding. PMID:8613894

  8. Trans-splicing Into Highly Abundant Albumin Transcripts for Production of Therapeutic Proteins In Vivo

    PubMed Central

    Wang, Jun; Mansfield, S Gary; Cote, Colette A; Jiang, Ping Du; Weng, Ke; Amar, Marcelo JA; Brewer, Bryan H; Remaley, Alan T; McGarrity, Gerard J; Garcia-Blanco, Mariano A; Puttaraju, M

    2008-01-01

    Spliceosome-mediated RNA trans-splicing has emerged as an exciting mode of RNA therapy. Here we describe a novel trans-splicing strategy, which targets highly abundant pre-mRNAs, to produce therapeutic proteins in vivo. First, we used a pre-trans-splicing molecule (PTM) that mediated trans-splicing of human apolipoprotein A-I (hapoA-I) into the highly abundant mouse albumin exon 1. Hydrodynamic tail vein injection of the hapoA-I PTM plasmid in mice followed by analysis of the chimeric transcripts and protein, confirmed accurate and efficient trans-splicing into albumin pre-mRNA and production of hapoA-I protein. The versatility of this approach was demonstrated by producing functional human papillomavirus type-16 E7 (HPV16-E7) single-chain antibody in C57BL/6 mice and functional factor VIII (FVIII) and phenotypic correction in hemophilia A mice. Altogether, these studies demonstrate that trans-splicing to highly abundant albumin transcripts can be used as a general platform to produce therapeutic proteins in vivo. PMID:19066600

  9. Unfoldomics of prostate cancer: on the abundance and roles of intrinsically disordered proteins in prostate cancer.

    PubMed

    Landau, Kevin S; Na, Insung; Schenck, Ryan O; Uversky, Vladimir N

    2016-01-01

    Prostatic diseases such as prostate cancer and benign prostatic hyperplasia are highly prevalent among men. The number of studies focused on the abundance and roles of intrinsically disordered proteins in prostate cancer is rather limited. The goal of this study is to analyze the prevalence and degree of disorder in proteins that were previously associated with the prostate cancer pathogenesis and to compare these proteins to the entire human proteome. The analysis of these datasets provides means for drawing conclusions on the roles of disordered proteins in this common male disease. We also hope that the results of our analysis can potentially lead to future experimental studies of these proteins to find novel pathways associated with this disease. PMID:27453073

  10. Unfoldomics of prostate cancer: on the abundance and roles of intrinsically disordered proteins in prostate cancer

    PubMed Central

    Landau, Kevin S; Na, Insung; Schenck, Ryan O; Uversky, Vladimir N

    2016-01-01

    Prostatic diseases such as prostate cancer and benign prostatic hyperplasia are highly prevalent among men. The number of studies focused on the abundance and roles of intrinsically disordered proteins in prostate cancer is rather limited. The goal of this study is to analyze the prevalence and degree of disorder in proteins that were previously associated with the prostate cancer pathogenesis and to compare these proteins to the entire human proteome. The analysis of these datasets provides means for drawing conclusions on the roles of disordered proteins in this common male disease. We also hope that the results of our analysis can potentially lead to future experimental studies of these proteins to find novel pathways associated with this disease. PMID:27453073

  11. Fpk1/2 kinases regulate cellular sphingoid long-chain base abundance and alter cellular resistance to LCB elevation or depletion

    PubMed Central

    Yamane-Sando, Yukari; Shimobayashi, Etsuko; Shimobayashi, Mitsugu; Kozutsumi, Yasunori; Oka, Shogo; Takematsu, Hiromu

    2014-01-01

    Sphingolipids are a family of eukaryotic lipids biosynthesized from sphingoid long-chain bases (LCBs). Sphingolipids are an essential class of lipids, as their depletion results in cell death. However, acute LCB supplementation is also toxic; thus, proper cellular LCB levels should be maintained. To characterize the “sphingolipid-signaling intercross,” we performed a kinome screening assay in which budding yeast protein kinase-knockout strains were screened for resistance to ISP-1, a potent inhibitor of LCB biosynthesis. Here, one pair of such DIR (deletion-mediated ISP-1 resistance) genes, FPK1 and FPK2, was further characterized. Cellular LCB levels increased in the fpk1/2Δ strain, which was hypersensitive to phytosphingosine (PHS), a major LCB species of yeast cells. Concomitantly, this strain acquired resistance to ISP-1. Fpk1 and Fpk2 were involved in two downstream events; that is, ISP-1 uptake due to aminophospholipid flippase and LCB degradation due to LCB4 expression. RSK3, which belongs to the p90-S6K subfamily, was identified as a functional counterpart of Fpk1/2 in mammalian cells as the RSK3 gene functionally complemented the ISP-1-resistant phenotype of fpk1/2Δ cells. PMID:24510621

  12. Influence of Acute High Glucose on Protein Abundance Changes in Murine Glomerular Mesangial Cells

    PubMed Central

    Barati, Michelle T.; Gould, James C.; Salyer, Sarah A.; Isaacs, Susan; Wilkey, Daniel W.; Merchant, Michael L.

    2016-01-01

    The effects of acute exposure to high glucose levels as experienced by glomerular mesangial cells in postprandial conditions and states such as in prediabetes were investigated using proteomic methods. Two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry methods were used to identify protein expression patterns in immortalized rat mesangial cells altered by 2 h high glucose (HG) growth conditions as compared to isoosmotic/normal glucose control (NG⁎) conditions. Unique protein expression changes at 2 h HG treatment were measured for 51 protein spots. These proteins could be broadly grouped into two categories: (1) proteins involved in cell survival/cell signaling and (2) proteins involved in stress response. Immunoblot experiments for a protein belonging to both categories, prohibitin (PHB), supported a trend for increased total expression as well as significant increases in an acidic PHB isoform. Additional studies confirmed the regulation of proteasomal subunit alpha-type 2 and the endoplasmic reticulum chaperone and oxidoreductase PDI (protein disulfide isomerase), suggesting altered ER protein folding capacity and proteasomal function in response to acute HG. We conclude that short term high glucose induces subtle changes in protein abundances suggesting posttranslational modifications and regulation of pathways involved in proteostasis. PMID:26839892

  13. Influence of Acute High Glucose on Protein Abundance Changes in Murine Glomerular Mesangial Cells.

    PubMed

    Barati, Michelle T; Gould, James C; Salyer, Sarah A; Isaacs, Susan; Wilkey, Daniel W; Merchant, Michael L

    2016-01-01

    The effects of acute exposure to high glucose levels as experienced by glomerular mesangial cells in postprandial conditions and states such as in prediabetes were investigated using proteomic methods. Two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry methods were used to identify protein expression patterns in immortalized rat mesangial cells altered by 2 h high glucose (HG) growth conditions as compared to isoosmotic/normal glucose control (NG(⁎)) conditions. Unique protein expression changes at 2 h HG treatment were measured for 51 protein spots. These proteins could be broadly grouped into two categories: (1) proteins involved in cell survival/cell signaling and (2) proteins involved in stress response. Immunoblot experiments for a protein belonging to both categories, prohibitin (PHB), supported a trend for increased total expression as well as significant increases in an acidic PHB isoform. Additional studies confirmed the regulation of proteasomal subunit alpha-type 2 and the endoplasmic reticulum chaperone and oxidoreductase PDI (protein disulfide isomerase), suggesting altered ER protein folding capacity and proteasomal function in response to acute HG. We conclude that short term high glucose induces subtle changes in protein abundances suggesting posttranslational modifications and regulation of pathways involved in proteostasis. PMID:26839892

  14. Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3

    SciTech Connect

    Laiko, Marina; Murtazina, Rakhilya; Malyukova, Irina; Zhu, Chengru; Boedeker, Edgar C.; Gutsal, Oksana; O'Malley, Robert; Cole, Robert N.; Tarr, Phillip I.; Murray, Karen F.; Kane, Anne; Donowitz, Mark; Kovbasnjuk, Olga

    2010-02-15

    Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.

  15. Mature adipocyte proteome reveals differentially altered protein abundances between lean, overweight and morbidly obese human subjects.

    PubMed

    Benabdelkamel, Hicham; Masood, Afshan; Almidani, Ghaith M; Alsadhan, Abdulmajeed A; Bassas, Abdulelah F; Duncan, Mark W; Alfadda, Assim A

    2015-02-01

    Overweight (OW) and obese individuals are considered to be graded parts of the scale having increasing weight as a common feature. They may not, however, be part of the same continuum and may differ metabolically. In this study we applied an untargeted proteomic approach to compare protein abundances in mature adipocytes derived from the subcutaneous adipose tissue of overweight and morbidly obese female subjects to those of lean age matched controls. Mature adipocytes were isolated from liposuction samples of abdominal subcutaneous adipose tissue collected from both lean (L; n = 7, 23.3 ± 0.4 kg/m(2); mean BMI ± SD), overweight (OW; n = 8, 27.9 ± 0.6 kg/m(2); mean BMI ± SD) and morbidly obese (MOB; n = 7, 44.8 ± 3.8 kg/m(2); mean BMI ± SD) individuals. Total protein extracts were then compared by two-dimensional difference in gel electrophoresis (2D DIGE). One hundred and ten differentially expressed protein spots (i.e., fitting the statistical criteria ANOVA test, p < 0.05; fold-change ≥1.5) were detected, and of these, 89 were identified by MALDI-TOF mass spectrometry. Of these, 66 protein spots were common to both groups whereas 23 were unique to the MOB group. Significant differences were evident in the abundances of key proteins involved in glucose and lipid metabolism, energy regulation, cytoskeletal structure and redox control signaling pathways. Differences in the abundance of some chaperones were also evident. The differentially abundant proteins were investigated using Ingenuity Pathway Analysis (IPA) to establish their associations with known biological functions. The network identified in the OW group with the highest score relates to-: cell-to-cell signaling and interaction; in contrast, in the MOB group the major interacting pathways are associated with lipid metabolism, small molecule biochemistry and cancer. The differences in abundance of the differentially regulated proteins were validated by

  16. Relationships between protein-encoding gene abundance and corresponding process are commonly assumed yet rarely observed.

    PubMed

    Rocca, Jennifer D; Hall, Edward K; Lennon, Jay T; Evans, Sarah E; Waldrop, Mark P; Cotner, James B; Nemergut, Diana R; Graham, Emily B; Wallenstein, Matthew D

    2015-08-01

    For any enzyme-catalyzed reaction to occur, the corresponding protein-encoding genes and transcripts are necessary prerequisites. Thus, a positive relationship between the abundance of gene or transcripts and corresponding process rates is often assumed. To test this assumption, we conducted a meta-analysis of the relationships between gene and/or transcript abundances and corresponding process rates. We identified 415 studies that quantified the abundance of genes or transcripts for enzymes involved in carbon or nitrogen cycling. However, in only 59 of these manuscripts did the authors report both gene or transcript abundance and rates of the appropriate process. We found that within studies there was a significant but weak positive relationship between gene abundance and the corresponding process. Correlations were not strengthened by accounting for habitat type, differences among genes or reaction products versus reactants, suggesting that other ecological and methodological factors may affect the strength of this relationship. Our findings highlight the need for fundamental research on the factors that control transcription, translation and enzyme function in natural systems to better link genomic and transcriptomic data to ecosystem processes. PMID:25535936

  17. Relationships between protein-encoding gene abundance and corresponding process are commonly assumed yet rarely observed

    PubMed Central

    Rocca, Jennifer D; Hall, Edward K; Lennon, Jay T; Evans, Sarah E; Waldrop, Mark P; Cotner, James B; Nemergut, Diana R; Graham, Emily B; Wallenstein, Matthew D

    2015-01-01

    For any enzyme-catalyzed reaction to occur, the corresponding protein-encoding genes and transcripts are necessary prerequisites. Thus, a positive relationship between the abundance of gene or transcripts and corresponding process rates is often assumed. To test this assumption, we conducted a meta-analysis of the relationships between gene and/or transcript abundances and corresponding process rates. We identified 415 studies that quantified the abundance of genes or transcripts for enzymes involved in carbon or nitrogen cycling. However, in only 59 of these manuscripts did the authors report both gene or transcript abundance and rates of the appropriate process. We found that within studies there was a significant but weak positive relationship between gene abundance and the corresponding process. Correlations were not strengthened by accounting for habitat type, differences among genes or reaction products versus reactants, suggesting that other ecological and methodological factors may affect the strength of this relationship. Our findings highlight the need for fundamental research on the factors that control transcription, translation and enzyme function in natural systems to better link genomic and transcriptomic data to ecosystem processes. PMID:25535936

  18. RNA-dependent protein kinase (PKR) depletes nutrients, inducing phosphorylation of AMP-activated kinase in lung cancer.

    PubMed

    Guo, Chengcheng; Hao, Chuncheng; Shao, RuPing; Fang, Bingliang; Correa, Arlene M; Hofstetter, Wayne L; Roth, Jack A; Behrens, Carmen; Kalhor, Neda; Wistuba, Ignacio I; Swisher, Stephen G; Pataer, Apar

    2015-05-10

    We have demonstrated that RNA-dependent protein kinase (PKR) and its downstream protein p-eIF2α are independent prognostic markers for overall survival in lung cancer. In the current study, we further investigate the interaction between PKR and AMPK in lung tumor tissue and cancer cell lines. We examined PKR protein expression in 55 frozen primary lung tumor tissues by Western blotting and analyzed the association between PKR expression and expression of 139 proteins on tissue samples examined previously by Reverse Phase Protein Array (RPPA) from the same 55 patients. We observed that biomarkers were either positively (phosphorylated AMP-activated kinase(T172) [p-AMPK]) or negatively (insulin receptor substrate 1, meiotic recombination 11, ATR interacting protein, telomerase, checkpoint kinase 1, and cyclin E1) correlated with PKR. We further confirmed that induction of PKR with expression vectors in lung cancer cells causes activation of the AMPK protein independent of the LKB1, TAK1, and CaMKKβ pathway. We found that PKR causes nutrient depletion, which increases AMP levels and decreases ATP levels, causing AMPK phosphorylation. We further demonstrated that inhibiting AMPK expression with compound C or siRNA enhanced PKR-mediated cell death. We next explored the combination of PKR and p-AMPK expression in NSCLC patients and observed that expression of p-AMPK predicted a poor outcome for adenocarcinoma patients with high PKR expression and a better prognosis for those with low PKR expression. These findings were consistent with our in vitro results. AMPK might rescue cells facing metabolic stresses, such as ATP depletion caused by PKR. Our data indicate that PKR causes nutrient depletion, which induces the phosphorylation of AMPK. AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation. PMID:25798539

  19. Familial prion protein mutants inhibit Hrd1-mediated retrotranslocation of misfolded proteins by depleting misfolded protein sensor BiP.

    PubMed

    Peters, Sarah L; Déry, Marc-André; LeBlanc, Andrea C

    2016-03-01

    Similar to many proteins trafficking through the secretory pathway, cellular prion protein (PrP) partly retrotranslocates from the endoplasmic reticulum to the cytosol through the endoplasmic reticulum-associated degradation (ERAD) pathway in an attempt to alleviate accumulation of cellular misfolded PrP. Surprisingly, familial PrP mutants fail to retrotranslocate and simultaneously block normal cellular PrP retrotranslocation. That impairments in retrotranslocation of misfolded proteins could lead to global disruptions in cellular homeostasis prompted further investigations into PrP mutant retrotranslocation defects. A gain- and loss-of-function approach identified human E3 ubiquitin ligase, Hrd1, as a critical regulator of PrP retrotranslocation in mammalian cells. Expression of familial human PrP mutants, V210I(129V) and M232R(129V), not only abolished PrP retrotranslocation, but also that of Hrd1-dependent ERAD substrates, transthyretin TTR(D18G) and α1-anti-trypsin A1AT(NHK). Mutant PrP expression decreased binding immunoglobulin protein (BiP) levels by 50% and attenuated ER stress-induced BiP by increasing BiP turnover 6-fold. Overexpression of BiP with PrP mutants rescued retrotranslocation of PrP, TTR(D18G) and A1AT(NHK). PrP mutants-induced cell death was also rescued by co-expression of BiP. These results show that PrP mutants highjack the Hrd1-dependent ERAD pathway, an action that would result in misfolded protein accumulation especially in terminally differentiated neurons. This could explain the age-dependent neuronal degeneration in familial prion diseases. PMID:26740554

  20. Changes in protein abundance are observed in bacterial isolates from a natural host

    PubMed Central

    Rees, Megan A.; Stinear, Timothy P.; Goode, Robert J. A.; Coppel, Ross L.; Smith, Alexander I.; Kleifeld, Oded

    2015-01-01

    Bacterial proteomic studies frequently use strains cultured in synthetic liquid media over many generations. It is uncertain whether bacterial proteins expressed under these conditions will be the same as the repertoire found in natural environments, or when bacteria are infecting a host organism. Thus, genomic and proteomic characterization of bacteria derived from the host environment in comparison to reference strains grown in the lab, should aid understanding of pathogenesis. Isolates of Corynebacterium pseudotuberculosis were obtained from the lymph nodes of three naturally infected sheep and compared to a laboratory reference strain using bottom-up proteomics, after whole genome sequencing of each of the field isolates. These comparisons were performed following growth in liquid media that allowed us to reach the required protein amount for proteomic analysis. Over 1350 proteins were identified in the isolated strains, from which unique proteome features were revealed. Several of the identified proteins demonstrated a significant abundance difference in the field isolates compared to the reference strain even though there were no obvious differences in the DNA sequence of the corresponding gene or in nearby non-coding DNA. Higher abundance in the field isolates was observed for proteins related to hypoxia and nutrient deficiency responses as well as to thiopeptide biosynthesis. PMID:26528441

  1. LEDGF dominant interference proteins demonstrate prenuclear exposure of HIV-1 integrase and synergize with LEDGF depletion to destroy viral infectivity.

    PubMed

    Meehan, Anne M; Saenz, Dyana T; Morrison, James; Hu, Chunling; Peretz, Mary; Poeschla, Eric M

    2011-04-01

    Target cell overexpression of the integrase binding domain (IBD) of LEDGF/p75 (LEDGF) inhibits HIV-1 replication. The mechanism and protein structure requirements for this dominant interference are unclear. More generally, how and when HIV-1 uncoating occurs postentry is poorly defined, and it is unknown whether integrase within the evolving viral core becomes accessible to cellular proteins prior to nuclear entry. We used LEDGF dominant interference to address the latter question while characterizing determinants of IBD antiviral activity. Fusions of green fluorescent protein (GFP) with multiple C-terminal segments of LEDGF inhibited HIV-1 replication substantially, but minimal chimeras of either polarity (GFP-IBD or IBD-GFP) were most effective. Combining GFP-IBD expression with LEDGF depletion was profoundly antiviral. CD4(+) T cell lines were rendered virtually uninfectable, with single-cycle HIV-1 infectivity reduced 4 logs and high-input (multiplicity of infection = 5.0) replication completely blocked. We restricted GFP-IBD to specific intracellular locations and found that antiviral activity was preserved when the protein was confined to the cytoplasm or directed to the nuclear envelope. The life cycle block triggered by the cytoplasm-restricted protein manifested after nuclear entry, at the level of integration. We conclude that integrase within the viral core becomes accessible to host cell protein interaction in the cytoplasm. LEDGF dominant interference and depletion impair HIV-1 integration at distinct postentry stages. GFP-IBD may trigger premature or improper integrase oligomerization. PMID:21270171

  2. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces.

    PubMed

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota. PMID:27042829

  3. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces

    PubMed Central

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota. PMID:27042829

  4. A Systems Approach to Elucidate Heterosis of Protein Abundances in Yeast.

    PubMed

    Blein-Nicolas, Mélisande; Albertin, Warren; da Silva, Telma; Valot, Benoît; Balliau, Thierry; Masneuf-Pomarède, Isabelle; Bely, Marina; Marullo, Philippe; Sicard, Delphine; Dillmann, Christine; de Vienne, Dominique; Zivy, Michel

    2015-08-01

    Heterosis is a universal phenomenon that has major implications in evolution and is of tremendous agro-economic value. To study the molecular manifestations of heterosis and to find factors that maximize its strength, we implemented a large-scale proteomic experiment in yeast. We analyzed the inheritance of 1,396 proteins in 55 inter- and intraspecific hybrids obtained from Saccharomyces cerevisiae and S. uvarum that were grown in grape juice at two temperatures. We showed that the proportion of heterotic proteins was highly variable depending on the parental strain and on the temperature considered. For intraspecific hybrids, this proportion was higher at nonoptimal temperature. Unexpectedly, heterosis for protein abundance was strongly biased toward positive values in interspecific hybrids but not in intraspecific hybrids. Computer modeling showed that this observation could be accounted for by assuming concave relationships between protein abundances and their controlling factors, in line with the metabolic model of heterosis. These results point to nonlinear processes that could play a central role in heterosis. PMID:25971257

  5. A Systems Approach to Elucidate Heterosis of Protein Abundances in Yeast*

    PubMed Central

    Blein-Nicolas, Mélisande; Albertin, Warren; da Silva, Telma; Valot, Benoît; Balliau, Thierry; Masneuf-Pomarède, Isabelle; Bely, Marina; Marullo, Philippe; Sicard, Delphine; Dillmann, Christine; de Vienne, Dominique; Zivy, Michel

    2015-01-01

    Heterosis is a universal phenomenon that has major implications in evolution and is of tremendous agro-economic value. To study the molecular manifestations of heterosis and to find factors that maximize its strength, we implemented a large-scale proteomic experiment in yeast. We analyzed the inheritance of 1,396 proteins in 55 inter- and intraspecific hybrids obtained from Saccharomyces cerevisiae and S. uvarum that were grown in grape juice at two temperatures. We showed that the proportion of heterotic proteins was highly variable depending on the parental strain and on the temperature considered. For intraspecific hybrids, this proportion was higher at nonoptimal temperature. Unexpectedly, heterosis for protein abundance was strongly biased toward positive values in interspecific hybrids but not in intraspecific hybrids. Computer modeling showed that this observation could be accounted for by assuming concave relationships between protein abundances and their controlling factors, in line with the metabolic model of heterosis. These results point to nonlinear processes that could play a central role in heterosis. PMID:25971257

  6. Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains.

    PubMed

    Rychli, Kathrin; Grunert, Tom; Ciolacu, Luminita; Zaiser, Andreas; Razzazi-Fazeli, Ebrahim; Schmitz-Esser, Stephan; Ehling-Schulz, Monika; Wagner, Martin

    2016-02-01

    The foodborne pathogen Listeria monocytogenes, responsible for listeriosis a rare but severe infection disease, can survive in the food processing environment for month or even years. So-called persistent L. monocytogenes strains greatly increase the risk of (re)contamination of food products, and are therefore a great challenge for food safety. However, our understanding of the mechanism underlying persistence is still fragmented. In this study we compared the exoproteome of three persistent strains with the reference strain EGDe under mild stress conditions using 2D differential gel electrophoresis. Principal component analysis including all differentially abundant protein spots showed that the exoproteome of strain EGDe (sequence type (ST) 35) is distinct from that of the persistent strain R479a (ST8) and the two closely related ST121 strains 4423 and 6179. Phylogenetic analyses based on multilocus ST genes showed similar grouping of the strains. Comparing the exoproteome of strain EGDe and the three persistent strains resulted in identification of 22 differentially expressed protein spots corresponding to 16 proteins. Six proteins were significantly increased in the persistent L. monocytogenes exoproteomes, among them proteins involved in alkaline stress response (e.g. the membrane anchored lipoprotein Lmo2637 and the NADPH dehydrogenase NamA). In parallel the persistent strains showed increased survival under alkaline stress, which is often provided during cleaning and disinfection in the food processing environments. In addition, gene expression of the proteins linked to stress response (Lmo2637, NamA, Fhs and QoxA) was higher in the persistent strain not only at 37 °C but also at 10 °C. Invasion efficiency of EGDe was higher in intestinal epithelial Caco2 and macrophage-like THP1 cells compared to the persistent strains. Concurrently we found higher expression of proteins involved in virulence in EGDe e.g. the actin-assembly-inducing protein ActA and the

  7. Structure and function of a mitochondrial late embryogenesis abundant protein are revealed by desiccation.

    PubMed

    Tolleter, Dimitri; Jaquinod, Michel; Mangavel, Cécile; Passirani, Catherine; Saulnier, Patrick; Manon, Stephen; Teyssier, Emeline; Payet, Nicole; Avelange-Macherel, Marie-Hélène; Macherel, David

    2007-05-01

    Few organisms are able to withstand desiccation stress; however, desiccation tolerance is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. These hydrophilic proteins are prominent in plant seeds but also found in desiccation-tolerant organisms. In spite of many theories and observations, LEA protein function remains unclear. Here, we show that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into alpha-helices upon desiccation. Structural modeling revealed an analogy with class A amphipathic helices of apolipoproteins that coat low-density lipoprotein particles in mammals. LEAM appears spontaneously modified by deamidation and oxidation of several residues that contribute to its structural features. LEAM interacts with membranes in the dry state and protects liposomes subjected to drying. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. According to sequence analyses of several homologous proteins from various desiccation-tolerant organisms, a similar protection mechanism likely acts with other types of cellular membranes. PMID:17526751

  8. Mass Spectrometry and Next-Generation Sequencing Reveal an Abundant and Rapidly Evolving Abalone Sperm Protein

    PubMed Central

    Palmer, Melody R.; McDowall, Margo H.; Stewart, Lia; Ouaddi, Aleena; MacCoss, Michael J.; Swanson, Willie J.

    2014-01-01

    SUMMARY Abalone, a broadcast spawning marine mollusk, is an important model for molecular interactions and positive selection in fertilization, but the focus has previously been on only two sperm proteins, lysin and sp18.We used genomic and proteomic techniques to bring new insights to this model by characterizing the testis transcriptome and sperm proteome of the Red abalone Haliotis rufescens. One pair of homologous, testis-specific proteins contains a secretion signal and is small, abundant, and associated with the acrosome. Comparative analysis revealed that homologs are extremely divergent between species, and show strong evidence for positive selection. The acrosomal localization and rapid evolution of these proteins indicates that they play an important role in fertilization, and could be involved in the species-specificity of sperm-egg interactions in abalone. Our genomic and proteomic characterization of abalone fertilization resulted in the identification of interesting, novel peptides that have eluded detection in this important model system for 20 years. PMID:23585193

  9. Changes in Relative Thylakoid Protein Abundance Induced by Fluctuating Light in the Diatom Thalassiosira pseudonana.

    PubMed

    Grouneva, Irina; Muth-Pawlak, Dorota; Battchikova, Natalia; Aro, Eva-Mari

    2016-05-01

    One of the hallmarks of marine diatom biology is their ability to cope with rapid changes in light availability due to mixing of the water column and the lens effect. We investigated how irradiance fluctuations influence the relative abundance of key photosynthetic proteins in the centric diatom Thalassiosira pseudonana by means of mass-spectrometry-based approaches for relative protein quantitation. Most notably, fluctuating-light conditions lead to a substantial overall up-regulation of light-harvesting complex proteins as well as several subunits of photosystems II and I. Despite an initial delay in growth under FL, there were no indications of FL-induced photosynthesis limitation, in contrast to other photosynthetic organisms. Our findings further strengthen the notion that diatoms use a qualitatively different mechanism of photosynthetic regulation in which chloroplast-mitochondria interaction has overtaken crucial regulatory processes of photosynthetic light reactions that are typical for the survival of land plants, green algae, and cyanobacteria. PMID:27025989

  10. Three abundant germ line-specific transcripts in Volvox carteri encode photosynthetic proteins.

    PubMed

    Choi, G; Przybylska, M; Straus, D

    1996-09-01

    Volvox carteri is a multicellular eukaryotic green alga composed of about 2000 cells of only two differentiated types: somatic and germ line. To understand how embryonic cells are assigned either to somatic or germ line fates, we are investigating the regulation of transcripts that are abundant in only one cell type. Here we report the identity of three transcripts that are coordinately expressed at high levels in germ line cells but not in somatic cells. Surprisingly, all three transcripts encode photosynthetic chloroplast proteins (light-harvesting complex protein, oxygen-evolving enhancer protein 3, and ferredoxin-NADP+ reductase) that are transcribed from nuclear genes. We discuss why these mRNAs might be required at high levels in germ line cells and present a hypothesis, suggested by our results, on the evolution of cell specialization in the Volvocales. PMID:8781179

  11. Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A*

    PubMed Central

    Svozil, Julia; Hirsch-Hoffmann, Matthias; Dudler, Robert; Gruissem, Wilhelm; Baerenfaller, Katja

    2014-01-01

    As proteins are the main effectors inside cells, their levels need to be tightly regulated. This is partly achieved by specific protein degradation via the Ubiquitin-26S proteasome system (UPS). In plants, an exceptionally high number of proteins are involved in Ubiquitin-26S proteasome system-mediated protein degradation and it is known to regulate most, if not all, important cellular processes. Here, we investigated the response to the inhibition of the proteasome at the protein level treating leaves with the specific inhibitor Syringolin A (SylA) in a daytime specific manner and found 109 accumulated and 140 decreased proteins. The patterns of protein level changes indicate that the accumulating proteins cause proteotoxic stress that triggers various responses. Comparing protein level changes in SylA treated with those in a transgenic line over-expressing a mutated ubiquitin unable to form polyubiquitylated proteins produced little overlap pointing to different response pathways. To distinguish between direct and indirect targets of the UPS we also enriched and identified ubiquitylated proteins after inhibition of the proteasome, revealing a total of 1791 ubiquitylated proteins in leaves and roots and 1209 that were uniquely identified in our study. The comparison of the ubiquitylated proteins with those changing in abundance after SylA-mediated inhibition of the proteasome confirmed the complexity of the response and revealed that some proteins are regulated both at transcriptional and post-transcriptional level. For the ubiquitylated proteins that accumulate in the cytoplasm but are targeted to the plastid or the mitochondrion, we often found peptides in their target sequences, demonstrating that the UPS is involved in controlling organellar protein levels. Attempts to identify the sites of ubiquitylation revealed that the specific properties of this post-translational modification can lead to incorrect peptide spectrum assignments in complex peptide mixtures

  12. Simplified and efficient quantification of low-abundance proteins at very high multiplex via targeted mass spectrometry.

    PubMed

    Burgess, Michael W; Keshishian, Hasmik; Mani, D R; Gillette, Michael A; Carr, Steven A

    2014-04-01

    Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) of plasma that has been depleted of abundant proteins and fractionated at the peptide level into six to eight fractions is a proven method for quantifying proteins present at low nanogram-per-milliliter levels. A drawback of fraction-MRM is the increased analysis time due to the generation of multiple fractions per biological sample. We now report that the use of heated, long, fused silica columns (>30 cm) packed with 1.9 μm of packing material can reduce or eliminate the need for fractionation prior to LC-MRM-MS without a significant loss of sensitivity or precision relative to fraction-MRM. We empirically determined the optimal column length, temperature, gradient duration, and sample load for such assays and used these conditions to study detection sensitivity and assay precision. In addition to increased peak capacity, longer columns packed with smaller beads tolerated a 4- to 6-fold increase in analyte load without a loss of robustness or reproducibility. The longer columns also provided a 4-fold improvement in median limit-of-quantitation values with increased assay precision relative to the standard 12 cm columns packed with 3 μm material. Overall, the optimized chromatography provided an approximately 3-fold increase in analysis throughput with excellent robustness and less than a 2-fold reduction in quantitative sensitivity relative to fraction-MRM. The value of the system for increased multiplexing was demonstrated by the ability to configure an 800-plex MRM-MS assay, run in a single analysis, comprising 2400 transitions with retention time scheduling to monitor 400 unlabeled and heavy labeled peptide pairs. PMID:24522978

  13. Selectable one-step PCR-mediated integration of a degron for rapid depletion of endogenous human proteins

    PubMed Central

    Sheridan, Ryan M.; Bentley, David L.

    2016-01-01

    Manipulation of protein stability with ligand-regulated degron fusions is a powerful method for investigating gene function. We developed a selectable cassette for easy C-terminal tagging of endogenous human proteins with the E. coli dihydrofolate reductase (eDHFR) degron using CRISPR/Cas9 genome editing. This cassette permits high-efficiency recovery of correct integration events using an in-frame self-cleaving 2A peptide and the puromycin resistance gene. PCR amplified donor eDHFR cassette fragments with 100 bases of homology on each end are integrated by homology-directed repair (HDR) of guide RNA (gRNA)-targeted double-stranded DNA breaks at the 3′ ends of open reading frames (ORFs). As proof of principle, we generated cell lines in which three endogenous proteins were tagged with the eDHFR degron. When the antibiotic trimethoprim is removed from the media, each of the eDHFR-tagged proteins was depleted by >90% within 2–4 h, and this depletion was reversed by re-addition of trimethoprim. Since puromycin selection permits recovery of in-frame degron fusions with high efficiency using only 100-bp long regions of homology, this method should be applicable on a genome-wide scale for generating libraries of conditional mutant cell lines. PMID:26842351

  14. Traditional reactive carbonyl scavengers do not prevent the carbonylation of brain proteins induced by acute glutathione depletion.

    PubMed

    Zheng, J; Bizzozero, O A

    2010-03-01

    This study investigated the effect of reactive carbonyl species (RCS)-trapping agents on the formation of protein carbonyls during depletion of brain glutathione (GSH). To this end, rat brain slices were incubated with the GSH-depletor diethyl maleate in the absence or presence of chemically different RCS scavengers (hydralazine, methoxylamine, aminoguanidine, pyridoxamine, carnosine, taurine and z-histidine hydrazide). Despite their strong reactivity towards the most common RCS, none of the scavengers tested, with the exception of hydralazine, prevented protein carbonylation. These findings suggest that the majority of protein-associated carbonyl groups in this oxidative stress paradigm do not derive from stable lipid peroxidation products like malondialdehyde (MDA), acrolein and 4-hydroxynonenal (4-HNE). This conclusion was confirmed by the observation that the amount of MDA-, acrolein- and 4-HNE-protein adducts does not increase upon GSH depletion. Additional studies revealed that the efficacy of hydralazine at preventing carbonylation was due to its ability to reduce oxidative stress, most likely by inhibiting mitochondrial production of superoxide and/or by scavenging lipid free radicals. PMID:20001647

  15. Isolation and characterization of haemoporin, an abundant haemolymph protein from Aplysia californica.

    PubMed Central

    Jaenicke, Elmar; Walsh, Patrick J; Decker, Heinz

    2003-01-01

    In the present study, we show the isolation and characterization of the protein haemoporin, which constitutes the second most abundant protein fraction in the haemolymph of the marine gastropod Aplysia californica. Although Aplysia is commonly used to investigate the molecular basis of learning, not much is known about the proteins in its haemolymph, which is in contact with the neurons owing to the open circulatory system of molluscs. In the native state, haemoporin is a macromolecular complex forming a cylinder with a central solvent-filled pore. The native complex most probably is a homopentamer made up from 70 kDa subunits with a molecular mass of 360 kDa and a sedimentation coefficient of 11.7 S. Prediction of the secondary structure by CD spectroscopy revealed that haemoporin contains 36% alpha-helices and 19% beta-strands. An absorption band in the 300-400 nm region indicates that haemoporin probably contains a bound substance. Haemoporin also contains a below average amount of tryptophan as evident from absorption and fluorescence spectra. The specific absorption coefficient at 280 nm (a (280 nm, 1 mg/ml)) varies between 0.42 and 0.59 l x g(-1) x cm(-1) depending on the method. The function of the protein is not yet known, but there are structural parallels between haemoporin and a pore protein reported previously in the haemolymph of another marine gastropod Megathura crenulata. The alanine-rich N-terminal sequence (AAVPEAAAEATAEAAPVSEF) is unique among protein sequences and indicates an alpha-helical structure. Whereas one side of the helix is hydrophobic and faces the interior of the protein, the other side contains a glutamic cluster, which may form the channel of the pore in the quaternary structure. Thus both proteins might belong to a new class of haemolymph proteins present in the haemolymph of marine gastropods. PMID:12889987

  16. Depletion of Amyloid Precursor Protein (APP) causes G0 arrest in non-small cell lung cancer (NSCLC) cells

    PubMed Central

    Sobol, Anna; Galluzzo, Paola; Weber, Megan J.; Alani, Sara; Bocchetta, Maurizio

    2015-01-01

    We recently reported that Amyloid Precursor Protein (APP) regulates global protein synthesis in a variety of human dividing cells, including non-small cell lung cancer (NSCLC) cells. More specifically, APP depletion causes an increase of both cap- and IRES-dependent translation. Since growth and proliferation are tightly coupled processes, here we asked what effects artificial downregulation of APP could have elicited in NSCLC cells proliferation. APP depletion caused a G0/G1 arrest through destabilization of the cyclin-C protein and reduced pRb phosphorylation at residues Ser802/811. siRNA to cyclin-C mirrored the cell cycle distribution observed when silencing APP. Cells arrested in G0/G1 (and with augmented global protein synthesis) increased their size and underwent a necrotic cell death due to cell membrane permeabilization. These phenotypes were reversed by overexpression of the APP C-terminal domain, indicating a novel role for APP in regulating early cell cycle entry decisions. It is seems that APP moderates the rate of protein synthesis before the cell clears growth factors- and nutrients-dependent checkpoint in mid G1. Our results raise questions on how such processes interact in the context of (at least) dividing NSCLC cells. The data presented here suggest that APP, although required for G0/G1 transitions, moderates the rate of protein synthesis before the cell fully commits to cell cycle progression following mechanisms, which seem additional to concurrent signals deriving from the PI3-K/Akt/mTORC-1 axis. APP appears to play a central role in regulating cell cycle entry with the rate of protein synthesis; and its loss-of-function causes cell size abnormalities and death. PMID:25502341

  17. Depletion of Amyloid Precursor Protein (APP) causes G0 arrest in non-small cell lung cancer (NSCLC) cells.

    PubMed

    Sobol, Anna; Galluzzo, Paola; Weber, Megan J; Alani, Sara; Bocchetta, Maurizio

    2015-06-01

    We recently reported that Amyloid Precursor Protein (APP) regulates global protein synthesis in a variety of human dividing cells, including non-small cell lung cancer (NSCLC) cells. More specifically, APP depletion causes an increase of both cap- and IRES-dependent translation. Since growth and proliferation are tightly coupled processes, here, we asked what effects artificial downregulation of APP could have elicited in NSCLC cells proliferation. APP depletion caused a G0/G1 arrest through destabilization of the cyclin-C protein and reduced pRb phosphorylation at residues Ser802/811. siRNA to cyclin-C mirrored the cell cycle distribution observed when silencing APP. Cells arrested in G0/G1 (and with augmented global protein synthesis) increased their size and underwent a necrotic cell death due to cell membrane permeabilization. These phenotypes were reversed by overexpression of the APP C-terminal domain, indicating a novel role for APP in regulating early cell cycle entry decisions. It is seems that APP moderates the rate of protein synthesis before the cell clears growth factors- and nutrients-dependent checkpoint in mid G1. Our results raise questions on how such processes interact in the context of (at least) dividing NSCLC cells. The data presented here suggest that APP, although required for G0/G1 transitions, moderates the rate of protein synthesis before the cell fully commits to cell cycle progression following mechanisms, which seem additional to concurrent signals deriving from the PI3-K/Akt/mTORC-1 axis. APP appears to play a central role in regulating cell cycle entry with the rate of protein synthesis; and its loss-of-function causes cell size abnormalities and death. PMID:25502341

  18. Depletion of three combined THOC5 mRNA export protein target genes synergistically induces human hepatocellular carcinoma cell death.

    PubMed

    Saran, S; Tran, D D H; Ewald, F; Koch, A; Hoffmann, A; Koch, M; Nashan, B; Tamura, T

    2016-07-21

    Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis and with limited possibilities of medical intervention. It has been shown that over 100 putative driver genes are associated with multiple recurrently altered pathways in HCC, suggesting that multiple pathways will need to be inhibited for any therapeutic method. mRNA processing is regulated by a complex RNA-protein network that is essential for the maintenance of homeostasis. THOC5, a member of mRNA export complex, has a role in less than 1% of mRNA processing, and is required for cell growth and differentiation, but not for cell survival in normal fibroblasts, hepatocytes and macrophages. In this report, we show that 50% depletion of THOC5 in human HCC cell lines Huh7 and HepG2 induced apoptosis. Transcriptome analysis using THOC5-depleted cells revealed that 396 genes, such as transmembrane BAX inhibitor motif containing 4 (TMBIM4), transmembrane emp24-like trafficking protein 10 (Tmed10) and D-tyrosyl-tRNA deacylase 2 (Dtd2) genes were downregulated in both cell lines. The depletion of one of these THOC5 target genes in Huh7 or HepG2 did not significantly induce cell death, suggesting that these may be fine tuners for HCC cell survival. However, the depletion of a combination of these genes synergistically increased the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive HCC. It must be noted that the depletion of these genes did not induce cell death in the hepatocyte cell line, THLE-2 cells. THOC5 expression was enhanced in 78% of cytological differentiation grading G2 and G3 tumor in primary HCC. Furthermore, the expression of a putative glycoprotein, Tmed10, is correlated to THOC5 expression level in primary HCCs, suggesting that this protein may be a novel biomarker for HCC. These data imply that the suppression of the multiple THOC5 target genes may represent a novel strategy for HCC therapy. PMID:26549021

  19. The highly abundant protein Ag-lbp55 from Ascaridia galli represents a novel type of lipid-binding proteins.

    PubMed

    Jordanova, Rositsa; Radoslavov, Georgi; Fischer, Peter; Torda, Andrew; Lottspeich, Friedrich; Boteva, Raina; Walter, Rolf D; Bankov, Ilia; Liebau, Eva

    2005-12-16

    Lipid-binding proteins exhibit important functions in lipid transport, cellular signaling, gene transcription, and cytoprotection. Their functional analogues in nematodes are nematode polyprotein allergens/antigens and fatty acid and retinoid-binding proteins. This work describes a novel 55-kDa protein, Ag-lbp55, purified from the parasitic nematode Ascaridia galli. By direct N-terminal sequencing, a partial amino acid sequence was obtained that allowed the design of oligonucleotide primers to obtain the full-length cDNA sequence. Sequence analysis revealed the presence of an N-terminal signal peptide of 25 amino acid residues and a FAR domain at the C terminus. Data base searches showed almost no significant homologies to other described proteins. The secondary structure of Ag-lbp55 was predominantly alpha-helical (65%) as shown by CD spectroscopy. It was found to bind with high affinity fatty acids (caprylic, oleic, and palmitic acid) and their fluorescent analogue dansylaminoundecanic acid. Immunolocalization showed that Ag-lbp55 is a highly abundant protein, mainly distributed in the inner hypodermis and extracellularly in the pseudocoelomatic fluid. A similar staining pattern was observed in other pathogenic nematodes, indicating the existence of similar proteins in these species. PMID:16210327

  20. Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins. Molecular characterization and functional analyses.

    PubMed

    NDong, Christian; Danyluk, Jean; Wilson, Kenneth E; Pocock, Tessa; Huner, Norman P A; Sarhan, Fathey

    2002-07-01

    Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, and photosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated with PSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and data mining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, and LEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEA proteins. They share important characteristics such as a sorting signal that is predicted to target them to either the chloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellular fractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene are consistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale). Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of different wheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenic plants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. The putative function of this protein in the enhancement of freezing tolerance is discussed. PMID:12114590

  1. Cold-Regulated Cereal Chloroplast Late Embryogenesis Abundant-Like Proteins. Molecular Characterization and Functional Analyses

    PubMed Central

    NDong, Christian; Danyluk, Jean; Wilson, Kenneth E.; Pocock, Tessa; Huner, Norman P.A.; Sarhan, Fathey

    2002-01-01

    Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, and photosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated with PSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and data mining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, and LEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEA proteins. They share important characteristics such as a sorting signal that is predicted to target them to either the chloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellular fractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene are consistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale). Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of different wheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenic plants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. The putative function of this protein in the enhancement of freezing tolerance is discussed. PMID:12114590

  2. Ultrasensitive Detection of Low-Abundance Protein Biomarkers by Mass Spectrometry Signal Amplification Assay.

    PubMed

    Du, Ruijun; Zhu, Lina; Gan, Jinrui; Wang, Yuning; Qiao, Liang; Liu, Baohong

    2016-07-01

    A mass spectrometry signal amplification method is developed for the ultrasensitive and selective detection of low-abundance protein biomarkers by utilizing tag molecules on gold nanoparticles (AuNPs). EpCAM and thrombin as model targets are captured by specific aptamers immobilized on the AuNPs. With laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS), the mass tag molecules are detected to represent the protein biomarkers. Benefiting from the MS signal amplification, the assay can achieve a limit of detection of 100 aM. The method is further applied to detect thrombin in fetal bovine serum and EpCAM in cell lysates to demonstrate its selectivity and feasibility in complex biological samples. With the high sensitivity and specificity, the protocol shows great promise for providing a new route to single-cell analysis and early disease diagnosis. PMID:27253396

  3. An Ultra-Depleted Mantle Component in the Ontong Java Plateau Revealed by Major, Trace and Volatile Element Abundances in Olivine-Hosted Melt Inclusions

    NASA Astrophysics Data System (ADS)

    Jackson, M. G.; Cabral, R. A.; Rose-Koga, E. F.; Koga, K. T.; Price, A. A.; Hauri, E. H.; Michael, P. J.

    2014-12-01

    The Ontong Java Plateau (OJP) represents the most voluminous large igneous province (LIP) preserved in the geologic record. The most voluminous volcanic stages of the OJP—the Kroenke and Kwaimbaita stages, which dominate the accessible portions of the plateau—have relatively flat primitive mantle normalized rare earth element (REE) patterns, or spidergrams. With the exception of relatively small volumes of late-stage melts—referred to as the Singgalo stage—that are characterized by slightly enriched REE spidergrams, the volcanic stages that dominated the eruptive history of the OJP exhibit remarkably homogeneous, flat REE patterns. Here we isolate, for the first time, olivine-hosted melt inclusions from OJP. We show that the melt inclusions have two clear populations defined by having distinct trace element characteristics. The first population has relatively flat trace element patterns that are similar to that observed in whole rock lavas from the most voluminous volcanic stages (Kroenke and Kwaimbaita stages) recorded in the OJP. In contrast, a second group of melt inclusions, referred to as UDM (ultra-depleted melt) inclusions, exhibit strikingly depleted REE spidergrams; these trace element patterns are far more depleted than any previously reported lava from OJP. The UDM have unique trace element signatures that preclude an origin by assimilation of hydrothermally-altered oceanic crust or re-melting the depleted mantle source left over after melt extraction during construction of the OJP. We interpret the new UDM compositions to be the result of melting of a previously unrecognized ultra-depleted component hosted in the OJP mantle source.

  4. Spatial Mapping of Protein Abundances in the Mouse Brain by Voxelation Integrated with High-Throughput Liquid Chromatography - Mass Spectrometry

    SciTech Connect

    Petyuk, Vladislav A; Qian, Weijun; Chin, Mark H; Wang, Haixing H; Livesay, Eric A; Monroe, Matthew E; Adkins, Joshua N; Jaitly, Navdeep; Anderson, David J; Camp, David G; Smith, Desmond J; Smith, Richard D

    2007-01-25

    Temporally and spatially resolved mapping of protein abundance patterns within the mammalian brain is of significant interest for understanding brain function and molecular etiologies of neurodegenerative diseases; however, such imaging efforts have been greatly challenged by complexity of the proteome, throughput and sensitivity of applied analytical methodologies, and accurate quantitation of protein abundances across the brain. Here, we describe a methodology for comprehensive spatial proteome mapping that addresses these challenges by employing voxelation integrated with automated microscale sample processing, high-throughput LC system coupled with high resolution Fourier transform ion cyclotron mass spectrometer and a “universal” stable isotope labeled reference sample approach for robust quantitation. We applied this methodology as a proof-of-concept trial for the analysis of protein distribution within a single coronal slice of a C57BL/6J mouse brain. For relative quantitation of the protein abundances across the slice, an 18O-isotopically labeled reference sample, derived from a whole control coronal slice from another mouse, was spiked into each voxel sample and stable isotopic intensity ratios were used to obtain measures of relative protein abundances. In total, we generated maps of protein abundance patterns for 1,028 proteins. The significant agreement of the protein distributions with previously reported data supports the validity of this methodology, which opens new opportunities for studying the spatial brain proteome and its dynamics during the course of disease progression and other important biological and associated health aspects in a discovery-driven fashion.

  5. Regression of lung and colon cancer xenografts by depleting or inhibiting RLIP76 (Ral-binding protein 1).

    PubMed

    Singhal, Sharad S; Singhal, Jyotsana; Yadav, Sushma; Dwivedi, Seema; Boor, Paul J; Awasthi, Yogesh C; Awasthi, Sanjay

    2007-05-01

    Ral-binding protein 1 (RALBP1) is a stress-responsive and stress-protective multispecific transporter of glutathione conjugates (GS-E) and xenobiotic toxins. It is frequently overexpressed in malignant cells and plays a prominent antiapoptotic role selectively in cancer cells through its ability to control cellular concentration of proapoptotic oxidized lipid byproducts. In the absence of chemotherapy, depletion or inhibition of RALBP1 causes regression of syngeneic mouse B16 melanoma. Because RALBP1 transports anthracycline and Vinca alkaloid drugs, as well as GS-E, and because it confers resistance to these drugs, we proposed that depletion or inhibition of RALBP1 should cause regression of human solid tumors that overexpress RALBP1 and augment chemotherapy efficacy. Non-small-cell lung cancer (NSCLC) H358 and H520 and colon SW480 cell lines were used. Cytotoxic synergy between anti-RALBP1 immunoglobulin G (IgG), cis-diammine-dichloroplatinum (II) [CDDP], and vinorelbine was examined in cell culture and xenografts of NSCLC cells. Effects of RALBP1 depletion by antisense were examined in xenografts of NSCLC H358, NSCLC H520, and colon SW480 cells. RALBP1 depletion by phosphorothioate antisense was confirmed and was associated with rapid, complete, and sustained remissions in established s.c. human lung and colon xenografts. RALBP1 inhibition by anti-RALBP1 IgG was equally as effective as antisense and enhanced CDDP-vinorelbine in lung cancer xenografts. These studies show that RALBP1 is a transporter that serves as a key effector function in cancer cell survival and is a valid target for cancer therapy, and confirm that inhibitory modulation of RALBP1 transport activity at the cell surface is sufficient for antitumor effects. PMID:17483352

  6. Identification and cDNA cloning of a protein abundantly expressed during apple fruit development.

    PubMed

    Yamada, K; Mori, H; Yamaki, S

    1999-02-01

    A 60 kDa protein (MF-60) abundantly appearing in matured apple fruit was detected by SDS-PAGE of the soluble protein. It was partially purified through Butyl-Toyopearl and DEAE-cellulose. Its partial amino acid sequences were determined to isolate a full-length cDNA. MF-60 cDNA (mf-60) consisting of 1,825 bp containing an open reading frame of 1,524 bp and encoding a 54.2 kDa polypeptide. The deduced polypeptide of mf-60 has 81.1% identity to turgor-responsive protein 26 g from wilted garden pea shoot. Northern blot and Western blot analyses showed that the levels of the protein and the transcript of MF-60 changed in parallel through the developmental season; they were very low in young fruit at 36 DAF and 60 DAF, started to increase at 85 DAF, and then remained at a higher level from 114 DAF to 176 DAF. These results suggested that MF-60 functions are connected with fruit development but not with the fruit ripening induced by ethylene. PMID:10202815

  7. Conditional depletion of methyl-CpG-binding protein 2 in astrocytes depresses the hypercapnic ventilatory response in mice.

    PubMed

    Garg, Saurabh K; Lioy, Daniel T; Knopp, Sharon J; Bissonnette, John M

    2015-09-15

    Mice that are deficient in the transcription factor methyl-CpG-binding protein 2 (MeCP2) have a depressed hypercapnic ventilatory response (HCVR). The expression of MeCP2 can be selectively removed from astrocytes or neurons, thus offering a tool to dissect the role of this transcription factor in astrocytes from that in neurons. Studies were carried out in the progeny of mice that were a cross between those harboring a tamoxifen (TAM)-inducible Cre recombinase transgene driven by the human astrocytic glial fibrillary acidic protein (hGFAP) promoter, or Cre recombinase under control of the synapsin promoter, with mice containing a Cre-excisable exon III in the Mecp2 gene. The TAM-conditional excision of the Mecp2 exon allowed the respiratory CO2 response to be studied in the same animals before and after selective depletion of MeCP2 in astrocytes. Immunohistochemistry showed that following TAM treatment only ∼20% of GFAP-labeled cells in the retrotrapazoid nucleus and in the raphé magnus were positive for MeCP2. The slope of the relative increase in minute ventilation as a function of 1, 3, and 5% inspired CO2 was depressed in mice with depleted astrocyte MeCP2 compared with wild-type littermates. In contrast, selective depletion of MeCP2 in neurons did not significantly affect slope. While neurons which constitute the respiratory network ultimately determine the ventilatory response to CO2, this study demonstrates that loss of MeCP2 in astrocytes alone is sufficient to result in a dramatic attenuation of the HCVR. We propose that the glial contribution to HCVR is under the control of the MeCP2 gene. PMID:26205541

  8. Growth Hormone Induces Transforming Growth Factor-Beta-Induced Protein in Podocytes: Implications for Podocyte Depletion and Proteinuria.

    PubMed

    Chitra, P Swathi; Swathi, T; Sahay, Rakesh; Reddy, G Bhanuprakash; Menon, Ram K; Kumar, P Anil

    2015-09-01

    The glomerular podocytes form a major size selective barrier for the filtration of serum proteins and reduced podocyte number is a critical event in the pathogenesis of proteinuria during diabetic nephropathy (DN). An elevated level of growth hormone (GH) is implicated as a causative factor in the development of nephropathy in patients with type 1 diabetes mellitus. We have previously shown that podocytes express GH receptor and are a target for GH action. To elucidate the molecular basis for the effects of GH on podocyte depletion, we conducted PCR-array analyses for extracellular matrix and adhesion molecules in podocytes. Our studies reveal that GH increases expression of a gene that encodes transforming growth factor-beta-induced protein (TGFBIp) expression. Similarly, microarray data retrieved from the Nephromine database revealed elevation of TGFBIp in patients with DN. Treatment with GH results in increased secretion of extracellular TGFBIp by podocytes. Both GH and TGFBIp induced apoptosis and epithelial mesenchymal transition (EMT) of podocytes. Exposure of podocytes to GH and TGFBIp resulted in increased migration of cells and altered podocyte permeability to albumin across podocyte monolayer. Administration of GH to rats induced EMT and apoptosis in the glomerular fraction of the kidney. Therefore, we conclude that the GH-dependent increase in TGFBIp in the podocyte is one of the mechanisms responsible for podocyte depletion in DN. PMID:25740786

  9. Differential Gene Expression and Protein Abundance Evince Ontogenetic Bias toward Castes in a Primitively Eusocial Wasp

    PubMed Central

    Hunt, James H.; Wolschin, Florian; Henshaw, Michael T.; Newman, Thomas C.; Toth, Amy L.; Amdam, Gro V.

    2010-01-01

    Polistes paper wasps are models for understanding conditions that may have characterized the origin of worker and queen castes and, therefore, the origin of paper wasp sociality. Polistes is “primitively eusocial” by virtue of having context-dependent caste determination and no morphological differences between castes. Even so, Polistes colonies have a temporal pattern in which most female larvae reared by the foundress become workers, and most reared by workers become future-reproductive gynes. This pattern is hypothesized to reflect development onto two pathways, which may utilize mechanisms that regulate diapause in other insects. Using expressed sequence tags (ESTs) for Polistes metricus we selected candidate genes differentially expressed in other insects in three categories: 1) diapause vs. non-diapause phenotypes and/or worker vs. queen differentiation, 2) behavioral subcastes of worker honey bees, and 3) no a priori expectation of a role in worker/gyne development. We also used a non-targeted proteomics screen to test for peptide/protein abundance differences that could reflect larval developmental divergence. We found that foundress-reared larvae (putative worker-destined) and worker-reared larvae (putative gyne-destined) differed in quantitative expression of sixteen genes, twelve of which were associated with caste and/or diapause in other insects, and they also differed in abundance of nine peptides/proteins. Some differentially-expressed genes are involved in diapause regulation in other insects, and other differentially-expressed genes and proteins are involved in the insulin signaling pathway, nutrient metabolism, and caste determination in highly social bees. Differential expression of a gene and a peptide encoding hexameric storage proteins is especially noteworthy. Although not conclusive, our results support hypotheses of 1) larval developmental pathway divergence that can lead to caste bias in adults and 2) nutritional differences as the

  10. Proteome analysis of abundant proteins extracted from the leaf of Gynura procumbens (Lour.) Merr.

    PubMed

    Hew, Chaw-Sen; Gam, Lay-Harn

    2011-12-01

    Gynura procumbens (Lour.) Merr. is a traditionally used medicinal plant to decrease cholesterol level, reduce high blood pressure, control diabetics, and for treatment of cancer. In our present study, a proteomic approach was applied to study the proteome of the plant that had never analyzed before. We have identified 92 abundantly expressed proteins from the leaves of G. procumbens (Lour.) Merr. Amongst the identified proteins was miraculin, a taste-masking agent with high commercial value. Miraculin made up ∼0.1% of the total protein extracted; the finding of miraculin gave a great commercial value to G. procumbens (Lour.) Merr. as miraculin's natural source is limited while the production of recombinant miraculin faced challenges of not being able to exhibit the taste-masking effect as in the natural miraculin. We believe the discovery of miraculin in G. procumbens (Lour.) Merr., provides commercial feasibility of miraculin in view of the availability of G. procumbens (Lour.) Merr. that grow wildly and easily in tropical climate. PMID:21938418

  11. Increased abundance of proteins involved in phytosiderophore production in boron-tolerant barley.

    PubMed

    Patterson, John; Ford, Kris; Cassin, Andrew; Natera, Siria; Bacic, Antony

    2007-07-01

    Boron (B) phytotoxicity affects cereal-growing regions worldwide. Although B-tolerant barley (Hordeum vulgare) germplasm is available, molecules responsible for this tolerance mechanism have not been defined. We describe and use a new comparative proteomic technique, iTRAQ peptide tagging (iTRAQ), to compare the abundances of proteins from B-tolerant and -intolerant barley plants from a 'Clipper' x 'Sahara' doubled-haploid population selected on the basis of a presence or absence of two B-tolerance quantitative trait loci. iTRAQ was used to identify three enzymes involved in siderophore production (Iron Deficiency Sensitive2 [IDS2], IDS3, and a methylthio-ribose kinase) as being elevated in abundance in the B-tolerant plants. Following from this result, we report a potential link between iron, B, and the siderophore hydroxymugineic acid. We believe that this study highlights the potency of the iTRAQ approach to better understand mechanisms of abiotic stress tolerance in cereals, particularly when applied in conjunction with bulked segregant analysis. PMID:17478636

  12. A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells.

    PubMed

    Tichon, Ailone; Gil, Noa; Lubelsky, Yoav; Havkin Solomon, Tal; Lemze, Doron; Itzkovitz, Shalev; Stern-Ginossar, Noam; Ulitsky, Igor

    2016-01-01

    Thousands of long noncoding RNA (lncRNA) genes are encoded in the human genome, and hundreds of them are evolutionarily conserved, but their functions and modes of action remain largely obscure. Particularly enigmatic lncRNAs are those that are exported to the cytoplasm, including NORAD-an abundant and highly conserved cytoplasmic lncRNA. Here we show that most of the sequence of NORAD is comprised of repetitive units that together contain at least 17 functional binding sites for the two mammalian Pumilio homologues. Through binding to PUM1 and PUM2, NORAD modulates the mRNA levels of their targets, which are enriched for genes involved in chromosome segregation during cell division. Our results suggest that some cytoplasmic lncRNAs function by modulating the activities of RNA-binding proteins, an activity which positions them at key junctions of cellular signalling pathways. PMID:27406171

  13. A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells

    PubMed Central

    Tichon, Ailone; Gil, Noa; Lubelsky, Yoav; Havkin Solomon, Tal; Lemze, Doron; Itzkovitz, Shalev; Stern-Ginossar, Noam; Ulitsky, Igor

    2016-01-01

    Thousands of long noncoding RNA (lncRNA) genes are encoded in the human genome, and hundreds of them are evolutionarily conserved, but their functions and modes of action remain largely obscure. Particularly enigmatic lncRNAs are those that are exported to the cytoplasm, including NORAD—an abundant and highly conserved cytoplasmic lncRNA. Here we show that most of the sequence of NORAD is comprised of repetitive units that together contain at least 17 functional binding sites for the two mammalian Pumilio homologues. Through binding to PUM1 and PUM2, NORAD modulates the mRNA levels of their targets, which are enriched for genes involved in chromosome segregation during cell division. Our results suggest that some cytoplasmic lncRNAs function by modulating the activities of RNA-binding proteins, an activity which positions them at key junctions of cellular signalling pathways. PMID:27406171

  14. Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

    PubMed Central

    Bernfur, Katja; Larsson, Olaf; Larsson, Christer; Gustavsson, Niklas

    2013-01-01

    Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome. PMID:23990937

  15. The COG and COPI Complexes Interact to Control the Abundance of GEARs, a Subset of Golgi Integral Membrane ProteinsD⃞

    PubMed Central

    Oka, Toshihiko; Ungar, Daniel; Hughson, Frederick M.; Krieger, Monty

    2004-01-01

    The conserved oligomeric Golgi (COG) complex is a soluble hetero-octamer associated with the cytoplasmic surface of the Golgi. Mammalian somatic cell mutants lacking the Cog1 (ldlB) or Cog2 (ldlC) subunits exhibit pleiotropic defects in Golgi-associated glycoprotein and glycolipid processing that suggest COG is involved in the localization, transport, and/or function of multiple Golgi processing proteins. We have identified a set of COG-sensitive, integral membrane Golgi proteins called GEARs (mannosidase II, GOS-28, GS15, GPP130, CASP, giantin, and golgin-84) whose abundances were reduced in the mutant cells and, in some cases, increased in COG-overexpressing cells. In the mutants, some GEARs were abnormally localized in the endoplasmic reticulum and were degraded by proteasomes. The distributions of the GEARs were altered by small interfering RNA depletion of ε-COP in wild-type cells under conditions in which COG-insensitive proteins were unaffected. Furthermore, synthetic phenotypes arose in mutants deficient in both ε-COP and either Cog1 or Cog2. COG and COPI may work in concert to ensure the proper retention or retrieval of a subset of proteins in the Golgi, and COG helps prevent the endoplasmic reticulum accumulation and degradation of some GEARs. PMID:15004235

  16. Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins.

    PubMed

    Armenti, Stephen T; Lohmer, Lauren L; Sherwood, David R; Nance, Jeremy

    2014-12-01

    The capability to conditionally inactivate gene function is essential for understanding the molecular basis of development. In gene and mRNA targeting approaches, protein products can perdure, complicating genetic analysis. Current methods for selective protein degradation require drug treatment or take hours for protein removal, limiting their utility in studying rapid developmental processes in vivo. Here, we repurpose an endogenous protein degradation system to rapidly remove targeted C. elegans proteins. We show that upon expression of the E3 ubiquitin ligase substrate-recognition subunit ZIF-1, proteins tagged with the ZF1 zinc-finger domain can be quickly degraded in all somatic cell types examined with temporal and spatial control. We demonstrate that genes can be engineered to become conditional loss-of-function alleles by introducing sequences encoding the ZF1 tag into endogenous loci. Finally, we use ZF1 tagging to establish the site of cdc-42 gene function during a cell invasion event. ZF1 tagging provides a powerful new tool for the analysis of dynamic developmental events. PMID:25377555

  17. Depletion of kinesin-12, a myosin-IIB-interacting protein, promotes migration of cortical astrocytes.

    PubMed

    Feng, Jie; Hu, Zunlu; Chen, Haijiao; Hua, Juan; Wu, Ronghua; Dong, Zhangji; Qiang, Liang; Liu, Yan; Baas, Peter W; Liu, Mei

    2016-06-15

    Kinesin-12 (also named Kif15) participates in important events during neuronal development, such as cell division of neuronal precursors, migration of young neurons and establishment of axons and dendritic arbors, by regulating microtubule organization. Little is known about the molecular mechanisms behind the functions of kinesin-12, and even less is known about its roles in other cell types of the nervous system. Here, we show that kinesin-12 depletion from cultured rat cortical astrocytes decreases cell proliferation but increases migration. Co-immunoprecipitation, GST pulldown and small interfering RNA (siRNA) experiments indicated that kinesin-12 directly interacts with myosin-IIB through their tail domains. Immunofluorescence analyses indicated that kinesin-12 and myosin-IIB colocalize in the lamellar region of astrocytes, and fluorescence resonance energy transfer analyses revealed an interaction between the two. The phosphorylation at Thr1142 of kinesin-12 was vital for their interaction. Loss of their interaction through expression of a phosphorylation mutant of kinesin-12 promoted astrocyte migration. We suggest that kinesin-12 and myosin-IIB can form a hetero-oligomer that generates force to integrate microtubules and actin filaments in certain regions of cells, and in the case of astrocytes, that this interaction can modulate their migration. PMID:27170353

  18. Regulated protein depletion by the auxin-inducible degradation system in Drosophila melanogaster.

    PubMed

    Trost, Martina; Blattner, Ariane C; Lehner, Christian F

    2016-01-01

    The analysis of consequences resulting after experimental elimination of gene function has been and will continue to be an extremely successful strategy in biological research. Mutational elimination of gene function has been widely used in the fly Drosophila melanogaster. RNA interference is used extensively as well. In the fly, exceptionally precise temporal and spatial control over elimination of gene function can be achieved in combination with sophisticated transgenic approaches and clonal analyses. However, the methods that act at the gene and transcript level cannot eliminate protein products which are already present at the time when mutant cells are generated or RNA interference is started. Targeted inducible protein degradation is therefore of considerable interest for controlled rapid elimination of gene function. To this end, a degradation system was developed in yeast exploiting TIR1, a plant F box protein, which can recruit proteins with an auxin-inducible degron to an E3 ubiquitin ligase complex, but only in the presence of the phytohormone auxin. Here we demonstrate that the auxin-inducible degradation system functions efficiently also in Drosophila melanogaster. Neither auxin nor TIR1 expression have obvious toxic effects in this organism, and in combination they result in rapid degradation of a target protein fused to the auxin-inducible degron. PMID:27010248

  19. Enrich: software for analysis of protein function by enrichment and depletion of variants

    PubMed Central

    Fowler, Douglas M.; Araya, Carlos L.; Gerard, Wayne; Fields, Stanley

    2011-01-01

    Summary: Measuring the consequences of mutation in proteins is critical to understanding their function. These measurements are essential in such applications as protein engineering, drug development, protein design and genome sequence analysis. Recently, high-throughput sequencing has been coupled to assays of protein activity, enabling the analysis of large numbers of mutations in parallel. We present Enrich, a tool for analyzing such deep mutational scanning data. Enrich identifies all unique variants (mutants) of a protein in high-throughput sequencing datasets and can correct for sequencing errors using overlapping paired-end reads. Enrich uses the frequency of each variant before and after selection to calculate an enrichment ratio, which is used to estimate fitness. Enrich provides an interactive interface to guide users. It generates user-accessible output for downstream analyses as well as several visualizations of the effects of mutation on function, thereby allowing the user to rapidly quantify and comprehend sequence–function relationships. Availability and Implementation: Enrich is implemented in Python and is available under a FreeBSD license at http://depts.washington.edu/sfields/software/enrich/. Enrich includes detailed documentation as well as a small example dataset. Contact: dfowler@uw.edu; fields@uw.edu Supplementary Information: Supplementary data is available at Bioinformatics online. PMID:22006916

  20. Ubiquitous Autofragmentation of Fluorescent Proteins Creates Abundant Defective Ribosomal Products (DRiPs) for Immunosurveillance.

    PubMed

    Wei, Jiajie; Gibbs, James S; Hickman, Heather D; Cush, Stephanie S; Bennink, Jack R; Yewdell, Jonathan W

    2015-06-26

    broadly, given the wide use of fluorescent proteins, their ubiquitous and abundant fragmentation must be considered when interpreting experiments using these extremely useful probes. PMID:25971973

  1. Ubiquitous Autofragmentation of Fluorescent Proteins Creates Abundant Defective Ribosomal Products (DRiPs) for Immunosurveillance*

    PubMed Central

    Wei, Jiajie; Gibbs, James S.; Hickman, Heather D.; Cush, Stephanie S.; Bennink, Jack R.; Yewdell, Jonathan W.

    2015-01-01

    broadly, given the wide use of fluorescent proteins, their ubiquitous and abundant fragmentation must be considered when interpreting experiments using these extremely useful probes. PMID:25971973

  2. Macrophage bone morphogenic protein receptor 2 depletion in idiopathic pulmonary fibrosis and Group III pulmonary hypertension.

    PubMed

    Chen, Ning-Yuan; D Collum, Scott; Luo, Fayong; Weng, Tingting; Le, Thuy-Trahn; M Hernandez, Adriana; Philip, Kemly; Molina, Jose G; Garcia-Morales, Luis J; Cao, Yanna; Ko, Tien C; Amione-Guerra, Javier; Al-Jabbari, Odeaa; Bunge, Raquel R; Youker, Keith; Bruckner, Brian A; Hamid, Rizwan; Davies, Jonathan; Sinha, Neeraj; Karmouty-Quintana, Harry

    2016-08-01

    Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology. The development of pulmonary hypertension (PH) is considered the single most significant predictor of mortality in patients with chronic lung diseases. The processes that govern the progression and development of fibroproliferative and vascular lesions in IPF are not fully understood. Using human lung explant samples from patients with IPF with or without a diagnosis of PH as well as normal control tissue, we report reduced BMPR2 expression in patients with IPF or IPF+PH. These changes were consistent with dampened P-SMAD 1/5/8 and elevated P-SMAD 2/3, demonstrating reduced BMPR2 signaling and elevated TGF-β activity in IPF. In the bleomycin (BLM) model of lung fibrosis and PH, we also report decreased BMPR2 expression compared with control animals that correlated with vascular remodeling and PH. We show that genetic abrogation or pharmacological inhibition of interleukin-6 leads to diminished markers of fibrosis and PH consistent with elevated levels of BMPR2 and reduced levels of a collection of microRNAs (miRs) that are able to degrade BMPR2. We also demonstrate that isolated bone marrow-derived macrophages from BLM-exposed mice show reduced BMPR2 levels upon exposure with IL6 or the IL6+IL6R complex that are consistent with immunohistochemistry showing reduced BMPR2 in CD206 expressing macrophages from lung sections from IPF and IPF+PH patients. In conclusion, our data suggest that depletion of BMPR2 mediated by a collection of miRs induced by IL6 and subsequent STAT3 phosphorylation as a novel mechanism participating to fibroproliferative and vascular injuries in IPF. PMID:27317687

  3. Dietary soy protein benefit in experimental kidney disease is preserved after isoflavone depletion of diet.

    PubMed

    Ogborn, Malcolm R; Nitschmann, Evan; Bankovic-Calic, Neda; Weiler, Hope A; Aukema, Harold M

    2010-11-01

    Soy diet ameliorates renal injury in the Han:SPRD-cy rat. The relative roles of protein, isoflavones and changes in polyunsaturated fatty acid (PUFA) status are not determined. We fed male Han:SPRD-cy heterozygotes casein (C), high isoflavone soy protein (HIS), alcohol-extracted low isoflavone soy protein (LIS) or mixed soy protein diet (MIS). LIS and MIS were associated with a small decrease in animal weight compared with HIS or C. Soy diets preserved normal renal function and reduced relative renal weight (10.9-14.6 g/kg, cf. 23.6, P < 0.001), scores for cystic change (0.168-0.239, cf. 0.386, P < 0.05), fibrosis (0.013-0.015, cf. 0.032, P < 0.05), tissue oxidized LDL content (0.012-0.021, cf. 0.048, P < 0.05), inflammation (8.5-12.9, cf. 31.2, P < 0.05) and epithelial cell proliferation (6.5-13.8, cf. 26.3, P < 0.05). In post hoc testing, LIS produced a greater reduction in relative renal weight, cystic change and epithelial proliferation, whereas HIS produced a significantly greater reduction in oxidized-LDL. Soy diets were associated with increased hepatic content of 18C PUFA (P < 0.001). LIS and HIS diets were associated with a small increase in body fat content (P < 0.001). Alcohol-extracted soy protein retains its major protective effects in this model with subtle differences attributable to isoflavones. PMID:20921276

  4. Liquid phase based separation systems for depletion, prefractionation, and enrichment of proteins in biological fluids and matrices for in-depth proteomics analysis-An update covering the period 2011-2014.

    PubMed

    Puangpila, Chanida; Mayadunne, Erandi; El Rassi, Ziad

    2015-01-01

    This review article expands on the previous one (S. Selvaraju and Z. El Rassi, Electrophoresis 2012, 33, 74-88) by reviewing pertinent literature in the period extending from early 2011 to present. As the previous review article, the present one is concerned with proteomic sample preparation (e.g., depletion of high-abundance proteins, reduction of the protein dynamic concentration range, enrichment of a particular subproteome), and the subsequent chromatographic and/or electrophoretic prefractionation prior to peptide separation and identification by LC-MS/MS. This review article, however, is distinguished from its earlier version by expanding on capturing/enriching subphosphoproteomes by immobilized metal affinity chromatography and metal oxide affinity chromatography. Seventy-seven papers published in the period extending from mid-2011 to the present have been reviewed. By no means this review article is exhaustive, given the fact that its aim is to give a concise treatment of the latest developments in the field. PMID:25287967

  5. Myristoyl-CoA:protein N-myristoyltransferase depletion in trypanosomes causes avirulence and endocytic defects

    PubMed Central

    Price, Helen P.; Güther, M. Lucia S.; Ferguson, Michael A.J.; Smith, Deborah F.

    2010-01-01

    The enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyses the co-translational covalent attachment of the fatty acid myristate to the N-terminus of target proteins. NMT is known to be essential for viability in Trypanosoma brucei and Leishmania major. Here we describe phenotypic analysis of T. brucei bloodstream form cells following knockdown of NMT expression by tetracycline-inducible RNA interference. Cell death occurs from 72 h post-induction, with approximately 50% of cells displaying a defect in endocytic uptake by this time. The majority of these induced cells do not have an enlarged flagellar pocket typical of a block in endocytosis but vesicle accumulation around the flagellar pocket indicates a defect in vesicular progression following endocytic fusion. Induced parasites have a wild-type or slightly enlarged Golgi apparatus, unlike the phenotype of cells with reduced expression of a major N-myristoylated protein, ARL1. Critically we show that following NMT knockdown, T. brucei bloodstream form cells are unable to establish an infection in a mouse model, therefore providing further validation of this enzyme as a target for drug development. PMID:19782106

  6. Module-based functional pathway enrichment analysis of a protein-protein interaction network to study the effects of intestinal microbiota depletion in mice

    PubMed Central

    JIA, ZHEN-YI; XIA, YANG; TONG, DANIAN; YAO, JING; CHEN, HONG-QI; YANG, JUN

    2014-01-01

    Complex communities of microorganisms play important roles in human health, and alterations in the intestinal microbiota may induce intestinal inflammation and numerous diseases. The purpose of this study was to identify the key genes and processes affected by depletion of the intestinal microbiota in a murine model. The Affymetrix microarray dataset GSE22648 was downloaded from the Gene Expression Omnibus database, and differentially expressed genes (DEGs) were identified using the limma package in R. A protein-protein interaction (PPI) network was constructed for the DEGs using the Cytoscape software, and the network was divided into several modules using the MCODE plugin. Furthermore, the modules were functionally annotated using the PiNGO plugin, and DEG-related pathways were retrieved and analyzed using the GenMAPP software. A total of 53 DEGs were identified, of which 26 were upregulated and 27 were downregulated. The PPI network of these DEGs comprised 3 modules. The most significant module-related DEGs were the cytochrome P450 (CYP) 4B1 isozyme gene (CYP4B1) in module 1, CYP4F14 in module 2 and the tachykinin precursor 1 gene (TAC1) in module 3. The majority of enriched pathways of module 1 and 2 were oxidation reduction pathways (metabolism of xenobiotics by CYPs) and lipid metabolism-related pathways, including linoleic acid and arachidonic acid metabolism. The neuropeptide signaling pathway was the most significantly enriched functional pathway of module 3. In conclusion, our findings strongly suggest that intestinal microbiota depletion affects cellular metabolism and oxidation reduction pathways. In addition, this is the first time, to the best of our knowledge, that the neuropeptide signaling pathway is reported to be affected by intestinal microbiota depletion in mice. The present study provides a list of candidate genes and processes related to the interaction of microbiota with the intestinal tract. PMID:24718810

  7. Module-based functional pathway enrichment analysis of a protein-protein interaction network to study the effects of intestinal microbiota depletion in mice.

    PubMed

    Jia, Zhen-Yi; Xia, Yang; Tong, Danian; Yao, Jing; Chen, Hong-Qi; Yang, Jun

    2014-06-01

    Complex communities of microorganisms play important roles in human health, and alterations in the intestinal microbiota may induce intestinal inflammation and numerous diseases. The purpose of this study was to identify the key genes and processes affected by depletion of the intestinal microbiota in a murine model. The Affymetrix microarray dataset GSE22648 was downloaded from the Gene Expression Omnibus database, and differentially expressed genes (DEGs) were identified using the limma package in R. A protein-protein interaction (PPI) network was constructed for the DEGs using the Cytoscape software, and the network was divided into several modules using the MCODE plugin. Furthermore, the modules were functionally annotated using the PiNGO plugin, and DEG-related pathways were retrieved and analyzed using the GenMAPP software. A total of 53 DEGs were identified, of which 26 were upregulated and 27 were downregulated. The PPI network of these DEGs comprised 3 modules. The most significant module-related DEGs were the cytochrome P450 (CYP) 4B1 isozyme gene (CYP4B1) in module 1, CYP4F14 in module 2 and the tachykinin precursor 1 gene (TAC1) in module 3. The majority of enriched pathways of module 1 and 2 were oxidation reduction pathways (metabolism of xenobiotics by CYPs) and lipid metabolism-related pathways, including linoleic acid and arachidonic acid metabolism. The neuropeptide signaling pathway was the most significantly enriched functional pathway of module 3. In conclusion, our findings strongly suggest that intestinal microbiota depletion affects cellular metabolism and oxidation reduction pathways. In addition, this is the first time, to the best of our knowledge, that the neuropeptide signaling pathway is reported to be affected by intestinal microbiota depletion in mice. The present study provides a list of candidate genes and processes related to the interaction of microbiota with the intestinal tract. PMID:24718810

  8. Brugia malayi abundant larval transcript 2 protein treatment attenuates experimentally-induced colitis in mice.

    PubMed

    Khatri, Vishal; Amdare, Nitin; Yadav, Ravi Shankar; Tarnekar, Aaditya; Goswami, Kalyan; Reddy, Maryada Venkata Rami

    2015-11-01

    Helminths are known to modulate host's immunity by suppressing host protective pro-inflammatory responses. Such immunomodulatory effects have been experimentally shown to have therapeutic implications in immune mediated disorders. In the present study, we have explored a filarial protein i.e. Brugia malayi recombinant abundant larval transcript 2 (rBmALT2) for its therapeutic effect in dextran sodium sulfate (DSS) induced colitis in mouse model. The immunomodulatory activity of rBmALT-2 was initially confirmed by demonstrating that it suppressed the lipopolysaccharide (LPS) induced nitric oxide synthesis and down-regulated the expression of pro-inflammatory cytokines in vitro by peritoneal exudate cells of mice. Treatment with rBmALT2 reduced severity of colitis associated with significant reduction in weight loss, disease activity, colon damage, mucosal edema and histopathological score including myeloperoxidase activity in colon tissues. rBmALT2 was comparatively more effective in attenuation of colitis when used in the preventive mode than when used for curative purpose. The therapeutic effect of rBmALT2 was found to be associated with downregulation of IFN-γ, IL-6, IL-17 and upregulation of IL-10 cytokines. These results provide strong experimental evidence that BmALT2 could be a potential alternative therapeutic agent in colitis. PMID:26669016

  9. Phylogenetic analysis of microalgae based on highly abundant proteins using mass spectrometry.

    PubMed

    Lee, Hae-Won; Roh, Seong Woon; Cho, Kichul; Kim, Kil-Nam; Cha, In-Tae; Yim, Kyung June; Song, Hye Seon; Nam, Young-Do; Oda, Tatsuya; Chung, Young-Ho; Kim, Soo Jung; Choi, Jong-Soon; Kim, Daekyung

    2015-01-01

    The blooms of toxic phototrophic microorganisms, such as microalgae and cyanobacteria, which are typically found in freshwater and marine environments, are becoming more frequent and problematic in aquatic systems. Due to accumulation of toxic algae, harmful algal blooms (HABs) exert negative effects on aquatic systems. Therefore, rapid detection of harmful microalgae is important for monitoring the occurrence of HABs. Mass spectrometry-based methods have become sensitive, specific techniques for the identification and characterization of microorganisms. Matrix-assisted laser desorption/ionization (MALDI) with time-of-flight (TOF) mass spectrometry (MS) allows us to measure a unique molecular fingerprint of highly abundant proteins in a microorganism and has been used for the rapid, accurate identification of bacteria and fungi in clinical microbiology. Here, we tested the specificity of MALDI-TOF MS using microalgal strains (Heterocapsa, Alexandrium, Nannochloropsis, Chaetoceros, Chlorella, and Dunaliella spp.). Our research suggested that this method was comparable in terms of the rapid identification of microalgea to conventional methods based on genetic information and morphology. Thus, this efficient mass spectrometry-based technique may have applications in the rapid identification of harmful microorganisms from aquatic environmental samples. PMID:25476355

  10. Partial calcium depletion during membrane filtration affects gelation of reconstituted milk protein concentrates.

    PubMed

    Eshpari, H; Jimenez-Flores, R; Tong, P S; Corredig, M

    2015-12-01

    Milk protein concentrate powders (MPC) with improved rehydration properties are often manufactured using processing steps, such as acidification and high-pressure processing, and with addition of other ingredients, such as sodium chloride, during their production. These steps are known to increase the amount of serum caseins or modify the mineral equilibrium, hence improving solubility of the retentates. The processing functionality of the micelles may be affected. The aim of this study was to investigate the effects of partial acidification by adding glucono-δ-lactone (GDL) to skim milk during membrane filtration on the structural changes of the casein micelles by observing their chymosin-induced coagulation behavior, as such coagulation is affected by both the supramolecular structure of the caseins and calcium equilibrium. Milk protein concentrates were prepared by preacidification with GDL to pH 6 using ultrafiltration (UF) and diafiltration (DF) followed by spray-drying. Reconstituted UF and DF samples (3.2% protein) treated with GDL showed significantly increased amounts of soluble calcium and nonsedimentable caseins compared with their respective controls, as measured by ion chromatography and sodium dodecyl sulfate-PAGE electrophoresis, respectively. The primary phase of chymosin-induced gelation was not significantly different between treatments as measured by the amount of caseino-macropeptide released. The rheological properties of the reconstituted MPC powders were determined immediately after addition of chymosin, both before and after dialysis against skim milk, to ensure similar serum composition for all samples. Reconstituted samples before dialysis showed no gelation (defined as tan δ=1), and after re-equilibration only control UF and DF samples showed gelation. The gelation properties of reconstituted MPC powders were negatively affected by the presence of soluble casein, and positively affected by the amount of both soluble and insoluble

  11. CYCLoPs: A Comprehensive Database Constructed from Automated Analysis of Protein Abundance and Subcellular Localization Patterns in Saccharomyces cerevisiae

    PubMed Central

    Koh, Judice L. Y.; Chong, Yolanda T.; Friesen, Helena; Moses, Alan; Boone, Charles; Andrews, Brenda J.; Moffat, Jason

    2015-01-01

    Changes in protein subcellular localization and abundance are central to biological regulation in eukaryotic cells. Quantitative measures of protein dynamics in vivo are therefore highly useful for elucidating specific regulatory pathways. Using a combinatorial approach of yeast synthetic genetic array technology, high-content screening, and machine learning classifiers, we developed an automated platform to characterize protein localization and abundance patterns from images of log phase cells from the open-reading frame−green fluorescent protein collection in the budding yeast, Saccharomyces cerevisiae. For each protein, we produced quantitative profiles of localization scores for 16 subcellular compartments at single-cell resolution to trace proteome-wide relocalization in conditions over time. We generated a collection of ∼300,000 micrographs, comprising more than 20 million cells and ∼9 billion quantitative measurements. The images depict the localization and abundance dynamics of more than 4000 proteins under two chemical treatments and in a selected mutant background. Here, we describe CYCLoPs (Collection of Yeast Cells Localization Patterns), a web database resource that provides a central platform for housing and analyzing our yeast proteome dynamics datasets at the single cell level. CYCLoPs version 1.0 is available at http://cyclops.ccbr.utoronto.ca. CYCLoPs will provide a valuable resource for the yeast and eukaryotic cell biology communities and will be updated as new experiments become available. PMID:26048563

  12. The sulfur depletion problem: upper limits on the H2S2, HS·2, and S2 gas-phase abundances toward the low-mass warm core IRAS 16293-2422

    NASA Astrophysics Data System (ADS)

    Martín-Doménech, R.; Jiménez-Serra, I.; Muñoz Caro, G. M.; Müller, H. S. P.; Occhiogrosso, A.; Testi, L.; Woods, P. M.; Viti, S.

    2016-01-01

    Context. A fraction of the missing sulfur in dense clouds and circumstellar regions could be in the form of three species not yet detected in the interstellar medium: H2S2, HS.2, and S2 according to experimental simulations performed under astrophysically relevant conditions. These S-S bonded molecules can be formed by the energetic processing of H2S-bearing ice mantles on dust grains, and subsequently desorb to the gas phase. Aims: The detection of these species could partially solve the sulfur depletion problem, and would help to improve our knowledge of the poorly known chemistry of sulfur in the interstellar medium. To this purpose we calculated the frequencies and expected intensities of the rotational transitions not previously reported, and performed dedicated ground-based observations toward the low-mass warm core IRAS 16293-2422, a region with one of the highest measured gas-phase H2S abundances. Methods: Observations in the submillimeter regime were obtained with the APEX 12 m telescope during 15 h of observation. A total of ~16 GHz were covered in a range of about 100 GHz, targeting a wide selection of the predicted rotational transitions of the three molecules. Results: The 1σ noise rms values were extracted in the spectral regions where the targeted species should have been detected. These values were a factor of 2-7 lower than those reached by previous observations toward the same source, and allowed us to estimate a 1σ upper limit to their molecular abundances of ≤8.1 × 10-9, ≤ 1.1 × 10-8, and ≤ 2.9 × 10-7 relative to H2, for H2S 2 , HS.2, and S2, respectively. Conclusions: The upper limit abundances of the three molecules containing the S2 unit are up to two orders of magnitude lower than the H2S abundance in the source, and one order of magnitude lower than the expected abundances from the experimental simulations using ice analogs. Subsequent gas-phase chemistry after desorption could lower the abundances of the three species to

  13. Alteration in abundance of specific membrane proteins of Aggregatibacter actinomycetemcomitans is attributed to deletion of the inner membrane protein MorC

    PubMed Central

    Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an important pathogen in the etiology of human periodontal and systemic diseases. Inactivation of the gene coding for the inner membrane protein, morphogenesis protein C (MorC), results is pleotropic effects pertaining to the membrane structure and function of this bacterium. The role of this protein in membrane biogenesis is unknown. To begin to understand the role of this conserved protein, stable isotope dimethyl labeling in conjunction with mass spectrometry was used to quantitatively analyze differences in the membrane proteomes of the isogenic mutant and wild-type strain. A total of 613 proteins were quantified and 601 of these proteins were found to be equal in abundance between the two strains. The remaining 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain compared with the wild-type strain. The 12 proteins were ascribed functions associated with protein quality control systems, oxidative stress responses, and protein secretion. The potential relationship between these proteins and the phenotypes of the morC mutant strain is discussed. PMID:25684173

  14. Poly(A) binding protein abundance regulates eukaryotic translation initiation factor 4F assembly in human cytomegalovirus-infected cells.

    PubMed

    McKinney, Caleb; Perez, Cesar; Mohr, Ian

    2012-04-10

    By commandeering cellular translation initiation factors, or destroying those dispensable for viral mRNA translation, viruses often suppress host protein synthesis. In contrast, cellular protein synthesis proceeds in human cytomegalovirus (HCMV)-infected cells, forcing viral and cellular mRNAs to compete for limiting translation initiation factors. Curiously, inactivating the host translational repressor 4E-BP1 in HCMV-infected cells stimulates synthesis of the cellular poly(A) binding protein (PABP), significantly increasing PABP abundance. Here, we establish that new PABP synthesis is translationally controlled by the HCMV-encoded UL38 mammalian target of rapamycin complex 1-activator. The 5' UTR within the mRNA encoding PABP contains a terminal oligopyrimidine (TOP) element found in mRNAs, the translation of which is stimulated in response to mitogenic, growth, and nutritional stimuli, and proteins encoded by TOP-containing mRNAs accumulated in HCMV-infected cells. Furthermore, UL38 expression was necessary and sufficient to regulate expression of a PABP TOP-containing reporter. Remarkably, preventing the rise in PABP abundance by RNAi impaired eIF4E binding to eIF4G, thereby reducing assembly of the multisubunit initiation factor eIF4F, viral protein production, and replication. This finding demonstrates that viruses can increase host translation initiation factor concentration to foster their replication and defines a unique mechanism whereby control of PABP abundance regulates eIF4F assembly. PMID:22431630

  15. Assessment of 24-hours Aldosterone Administration on Protein Abundances in Fluorescence-Sorted Mouse Distal Renal Tubules by Mass Spectrometry

    PubMed Central

    Jensen, Thomas B; Pisitkun, Trairak; Hoffert, Jason D; Jensen, Uffe B; Fenton, Robert A; Praetorius, Helle A; Knepper, Mark A; Praetorius, Jeppe

    2013-01-01

    Background/Aims Aldosterone exerts multiple long-term effects in the distal renal tubules. The aim of this study was to establish a method for identifying proteins in these tubules that change in abundance by only 24-hours aldosterone administration. Methods Mice endogenously expressing green fluorescent protein (eGFP) in the connecting tubule and cortical collecting ducts were treated with a subcutaneous injection of 2.0 mg/kg aldosterone or vehicle (n=5), and sacrificed 24 hours later. Suspensions of single cells were obtained enzymatically, and eGFP positive cells were isolated by fluorescence activated cell sorting (FACS). Samples of 100 μg proteins were digested with trypsin and labeled with 8-plex iTRAQ reagents and processed for liquid chromatography tandem mass spectrometry (LC-MS/MS). Results FACS yielded 1.4 million cells per mouse. The LC-MS/MS spectra were matched to peptides by the SEQUEST search algorithm, which identified 3002 peptides corresponding to 506 unique proteins of which 20 significantly changed abundance 24-hours after aldosterone injection. Conclusion We find the method suitable and useful for studying hormonal effects on protein abundance in distal tubular segments. PMID:23428628

  16. Global quantitative proteomics reveal up-regulation of endoplasmic reticulum stress response proteins upon depletion of eIF5A in HeLa cells

    PubMed Central

    Mandal, Ajeet; Mandal, Swati; Park, Myung Hee

    2016-01-01

    The eukaryotic translation factor, eIF5A, is a translation factor essential for protein synthesis, cell growth and animal development. By use of a adenoviral eIF5A shRNA, we have achieved an effective depletion of eIF5A in HeLa cells and undertook in vivo comprehensive proteomic analyses to examine the effects of eIF5A depletion on the total proteome and to identify cellular pathways influenced by eIF5A. The proteome of HeLa cells transduced with eIF5A shRNA was compared with that of scramble shRNA-transduced counterpart by the iTRAQ method. We identified 972 proteins consistently detected in three iTRAQ experiments and 104 proteins with significantly altered levels (protein ratio ≥1.5 or ≤0.66, p-value ≤0.05) at 72 h and/or 96 h of Ad-eIF5A-shRNA transduction. The altered expression levels of key pathway proteins were validated by western blotting. Integration of functional ontology with expression data of the 104 proteins revealed specific biological processes that are prominently up- or down-regulated. Heatmap analysis and Cytoscape visualization of biological networks identified protein folding as the major cellular process affected by depletion of eIF5A. Our unbiased, quantitative, proteomic data demonstrate that the depletion of eIF5A leads to endoplasmic reticulum stress, an unfolded protein response and up-regulation of chaperone expression in HeLa cells. PMID:27180817

  17. Global quantitative proteomics reveal up-regulation of endoplasmic reticulum stress response proteins upon depletion of eIF5A in HeLa cells.

    PubMed

    Mandal, Ajeet; Mandal, Swati; Park, Myung Hee

    2016-01-01

    The eukaryotic translation factor, eIF5A, is a translation factor essential for protein synthesis, cell growth and animal development. By use of a adenoviral eIF5A shRNA, we have achieved an effective depletion of eIF5A in HeLa cells and undertook in vivo comprehensive proteomic analyses to examine the effects of eIF5A depletion on the total proteome and to identify cellular pathways influenced by eIF5A. The proteome of HeLa cells transduced with eIF5A shRNA was compared with that of scramble shRNA-transduced counterpart by the iTRAQ method. We identified 972 proteins consistently detected in three iTRAQ experiments and 104 proteins with significantly altered levels (protein ratio ≥1.5 or ≤0.66, p-value ≤0.05) at 72 h and/or 96 h of Ad-eIF5A-shRNA transduction. The altered expression levels of key pathway proteins were validated by western blotting. Integration of functional ontology with expression data of the 104 proteins revealed specific biological processes that are prominently up- or down-regulated. Heatmap analysis and Cytoscape visualization of biological networks identified protein folding as the major cellular process affected by depletion of eIF5A. Our unbiased, quantitative, proteomic data demonstrate that the depletion of eIF5A leads to endoplasmic reticulum stress, an unfolded protein response and up-regulation of chaperone expression in HeLa cells. PMID:27180817

  18. Halo Star Lithium Depletion

    SciTech Connect

    Pinsonneault, M. H.; Walker, T. P.; Steigman, G.; Narayanan, Vijay K.

    1999-12-10

    The depletion of lithium during the pre-main-sequence and main-sequence phases of stellar evolution plays a crucial role in the comparison of the predictions of big bang nucleosynthesis with the abundances observed in halo stars. Previous work has indicated a wide range of possible depletion factors, ranging from minimal in standard (nonrotating) stellar models to as much as an order of magnitude in models that include rotational mixing. Recent progress in the study of the angular momentum evolution of low-mass stars permits the construction of theoretical models capable of reproducing the angular momentum evolution of low-mass open cluster stars. The distribution of initial angular momenta can be inferred from stellar rotation data in young open clusters. In this paper we report on the application of these models to the study of lithium depletion in main-sequence halo stars. A range of initial angular momenta produces a range of lithium depletion factors on the main sequence. Using the distribution of initial conditions inferred from young open clusters leads to a well-defined halo lithium plateau with modest scatter and a small population of outliers. The mass-dependent angular momentum loss law inferred from open cluster studies produces a nearly flat plateau, unlike previous models that exhibited a downward curvature for hotter temperatures in the 7Li-Teff plane. The overall depletion factor for the plateau stars is sensitive primarily to the solar initial angular momentum used in the calibration for the mixing diffusion coefficients. Uncertainties remain in the treatment of the internal angular momentum transport in the models, and the potential impact of these uncertainties on our results is discussed. The 6Li/7Li depletion ratio is also examined. We find that the dispersion in the plateau and the 6Li/7Li depletion ratio scale with the absolute 7Li depletion in the plateau, and we use observational data to set bounds on the 7Li depletion in main-sequence halo

  19. The ubiquitous distribution of late embryogenesis abundant proteins across cell compartments in Arabidopsis offers tailored protection against abiotic stress.

    PubMed

    Candat, Adrien; Paszkiewicz, Gaël; Neveu, Martine; Gautier, Romain; Logan, David C; Avelange-Macherel, Marie-Hélène; Macherel, David

    2014-07-01

    Late embryogenesis abundant (LEA) proteins are hydrophilic, mostly intrinsically disordered proteins, which play major roles in desiccation tolerance. In Arabidopsis thaliana, 51 genes encoding LEA proteins clustered into nine families have been inventoried. To increase our understanding of the yet enigmatic functions of these gene families, we report the subcellular location of each protein. Experimental data highlight the limits of in silico predictions for analysis of subcellular localization. Thirty-six LEA proteins localized to the cytosol, with most being able to diffuse into the nucleus. Three proteins were exclusively localized in plastids or mitochondria, while two others were found dually targeted to these organelles. Targeting cleavage sites could be determined for five of these proteins. Three proteins were found to be endoplasmic reticulum (ER) residents, two were vacuolar, and two were secreted. A single protein was identified in pexophagosomes. While most LEA protein families have a unique subcellular localization, members of the LEA_4 family are widely distributed (cytosol, mitochondria, plastid, ER, and pexophagosome) but share the presence of the class A α-helix motif. They are thus expected to establish interactions with various cellular membranes under stress conditions. The broad subcellular distribution of LEA proteins highlights the requirement for each cellular compartment to be provided with protective mechanisms to cope with desiccation or cold stress. PMID:25005920

  20. Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: Evidence for involvement of splicing regulatory proteins

    PubMed Central

    Huo, Qing; Kayikci, Melis; Odermatt, Philipp; Meyer, Kathrin; Michels, Olivia; Saxena, Smita; Ule, Jernej; Schümperli, Daniel

    2014-01-01

    Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations. PMID:25692239

  1. Specific protein 1 depletion attenuates glucose uptake and proliferation of human glioma cells by regulating GLUT3 expression

    PubMed Central

    ZHENG, CHUANYI; YANG, KUN; ZHANG, MAOYING; ZOU, MINGMING; BAI, ENQI; MA, QUANHONG; XU, RUXIANG

    2016-01-01

    It has been reported previously that the expression of glucose transporter member 3 (GLUT3) is increased in malignant glioma cells compared with normal glial cells. However, the regulating mechanism that causes this phenomenon remains unknown. The present study investigated the regulating role of transcription factor specific protein 1 (Sp1) in GLUT3 expression in a human glioma cell line. In the present study, Sp1 was identified to directly bind to the GLUT3 5′-untranslated region in human glioma U251 cells. Small interfering RNA- and the Sp1-inhibitor mithramycin A-mediated Sp1 knockdown experiments revealed that Sp1 depletion decreased glucose uptake and inhibited cell growth and invasion of U251 cells by downregulating GLUT3 expression. Therefore Sp1 is an important positive regulator for the expression of GLUT3 in human glioma cells, and may explain the overexpression of GLUT3 in U251 cells. These results suggest that Sp1 may have a role in glioma treatment. PMID:27347112

  2. Dexamethasone modulates rat renal brush border membrane phosphate transporter mRNA and protein abundance and glycosphingolipid composition.

    PubMed Central

    Levi, M; Shayman, J A; Abe, A; Gross, S K; McCluer, R H; Biber, J; Murer, H; Lötscher, M; Cronin, R E

    1995-01-01

    Glucocorticoids are important regulators of renal phosphate transport. This study investigates the role of alterations in renal brush border membrane (BBM) sodium gradient-dependent phosphate transport (Na-Pi cotransporter) mRNA and protein abundance in the dexamethasone induced inhibition of Na-Pi cotransport in the rat. Dexamethasone administration for 4 d caused a 1.5-fold increase in the Vmax of Na-Pi cotransport (1785 +/- 119 vs. 2759 +/- 375 pmol/5 s per mg BBM protein in control, P < 0.01), which was paralleled by a 2.5-fold decrease in the abundance of Na-Pi mRNA and Na-Pi protein. There was also a 1.7-fold increase in BBM glucosylceramide content (528 +/- 63 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02). To determine whether the alteration in glucosylceramide content per se played a functional role in the decrease in Na-Pi cotransport, control rats were treated with the glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1-propanol (PDMP). The resultant 1.5-fold decrease in BBM glucosylceramide content (199 +/- 19 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02) was associated with a 1.4-fold increase in Na-Pi cotransport activity (1422 +/- 73 vs. 1048 +/- 85 pmol/5 s per mg BBM protein in control, P < 0.01), and a 1.5-fold increase in BBM Na-Pi protein abundance. Thus, dexamethasone-induced inhibition of Na-Pi cotransport is associated with a decrease in BBM Na-Pi cotransporter abundance, and an increase in glucosylceramide. Since primary alteration in BBM glucosylceramide content per se directly and selectively modulates BBM Na-Pi cotransport activity and Na-Pi protein abundance, we propose that the increase in BBM glucosylceramide content plays an important role in mediating the inhibitory effect of dexamethasone on Na-Pi cotransport activity. Images PMID:7615789

  3. Unexpected depletion of plasma arachidonate and total protein in cats fed a low arachidonic acid diet due to peroxidation.

    PubMed

    Chamberlin, Amy; Mitsuhashi, Yuka; Bigley, Karen; Bauer, John E

    2011-10-01

    An opportunity to investigate a low-arachidonic acid (AA) feline diet possibly related to elevated peroxide value (PV) during storage on plasma phospholipid (PL) and reproductive tissue fatty acid (FA) profiles presented itself in the present study. Cats (nine animals per group) had been fed one of three dry extruded, complete and balanced diets for 300 d before spaying. The diets contained adequate AA (0.3 g/kg), similar concentration of antioxidants and were stored at ambient temperature, but differed in FA composition. The diets were designated as follows: diet A (high linoleic acid), diet B (high γ-linolenic acid) and diet C (adequate linoleic acid). Diet samples that were obtained the week before spaying revealed an elevated PV of diet A v. diets B and C (135 v. 5.80 and 2.12 meq/kg fat, respectively). Records revealed decreased food consumption of diet A cats beginning at 240 d but without weight loss; thus an opportunity presented to investigate diet PV effects. Total plasma protein and PL-AA concentrations in group A were significantly decreased at 140 and 300 d. Uterine and ovarian tissues collected at surgery revealed modest decrements of AA. Diet A was below minimum standards at 0.015 % (minimum 0.02 %), probably due to oxidation. The time at which diet A became unacceptable may have occurred between 60 and 140 d because plasma PL-AA was within our normal colony range (approximately 4-7 % relative) after 56 d of feeding. High-linoleic acid-containing diets may be more likely to be oxidised requiring additional antioxidants. The findings suggest that reduced plasma protein in combination with plasma AA concentrations may serve as biomarkers of diet peroxidation in cats before feed refusal, weight loss or tissue depletion. PMID:22005409

  4. Region-Specific Protein Abundance Changes in the Brain of MPTP-induced Parkinson’s Disease Mouse Model

    SciTech Connect

    Zhang, Xu; Zhou, Jianying; Chin, Mark H; Schepmoes, Athena A; Petyuk, Vladislav A; Weitz, Karl K; Petritis, Brianne O; Monroe, Matthew E; Camp, David G; Wood, Stephen A; Melega, William P; Bigelow, Diana J; Smith, Desmond J; Qian, Weijun; Smith, Richard D

    2010-02-15

    Parkinson’s disease (PD) is characterized by dopaminergic neurodegeneration in the nigrostriatal region of the brain; however, the neurodegeneration extends well beyond dopaminergic neurons. To gain a better understanding of the molecular changes relevant to PD, we applied two-dimensional LC-MS/MS to comparatively analyze the proteome changes in four brain regions (striatum, cerebellum, cortex, and the rest of brain) using a MPTP-induced PD mouse model with the objective to identify nigrostriatal-specific and other region-specific protein abundance changes. The combined analyses resulted in the identification of 4,895 non-redundant proteins with at least two unique peptides per protein. The relative abundance changes in each analyzed brain region were estimated based on the spectral count information. A total of 518 proteins were observed with significant MPTP-induced changes across different brain regions. 270 of these proteins were observed with specific changes occurring either only in the striatum and/or in the rest of the brain region that contains substantia nigra, suggesting that these proteins are associated with the underlying nigrostriatal pathways. Many of the proteins that exhibit significant abundance changes were associated with dopamine signaling, mitochondrial dysfunction, the ubiquitin system, calcium signaling, the oxidative stress response, and apoptosis. A set of proteins with either consistent change across all brain regions or with changes specific to the cortex and cerebellum regions were also detected. One of the interesting proteins is ubiquitin specific protease (USP9X), a deubiquination enzyme involved in the protection of proteins from degradation and promotion of the TGF-β pathway, which exhibited altered abundances in all brain regions. Western blot validation showed similar spatial changes, suggesting that USP9X is potentially associated with neurodegeneration. Together, this study for the first time presents an overall picture of

  5. Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment

    PubMed Central

    Zhou, Minghai; Ottenberg, Gregory; Sferrazza, Gian Franco; Hubbs, Christopher; Fallahi, Mohammad; Rumbaugh, Gavin; Brantley, Alicia F.

    2015-01-01

    The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer’s, Parkinson’s and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD+) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD+ followed by decreased ATP production, and are completely rescued by treatment with NAD+ or its precursor nicotinamide because of restoration of physiological NAD+ levels. Toxic prion protein-induced NAD+ depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD+. Intranasal NAD+ treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD+ starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD+ replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases. PMID:25678560

  6. Abundant class III acidic chitinase homologue in tamarind (Tamarindus indica) seed serves as the major storage protein.

    PubMed

    Rao, Devavratha H; Gowda, Lalitha R

    2008-03-26

    The phyla Leguminosae contains protease inhibitors, lectins, chitinases, and glycohydrolases as major defense proteins in their seeds. Electrophoretic analysis of the seed proteins of tamarind ( Tamarindus indica L.), an agri-waste material, indicated the unusual presence of two major proteins comparable to overexpression of recombinant proteins. These proteins were identified by amino-terminal analysis to be (1) Kunitz-type trypsin inhibitor and (2) class III endochitinase (34000 Da). These two proteins were purified to apparent homogeneity by a single-step chitin bead affinity chromatography and characterized. The Kunitz inhibitor was specific toward inhibiting trypsin with a stoichiometry of 1:1. The 33000 +/- 1000 Da protein, accounting for >50% of the total seed protein, is an acidic glycoprotein exhibiting a very low endotype hydrolytic activity toward chitin derivatives. SDS-PAGE followed by densitometry of tamarind seed germination indicates the disappearance of the chitinase with the concomitant appearance of a cysteine endopeptidase. On the basis of its abundance, accumulation without any pathogenesis-related stimulus, temporal regulation, amino acid composition, and very low enzyme activity, this 34000 Da protein designated "tamarinin" physiologically serves as the major storage protein. PMID:18298067

  7. Lactate dehydrogenase A as a highly abundant eye lens protein in platypus (Ornithorhynchus anatinus): upsilon (upsilon)-crystallin.

    PubMed

    van Rheede, Teun; Amons, Reinout; Stewart, Niall; de Jong, Wilfried W

    2003-06-01

    Vertebrate eye lenses mostly contain two abundant types of proteins, the alpha-crystallins and the beta/gamma-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (upsilon)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as epsilon-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/upsilon-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate ldh-a gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed. PMID:12716980

  8. Unbiased selective isolation of protein N-terminal peptides from complex proteome samples using phospho tagging (PTAG) and TiO(2)-based depletion.

    PubMed

    Mommen, Geert P M; van de Waterbeemd, Bas; Meiring, Hugo D; Kersten, Gideon; Heck, Albert J R; de Jong, Ad P J M

    2012-09-01

    A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO(2)) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with formaldehyde at the protein level, after which the proteins are digested and the newly formed internal peptides modified with the PTAG reagent glyceraldhyde-3-phosphate in nearly perfect yields (> 99%). The resulting phosphopeptides are depleted through binding onto TiO(2), keeping exclusively a set of N-acetylated and/or N-dimethylated terminal peptides for analysis by liquid chromatography-tandem MS. Analysis of peptides derivatized with differentially labeled isotopic analogs of the PTAG reagent revealed a high depletion efficiency (> 95%). The method enabled identification of 753 unique N-terminal peptides (428 proteins) in N. meningitidis and 928 unique N-terminal peptides (572 proteins) in S. cerevisiae. These included verified neo-N termini from subcellular-relocalized membrane and mitochondrial proteins. The presented PTAG approach is therefore a novel, versatile, and robust method for mass spectrometry-based N-proteome analysis and identification of protease-generated cleavage products. PMID:22729381

  9. Pharmacological zinc and phytase supplementation enhance metallothionein mRNA abundance and protein concentration in newly weaned pigs.

    PubMed

    Martínez, Michelle M; Hill, Gretchen M; Link, Jane E; Raney, Nancy E; Tempelman, Robert J; Ernst, Catherine W

    2004-03-01

    The swine industry feeds pharmacological zinc (Zn) to newly weaned pigs to improve health. Because most swine diets are plant-based with a high phytic acid content, we hypothesized that adding phytase to diets could reduce the amount of Zn required to obtain beneficial responses. The role of metallothionein (MT) in Zn homeostasis could be important in this positive response. Thus, the goal of this study was to investigate the effect of dietary Zn and phytase on relative MT mRNA abundance and protein concentration in newly weaned pigs. Diets containing adequate (150 mg Zn/kg) or pharmacological concentrations of Zn (1000 or 2000 mg Zn/kg), as zinc oxide, with or without phytase [0, 500 phytase units (FTU)/kg, Natuphos, BASF] were fed in a 3 x 2 factorial design. Plasma and tissue minerals were measured in pigs killed after 14 d of dietary intervention. Hepatic and renal relative MT mRNA abundance and protein were greater (P < 0.05) in pigs fed 1000 mg Zn/kg with phytase, or 2000 mg Zn/kg with or without phytase vs. the remaining treatments. Intestinal mucosa MT mRNA abundance and protein were greater (P < 0.05) in pigs fed 2000 mg Zn/kg with phytase than in pigs fed 2000 mg Zn/kg alone or 1000 mg Zn/kg with phytase. Pigs fed 1000 mg Zn/kg plus phytase or 2000 mg Zn/kg with or without phytase had higher plasma, hepatic, and renal Zn than those fed the adequate Zn diets or 1000 mg Zn/kg. We conclude that feeding 1000 mg Zn/kg with phytase enhances MT mRNA abundance and protein and Zn absorption to the same degree as 2000 mg Zn/kg with and without phytase. PMID:14988443

  10. Proteomics reveals differences in protein abundance and highly similar antigenic profiles between Besnoitia besnoiti and Besnoitia tarandi.

    PubMed

    García-Lunar, P; Regidor-Cerrillo, J; Ortega-Mora, L M; Gutiérrez-Expósito, D; Alvarez-García, G

    2014-10-15

    Besnoitia besnoiti and Besnoitia tarandi are two cyst-forming apicomplexan parasites of the genus Besnoitia. B. besnoiti uses cattle as an intermediate host, in which it causes a disease that progresses in two sequential phases: the acute anasarca stage and the chronic scleroderma stage. Reindeer and caribou act as intermediate hosts for B. tarandi, which causes clinical signs similar to those caused by B. besnoiti. Previous studies demonstrated high molecular similarity, as determined by 18S and ITS-1 RNA sequences, between these Besnoitia spp., and strong serological cross-reactivity between these species has recently been demonstrated. Thus, a difference gel electrophoresis approach and mass spectrometry analysis were used to describe the proteomes and explore differences in protein abundance between B. besnoiti and B. tarandi in tachyzoite extracts. Immunoproteomes were also compared using 2-DE immunoblotting with polyclonal sera from experimentally infected rabbits. From approximately 1400 spots detected in DIGE-gels, 28 and 29 spots were differentially abundant in B. besnoiti and B. tarandi tachyzoites, respectively (± 1.5-fold, p<0.05). Four and 13 spots were exclusively detected in B. besnoiti and B. tarandi, respectively. Of the 32 differentially abundant spots analyzed by MALDI-TOF/MS, 6 up-regulated B. besnoiti proteins (LDH; HSP90; purine nucleoside phosphorylase and 3 hypothetical proteins) and 6 up-regulated B. tarandi proteins (G3PDH; LDH; PDI; mRNA decapping protein and 2 hypothetical proteins) were identified. Interestingly, no specific antigen spots were recognized by sera on any of the Besnoitia species studied and a similar antigen profile has been observed for B. tarandi and B. besnoiti sera when cross reactions were studied. This fact corroborates the difficulty in discerning Besnoitia infections using current serological assays. The present study underscores the importance of sequencing the B. besnoiti genome for species diversity studies of

  11. A cherry protein and its gene, abundantly expressed in ripening fruit, have been identified as thaumatin-like.

    PubMed Central

    Fils-Lycaon, B R; Wiersma, P A; Eastwell, K C; Sautiere, P

    1996-01-01

    A 29-kD polypeptide is the most abundant soluble protein in ripe cherry fruit (Prunus avium L); accumulation begins at the onset of ripening as the fruit turns from yellow to red. This protein was extracted from ripe cherries and purified by size-exclusion and ion-exchange chromatography. Antibodies to the purified protein were used to screen a cDNA library from ripe cherries. Numerous recombinant plaques reacted positively with the antibodies; the DNA sequence of representative clones encoded a polypeptide of 245 amino acid residues. A signal peptide was indicated, and the predicted mature protein corresponded to the purified protein in size (23.3 kD, by mass spectrometry) and isoelectric point (4.2). A search of known protein sequences revealed a strong similarity between this polypeptide and the thaumatin family of pathogenesis-related proteins. The cherry thaumatin-like protein does not have a sweet taste, and no antifungal activity was seen in preliminary assays. Expression of the protein appears to be regulated at the gene level, with mRNA levels at their highest in the ripe fruit. PMID:8685266

  12. High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei.

    PubMed

    Juodka, B; Spiess, E; Angiolillo, A; Joswig, G; Rothbarth, K; Werner, D

    1995-04-25

    Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller, S.A., Dykes, D.D. and Polesky, H.F., Nucleic Acids Res., 16, 1215, 1988] efficiently resolves deproteinized DNA. However, this DNA is still associated with prominent polypeptides which remain stably attached to DNA during further treatments, e.g. during repeated salting-out steps, prolonged incubation of DNA in 1% SDS or 4 M urea at 56 degrees C and ethanol precipitation. The persistent polypeptides (62, 52 and 40 kDa) released from Ehrlich ascites cell DNA were further characterized. Microsequencing indicates that the DNA binding polypeptides are not yet characterized at the sequence level. Nuclease digestion of the DNA releases stable DNA-protein complexes with the shape of globular particles (12.8 +/- 0.8 nm) and their larger aggregates in which DNA remains protected from nuclease digestion. The isolated DNA-polypeptide complexes show ATPase (Km = 7.4 x 10(-4) M) and protein kinase activity. Antibodies reveal a parallel distribution of the complexes with chromatin, however, the complexes are retained in chromatin-depleted nuclei. PMID:7753627

  13. Cellular expression of human centromere protein C demonstrates a cyclic behavior with highest abundance in the G1 phase.

    PubMed Central

    Knehr, M; Poppe, M; Schroeter, D; Eickelbaum, W; Finze, E M; Kiesewetter, U L; Enulescu, M; Arand, M; Paweletz, N

    1996-01-01

    Centromere proteins are localized within the centromere-kinetochore complex, which can be proven by means of immunofluorescence microscopy and immunoelectron microscopy. In consequence, their putative functions seem to be related exclusively to mitosis, namely to the interaction of the chromosomal kinetochores with spindle microtubules. However, electron microscopy using immune sera enriched with specific antibodies against human centromere protein C (CENP-C) showed that it occurs not only in mitosis but during the whole cell cycle. Therefore, we investigated the cell cycle-specific expression of CENP-C systematically on protein and mRNA levels applying HeLa cells synchronized in all cell cycle phases. Immunoblotting confirmed protein expression during the whole cell cycle and revealed an increase of CENP-C from the S phase through the G2 phase and mitosis to highest abundance in the G1 phase. Since this was rather surprising, we verified it by quantifying phase-specific mRNA levels of CENP-C, paralleled by the amplification of suitable internal standards, using the polymerase chain reaction. The results were in excellent agreement with abundant protein amounts and confirmed the cyclic behavior of CENP-C during the cell cycle. In consequence, we postulate that in addition to its role in mitosis, CENP-C has a further role in the G1 phase that may be related to cell cycle control. Images Fig. 1 Fig. 2 Fig. 4 PMID:8816782

  14. Size-related variation in protein abundance in the brain and abdominal tissue of bumble bee workers.

    PubMed

    Wolschin, F; Shpigler, H; Amdam, G V; Bloch, G

    2012-06-01

    Female bumble bee workers of the same species often show a profound body size variation that is linked to a division of labour. Large individuals are more likely to forage whereas small individuals are more likely to perform in-nest activities. A higher sensory sensitivity, stronger circadian rhythms as well as better learning and memory performances appear to better equip large individuals for outdoor activities compared to their smaller siblings. The molecular mechanisms underlying worker functional polymorphism remain unclear. Proteins are major determinants of an individual's morphology and behaviour. We hypothesized that the abundance of proteins such as metabolic enzymes as well as proteins involved in neuronal functions would differ with body size and provide insights into the mechanisms underlying size-dependent physiological specialization in bumble bee workers. We conducted protein quantification measurements based on liquid chromatography coupled with tandem mass spectrometry on tissue samples derived from small and large Bombus impatiens and Bombus terrestris workers. Proteins found to differ significantly in abundance between small and large workers belong to the categories of structure, energy metabolism and stress response. These findings provide the first proteomic insight into mechanisms associated with size-based division of labour in social insects. PMID:22568679

  15. An in vivo detection system for transient and low‐abundant protein interactions and their kinetics in budding yeast

    PubMed Central

    Brezovich, Andrea; Schuschnig, Martina; Ammerer, Gustav

    2015-01-01

    Abstract Methylation tracking (M‐Track) is a protein‐proximity assay in Saccharomyces cerevisiae, allowing the detection of transient protein–protein interactions in living cells. The bait protein is fused to a histone lysine methyl transferase and the prey protein to a methylation acceptor peptide derived from histone 3. Upon interaction, the histone 3 fragment is stably methylated on lysine 9 and can be detected by methylation‐specific antibodies. Since methylation marking is irreversible in budding yeast and only takes place in living cells, the occurrence of artifacts during cell lysate preparation is greatly reduced, leading to a more accurate representation of native interactions. So far, this method has been limited to highly abundant or overexpressed proteins. However, many proteins of interest are low‐abundant, and overexpression of proteins may interfere with their function, leading to an artificial situation. Here we report the generation of a toolbox including a novel cleavage‐enrichment system for the analysis of very low‐abundant proteins at their native expression levels. In addition, we developed a system for the parallel analysis of two prey proteins in a single cell, as well as an inducible methylation system. The inducible system allows precise control over the time during which the interaction is detected and can be used to determine interaction kinetics. Furthermore, we generated a set of constructs facilitating the cloning‐free genomic tagging of proteins at their endogenous locus by homologous recombination, and their expression from centromeric plasmids. GenBank submissions: pCK900; KM407502, pCK901; KM407503, pCK902; KM407504, pCK903; KM407505, pCK904; KM407506, pCK905; KM407507, pCK906; KM407508, pCK907; KM407509, pCK908; KM407510, pCK909; KM407511, pCK910; KM407512, pCK911; KM407513. © 2015 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:25582094

  16. Application of an improved proteomics method for abundant protein cleanup: molecular and genomic mechanisms study in plant defense.

    PubMed

    Zhang, Yixiang; Gao, Peng; Xing, Zhuo; Jin, Shumei; Chen, Zhide; Liu, Lantao; Constantino, Nasie; Wang, Xinwang; Shi, Weibing; Yuan, Joshua S; Dai, Susie Y

    2013-11-01

    High abundance proteins like ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. To address this challenge, we developed and evaluated Polyethyleneimine Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shot-gun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. Insect resistance was induced by over-expressing either jacalin-like or cupin-like genes in rice. The results further highlighted that PARC can serve as an effective strategy for proteomics analysis and gene discovery. PMID:23943779

  17. Application of an Improved Proteomics Method for Abundant Protein Cleanup: Molecular and Genomic Mechanisms Study in Plant Defense*

    PubMed Central

    Zhang, Yixiang; Gao, Peng; Xing, Zhuo; Jin, Shumei; Chen, Zhide; Liu, Lantao; Constantino, Nasie; Wang, Xinwang; Shi, Weibing; Yuan, Joshua S.; Dai, Susie Y.

    2013-01-01

    High abundance proteins like ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. To address this challenge, we developed and evaluated Polyethyleneimine Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shot-gun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. Insect resistance was induced by over-expressing either jacalin-like or cupin-like genes in rice. The results further highlighted that PARC can serve as an effective strategy for proteomics analysis and gene discovery. PMID:23943779

  18. Comparison of Amino Acids Physico-Chemical Properties and Usage of Late Embryogenesis Abundant Proteins, Hydrophilins and WHy Domain

    PubMed Central

    Jaspard, Emmanuel; Hunault, Gilles

    2014-01-01

    Late Embryogenesis Abundant proteins (LEAPs) comprise several diverse protein families and are mostly involved in stress tolerance. Most of LEAPs are intrinsically disordered and thus poorly functionally characterized. LEAPs have been classified and a large number of their physico-chemical properties have been statistically analyzed. LEAPs were previously proposed to be a subset of a very wide family of proteins called hydrophilins, while a domain called WHy (Water stress and Hypersensitive response) was found in LEAP class 8 (according to our previous classification). Since little is known about hydrophilins and WHy domain, the cross-analysis of their amino acids physico-chemical properties and amino acids usage together with those of LEAPs helps to describe some of their structural features and to make hypothesis about their function. Physico-chemical properties of hydrophilins and WHy domain strongly suggest their role in dehydration tolerance, probably by interacting with water and small polar molecules. The computational analysis reveals that LEAP class 8 and hydrophilins are distinct protein families and that not all LEAPs are a protein subset of hydrophilins family as proposed earlier. Hydrophilins seem related to LEAP class 2 (also called dehydrins) and to Heat Shock Proteins 12 (HSP12). Hydrophilins are likely unstructured proteins while WHy domain is structured. LEAP class 2, hydrophilins and WHy domain are thus proposed to share a common physiological role by interacting with water or other polar/charged small molecules, hence contributing to dehydration tolerance. PMID:25296175

  19. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR).

    PubMed

    Gomez, Sandra; Adalid-Peralta, Laura; Palafox-Fonseca, Hector; Cantu-Robles, Vito Adrian; Soberón, Xavier; Sciutto, Edda; Fragoso, Gladis; Bobes, Raúl J; Laclette, Juan P; Yauner, Luis del Pozo; Ochoa-Leyva, Adrián

    2015-01-01

    Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest. PMID:25989346

  20. Comparison of amino acids physico-chemical properties and usage of late embryogenesis abundant proteins, hydrophilins and WHy domain.

    PubMed

    Jaspard, Emmanuel; Hunault, Gilles

    2014-01-01

    Late Embryogenesis Abundant proteins (LEAPs) comprise several diverse protein families and are mostly involved in stress tolerance. Most of LEAPs are intrinsically disordered and thus poorly functionally characterized. LEAPs have been classified and a large number of their physico-chemical properties have been statistically analyzed. LEAPs were previously proposed to be a subset of a very wide family of proteins called hydrophilins, while a domain called WHy (Water stress and Hypersensitive response) was found in LEAP class 8 (according to our previous classification). Since little is known about hydrophilins and WHy domain, the cross-analysis of their amino acids physico-chemical properties and amino acids usage together with those of LEAPs helps to describe some of their structural features and to make hypothesis about their function. Physico-chemical properties of hydrophilins and WHy domain strongly suggest their role in dehydration tolerance, probably by interacting with water and small polar molecules. The computational analysis reveals that LEAP class 8 and hydrophilins are distinct protein families and that not all LEAPs are a protein subset of hydrophilins family as proposed earlier. Hydrophilins seem related to LEAP class 2 (also called dehydrins) and to Heat Shock Proteins 12 (HSP12). Hydrophilins are likely unstructured proteins while WHy domain is structured. LEAP class 2, hydrophilins and WHy domain are thus proposed to share a common physiological role by interacting with water or other polar/charged small molecules, hence contributing to dehydration tolerance. PMID:25296175

  1. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR)

    PubMed Central

    Gomez, Sandra; Adalid-Peralta, Laura; Palafox-Fonseca, Hector; Cantu-Robles, Vito Adrian; Soberón, Xavier; Sciutto, Edda; Fragoso, Gladis; Bobes, Raúl J.; Laclette, Juan P.; Yauner, Luis del Pozo; Ochoa-Leyva, Adrián

    2015-01-01

    Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest. PMID:25989346

  2. An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication.

    PubMed

    Mauthe, Mario; Langereis, Martijn; Jung, Jennifer; Zhou, Xingdong; Jones, Alex; Omta, Wienand; Tooze, Sharon A; Stork, Björn; Paludan, Søren Riis; Ahola, Tero; Egan, Dave; Behrends, Christian; Mokry, Michal; de Haan, Cornelis; van Kuppeveld, Frank; Reggiori, Fulvio

    2016-08-29

    Autophagy is a catabolic process regulated by the orchestrated action of the autophagy-related (ATG) proteins. Recent work indicates that some of the ATG proteins also have autophagy-independent roles. Using an unbiased siRNA screen approach, we explored the extent of these unconventional functions of ATG proteins. We determined the effects of the depletion of each ATG proteome component on the replication of six different viruses. Our screen reveals that up to 36% of the ATG proteins significantly alter the replication of at least one virus in an unconventional fashion. Detailed analysis of two candidates revealed an undocumented role for ATG13 and FIP200 in picornavirus replication that is independent of their function in autophagy as part of the ULK complex. The high numbers of unveiled ATG gene-specific and pathogen-specific functions of the ATG proteins calls for caution in the interpretation of data, which rely solely on the depletion of a single ATG protein to specifically ablate autophagy. PMID:27573464

  3. Depletion of hepatoma-derived growth factor-related protein-3 induces apoptotic sensitization of radioresistant A549 cells via reactive oxygen species-dependent p53 activation

    SciTech Connect

    Yun, Hong Shik; Hong, Eun-Hee; Lee, Su-Jae; Baek, Jeong-Hwa; Lee, Chang-Woo; Yim, Ji-Hye; Um, Hong-Duck; Hwang, Sang-Gu

    2013-09-27

    Highlights: •HRP-3 is a radiation- and anticancer drug-responsive protein in A549 cells. •Depletion of HRP-3 induces apoptosis of radio- and chemoresistant A549 cells. •Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. •Depletion of HRP-3 enhances ROS-dependent p53 activation and PUMA expression. -- Abstract: Biomarkers based on functional signaling have the potential to provide greater insight into the pathogenesis of cancer and may offer additional targets for anticancer therapeutics. Here, we identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistance-related gene and characterized the molecular mechanism by which its encoded protein regulates the radio- and chemoresistant phenotype of lung cancer-derived A549 cells. Knockdown of HRP-3 promoted apoptosis of A549 cells and potentiated the apoptosis-inducing action of radio- and chemotherapy. This increase in apoptosis was associated with a substantial generation of reactive oxygen species (ROS) that was attributable to inhibition of the Nrf2/HO-1 antioxidant pathway and resulted in enhanced ROS-dependent p53 activation and p53-dependent expression of PUMA (p53 upregulated modulator of apoptosis). Therefore, the HRP-3/Nrf2/HO-1/ROS/p53/PUMA cascade is an essential feature of the A549 cell phenotype and a potential radiotherapy target, extending the range of targets in multimodal therapies against lung cancer.

  4. Proteomic analysis of formalin-fixed paraffin-embedded glomeruli suggests depletion of glomerular filtration barrier proteins in two-kidney, one-clip hypertensive rats

    PubMed Central

    Finne, Kenneth; Vethe, Heidrun; Skogstrand, Trude; Leh, Sabine; Dahl, Tone D.; Tenstad, Olav; Berven, Frode S.; Reed, Rolf K.; Vikse, Bjørn Egil

    2014-01-01

    Background It is well known that hypertension may cause glomerular damage, but the molecular mechanisms involved are still incompletely understood. Methods In the present study, we used formalin-fixed paraffin-embedded (FFPE) tissue to investigate changes in the glomerular proteome in the non-clipped kidney of two-kidney one-clip (2K1C) hypertensive rats, with special emphasis on the glomerular filtration barrier. 2K1C hypertension was induced in 6-week-old Wistar Hannover rats (n = 6) that were sacrificed 23 weeks later and compared with age-matched sham-operated controls (n = 6). Tissue was stored in FFPE tissue blocks and later prepared on tissue slides for laser microdissection. Glomeruli without severe morphological damage were isolated, and the proteomes were analysed using liquid chromatography–tandem mass spectrometry. Results 2K1C glomeruli showed reduced abundance of proteins important for slit diaphragm complex, such as nephrin, podocin and neph1. The podocyte foot process had a pattern of reduced abundance of transmembrane proteins but unchanged abundances of the podocyte cytoskeletal proteins synaptopodin and α-actinin-4. Lower abundance of important glomerular basement membrane proteins was seen. Possible glomerular markers of damage with increased abundance in 2K1C were transgelin, desmin and acyl-coenzyme A thioesterase 1. Conclusions Microdissection and tandem mass spectrometry could be used to investigate the proteome of isolated glomeruli from FFPE tissue. Glomerular filtration barrier proteins had reduced abundance in the non-clipped kidney of 2K1C hypertensive rats. PMID:25129444

  5. Biochemical and structural characterization of an endoplasmic reticulum-localized late embryogenesis abundant (LEA) protein from the liverwort Marchantia polymorpha.

    PubMed

    Hatanaka, Rie; Furuki, Takao; Shimizu, Tempei; Takezawa, Daisuke; Kikawada, Takahiro; Sakurai, Minoru; Sugawara, Yasutake

    2014-11-28

    Late embryogenesis abundant (LEA) proteins, which accumulate to high levels in seeds during late maturation, are associated with desiccation tolerance. A member of the LEA protein family was found in cultured cells of the liverwort Marchantia polymorpha; preculture treatment of these cells with 0.5M sucrose medium led to their acquisition of desiccation tolerance. We characterized this preculture-induced LEA protein, designated as MpLEA1. MpLEA1 is predominantly hydrophilic with a few hydrophobic residues that may represent its putative signal peptide. The protein also contains a putative endoplasmic reticulum (ER) retention sequence, HEEL, at the C-terminus. Microscopic observations indicated that GFP-fused MpLEA1 was mainly localized in the ER. The recombinant protein MpLEA1 is intrinsically disordered in solution. On drying, MpLEA1 shifted predominantly toward α-helices from random coils. Such changes in conformation are a typical feature of the group 3 LEA proteins. Recombinant MpLEA1 prevented the aggregation of α-casein during desiccation-rehydration events, suggesting that MpLEA1 exerts anti-aggregation activity against desiccation-sensitive proteins by functioning as a "molecular shield". Moreover, the anti-aggregation activity of MpLEA1 was ten times greater than that of BSA or insect LEA proteins, which are known to prevent aggregation on drying. Here, we show that an ER-localized LEA protein, MpLEA1, possesses biochemical and structural features specific to group 3 LEA proteins. PMID:25450698

  6. Pro-inflammatory flagellin proteins of prevalent motile commensal bacteria are variably abundant in the intestinal microbiome of elderly humans.

    PubMed

    Neville, B Anne; Sheridan, Paul O; Harris, Hugh M B; Coughlan, Simone; Flint, Harry J; Duncan, Sylvia H; Jeffery, Ian B; Claesson, Marcus J; Ross, R Paul; Scott, Karen P; O'Toole, Paul W

    2013-01-01

    Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute "cell motility" category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ(28). The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13-4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved

  7. Pro-Inflammatory Flagellin Proteins of Prevalent Motile Commensal Bacteria Are Variably Abundant in the Intestinal Microbiome of Elderly Humans

    PubMed Central

    Neville, B. Anne; Sheridan, Paul O.; Harris, Hugh M. B.; Coughlan, Simone; Flint, Harry J.; Duncan, Sylvia H.; Jeffery, Ian B.; Claesson, Marcus J.; Ross, R. Paul; Scott, Karen P.; O'Toole, Paul W.

    2013-01-01

    Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute “cell motility” category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ28. The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13–4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved

  8. Effects of Tamarindus indica fruit pulp extract on abundance of HepG2 cell lysate proteins and their possible consequential impact on metabolism and inflammation.

    PubMed

    Chong, Ursula R W; Abdul-Rahman, Puteri S; Abdul-Aziz, Azlina; Hashim, Onn H; Mat-Junit, Sarni

    2013-01-01

    The fruit pulp extract of Tamarindus indica has been reported for its antioxidant and hypolipidemic properties. In this study, the methanol extract of T. indica fruit pulp was investigated for its effects on the abundance of HepG2 cell lysate proteins. Cell lysate was extracted from HepG2 cells grown in the absence and presence of the methanol extract of T. indica fruit pulp. Approximately 2500 spots were resolved using two-dimensional gel electrophoresis and the abundance of 20 cellular proteins was found to be significantly reduced. Among the proteins of reduced abundance, fourteen, including six proteins involved in metabolism (including ethanolamine phosphate cytidylyltransferase), four mitochondrial proteins (including prohibitin and respiratory chain proteins), and four proteins involved in translation and splicing, were positively identified by mass spectrometry and database search. The identified HepG2 altered abundance proteins, when taken together and analyzed by Ingenuity Pathways Analysis (IPA) software, are suggestive of the effects of T. indica fruit pulp extract on metabolism and inflammation, which are modulated by LXR/RXR. In conclusion, the methanol fruit pulp extract of T. indica was shown to cause reduced abundance of HepG2 mitochondrial, metabolic, and regulatory proteins involved in oxidative phosphorylation, protein synthesis, and cellular metabolism. PMID:24455694

  9. Effects of Tamarindus indica Fruit Pulp Extract on Abundance of HepG2 Cell Lysate Proteins and Their Possible Consequential Impact on Metabolism and Inflammation

    PubMed Central

    Chong, Ursula R. W.; Abdul-Rahman, Puteri S.; Abdul-Aziz, Azlina; Hashim, Onn H.; Mat-Junit, Sarni

    2013-01-01

    The fruit pulp extract of Tamarindus indica has been reported for its antioxidant and hypolipidemic properties. In this study, the methanol extract of T. indica fruit pulp was investigated for its effects on the abundance of HepG2 cell lysate proteins. Cell lysate was extracted from HepG2 cells grown in the absence and presence of the methanol extract of T. indica fruit pulp. Approximately 2500 spots were resolved using two-dimensional gel electrophoresis and the abundance of 20 cellular proteins was found to be significantly reduced. Among the proteins of reduced abundance, fourteen, including six proteins involved in metabolism (including ethanolamine phosphate cytidylyltransferase), four mitochondrial proteins (including prohibitin and respiratory chain proteins), and four proteins involved in translation and splicing, were positively identified by mass spectrometry and database search. The identified HepG2 altered abundance proteins, when taken together and analyzed by Ingenuity Pathways Analysis (IPA) software, are suggestive of the effects of T. indica fruit pulp extract on metabolism and inflammation, which are modulated by LXR/RXR. In conclusion, the methanol fruit pulp extract of T. indica was shown to cause reduced abundance of HepG2 mitochondrial, metabolic, and regulatory proteins involved in oxidative phosphorylation, protein synthesis, and cellular metabolism. PMID:24455694

  10. Mastitomics, the integrated omics of bovine milk in an experimental model of Streptococcus uberis mastitis: 1. High abundance proteins, acute phase proteins and peptidomics.

    PubMed

    Thomas, Funmilola Clara; Mullen, William; Tassi, Riccardo; Ramírez-Torres, Adela; Mudaliar, Manikhandan; McNeilly, Tom N; Zadoks, Ruth N; Burchmore, Richard; David Eckersall, P

    2016-08-16

    A peptidomic investigation of milk from an experimental model of Streptococcus uberis mastitis in dairy cows has incorporated a study of milk high abundance and acute phase (APP) proteins as well as analysis of low molecular weight peptide biomarkers. Intramammary infection (IMI) with S. uberis caused a shift in abundance from caseins, β-lactoglobulin and α-lactalbumin to albumin, lactoferrin and IgG with the increase in lactoferrin occurring last. The APP response of haptoglobin, mammary associated serum amyloid A3 and C-reactive protein occurred between 30-48 hours post challenge with peak concentrations of APPs at 72-96 hours post challenge and declined thereafter at a rate resembling the fall in bacterial count rather than the somatic cell count. A peptide biomarker panel for IMI based on capillary electrophoresis and mass spectrometry was developed. It comprised 77 identified peptides (IMI77) composed mainly of casein derived peptides but also including peptides of glycosylation dependent cell adhesion molecule and serum amyloid A. The panel had a biomarker classification score that increased from 36 hour to 81 hour post challenge, significantly differentiating infected from non-infected milk, thus suggesting potential as a peptide biomarker panel of bovine mastitis and specifically that of S. uberis origin. The use of omic technology has shown a multifactorial cross system reaction in high and low abundance proteins and their peptide derivatives with changes of over a thousand fold in analyte levels in response to S. uberis infection. PMID:27412456

  11. An odorant-binding protein is abundantly expressed in the nose and in the seminal fluid of the rabbit.

    PubMed

    Mastrogiacomo, Rosa; D'Ambrosio, Chiara; Niccolini, Alberto; Serra, Andrea; Gazzano, Angelo; Scaloni, Andrea; Pelosi, Paolo

    2014-01-01

    We have purified an abundant lipocalin from the seminal fluid of the rabbit, which shows significant similarity with the sub-class of pheromone carriers "urinary" and "salivary" and presents an N-terminal sequence identical with that of an odorant-binding protein (rabOBP3) expressed in the nasal tissue of the same species. This protein is synthesised in the prostate and found in the seminal fluid, but not in sperm cells. The same protein is also expressed in the nasal epithelium of both sexes, but is completely absent in female reproductive organs. It presents four cysteines, among which two are arranged to form a disulphide bridge, and is glycosylated. This is the first report of an OBP identified at the protein level in the seminal fluid of a vertebrate species. The protein purified from seminal fluid is bound to some organic chemicals whose structure is currently under investigation. We reasonably speculate that, like urinary and salivary proteins reported in other species of mammals, this lipocalin performs a dual role, as carrier of semiochemicals in the seminal fluid and as detector of chemical signals in the nose. PMID:25391153

  12. An Odorant-Binding Protein Is Abundantly Expressed in the Nose and in the Seminal Fluid of the Rabbit

    PubMed Central

    Niccolini, Alberto; Serra, Andrea; Gazzano, Angelo; Scaloni, Andrea; Pelosi, Paolo

    2014-01-01

    We have purified an abundant lipocalin from the seminal fluid of the rabbit, which shows significant similarity with the sub-class of pheromone carriers “urinary” and “salivary” and presents an N-terminal sequence identical with that of an odorant-binding protein (rabOBP3) expressed in the nasal tissue of the same species. This protein is synthesised in the prostate and found in the seminal fluid, but not in sperm cells. The same protein is also expressed in the nasal epithelium of both sexes, but is completely absent in female reproductive organs. It presents four cysteines, among which two are arranged to form a disulphide bridge, and is glycosylated. This is the first report of an OBP identified at the protein level in the seminal fluid of a vertebrate species. The protein purified from seminal fluid is bound to some organic chemicals whose structure is currently under investigation. We reasonably speculate that, like urinary and salivary proteins reported in other species of mammals, this lipocalin performs a dual role, as carrier of semiochemicals in the seminal fluid and as detector of chemical signals in the nose. PMID:25391153

  13. Thousand and one ways to quantify and compare protein abundances in label-free bottom-up proteomics.

    PubMed

    Blein-Nicolas, Mélisande; Zivy, Michel

    2016-08-01

    How to process and analyze MS data to quantify and statistically compare protein abundances in bottom-up proteomics has been an open debate for nearly fifteen years. Two main approaches are generally used: the first is based on spectral data generated during the process of identification (e.g. peptide counting, spectral counting), while the second makes use of extracted ion currents to quantify chromatographic peaks and infer protein abundances based on peptide quantification. These two approaches actually refer to multiple methods which have been developed during the last decade, but were submitted to deep evaluations only recently. In this paper, we compiled these different methods as exhaustively as possible. We also summarized the way they address the different problems raised by bottom-up protein quantification such as normalization, the presence of shared peptides, unequal peptide measurability and missing data. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock. PMID:26947242

  14. Cell Cycle-Regulated Protein Abundance Changes in Synchronously Proliferating HeLa Cells Include Regulation of Pre-mRNA Splicing Proteins

    PubMed Central

    Lane, Karen R.; Yu, Yanbao; Lackey, Patrick E.; Chen, Xian; Marzluff, William F.; Cook, Jeanette Gowen

    2013-01-01

    Cell proliferation involves dramatic changes in DNA metabolism and cell division, and control of DNA replication, mitosis, and cytokinesis have received the greatest attention in the cell cycle field. To catalogue a wider range of cell cycle-regulated processes, we employed quantitative proteomics of synchronized HeLa cells. We quantified changes in protein abundance as cells actively progress from G1 to S phase and from S to G2 phase. We also describe a cohort of proteins whose abundance changes in response to pharmacological inhibition of the proteasome. Our analysis reveals not only the expected changes in proteins required for DNA replication and mitosis but also cell cycle-associated changes in proteins required for biological processes not known to be cell-cycle regulated. For example, many pre-mRNA alternative splicing proteins are down-regulated in S phase. Comparison of this dataset to several other proteomic datasets sheds light on global mechanisms of cell cycle phase transitions and underscores the importance of both phosphorylation and ubiquitination in cell cycle changes. PMID:23520512

  15. Sodium-pump gene-expression, protein abundance and enzyme activity in isolated nephron segments of the aging rat kidney

    PubMed Central

    Scherzer, Pnina; Gal-Moscovici, Anca; Sheikh-Hamad, David; Popovtzer, Mordecai M

    2015-01-01

    Aging is associated with alteration in renal tubular functions, including sodium handling and concentrating ability. Na-K-ATPase plays a key role in driving tubular transport, and we hypothesized that decreased concentrating ability of the aging kidney is due in part to downregulation of Na-K-ATPase. In this study, we evaluated Na and K balance, aldosterone levels, and Na-K-ATPase gene expression, protein abundance, and activity in aging rat kidney. Na-K-ATPase activity (assayed microfluorometrically), mRNA (RT-PCR), and protein abundance (immunoblotting) were quantitated in the following isolated nephron segments: PCT, PST, MTAL, DCT, and CCD from 2, 8, 15, and 24 month-old-rats. In the course of aging, creatinine clearance decreased from 0.48 ± 0.02 mL/min/100 g BW to 0.28 ± 0.06 (P < 0.001) and aldosterone decreased from 23.6 ± 0.8 ng/dL to 13.2 ± 0.6 (P < 0.001). Serum Na+ and K+ increased by 4.0% and 22.5%, respectively. Na-K-ATPase activity, mRNA, and protein abundance of the α1 subunit displayed similar trends in all assayed segments; increasing in PCT and PST; decreasing in MTAL and DCT; increasing in CCD: in PCT they increased by 40%, 75%, and 250%, respectively; while in PST they increased by 80%, 50%, and 100%, respectively (P < 0.001). In MTAL they declined by 36%, 24%, and 34%, respectively, and in DCT by 38%, 59%, and 60%, respectively (P < 0.001). They were higher in CCD by 110%, 115%, and 246%, respectively (P < 0.001). Rats maintained Na/K balance; however with a steady state elevated serum K+. These results reveal quantitative changes in axial distribution of Na-K-ATPase at the level of gene expression, protein abundance, and activity in the nephrons of aging animals and may explain, in part, the pathophysiology of the senescent kidney. PMID:26056060

  16. Uses of Phage Display in Agriculture: Sequence Analysis and Comparative Modeling of Late Embryogenesis Abundant Client Proteins Suggest Protein-Nucleic Acid Binding Functionality

    PubMed Central

    Kushwaha, Rekha; Downie, A. Bruce; Payne, Christina M.

    2013-01-01

    A group of intrinsically disordered, hydrophilic proteins—Late Embryogenesis Abundant (LEA) proteins—has been linked to survival in plants and animals in periods of stress, putatively through safeguarding enzymatic function and prevention of aggregation in times of dehydration/heat. Yet despite decades of effort, the molecular-level mechanisms defining this protective function remain unknown. A recent effort to understand LEA functionality began with the unique application of phage display, wherein phage display and biopanning over recombinant Seed Maturation Protein homologs from Arabidopsis thaliana and Glycine max were used to retrieve client proteins at two different temperatures, with one intended to represent heat stress. From this previous study, we identified 21 client proteins for which clones were recovered, sometimes repeatedly. Here, we use sequence analysis and homology modeling of the client proteins to ascertain common sequence and structural properties that may contribute to binding affinity with the protective LEA protein. Our methods uncover what appears to be a predilection for protein-nucleic acid interactions among LEA client proteins, which is suggestive of subcellular residence. The results from this initial computational study will guide future efforts to uncover the protein protective mechanisms during heat stress, potentially leading to phage-display-directed evolution of synthetic LEA molecules. PMID:23956788

  17. Delayed cell cycle progression in selenoprotein W depleted cells is regulated by a mitogen-activated protein kinase kinase 4–p38–p53 pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a p53- and p21Cip1-dependent G1-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser33 in p53, which is associated with decreased p53...

  18. A High-Content Imaging Screen for Cellular Regulators of β-Catenin Protein Abundance.

    PubMed

    Zeng, Xin; Montoute, Monica; Bee, Tiger W; Lin, Hong; Kallal, Lorena A; Liu, Yan; Agarwal, Pankaj; Wang, Dayuan; Lu, Quinn; Morrow, Dwight; Pope, Andrew J; Wu, Zining

    2016-03-01

    Abnormal accumulation of β-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, β-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the β-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to β-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/β-catenin is mainly mediated by protein-protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect β-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated β-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce β-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of β-catenin degradation. This study demonstrates that detecting cell-based β-catenin protein stability is a viable approach to identifying novel mechanisms of β-catenin regulation as well as small molecules of therapeutic potential. PMID:26656867

  19. Functional characterization of the late embryogenesis abundant (LEA) protein gene family from Pinus tabuliformis (Pinaceae) in Escherichia coli.

    PubMed

    Gao, Jie; Lan, Ting

    2016-01-01

    Late embryogenesis abundant (LEA) proteins are a large and highly diverse gene family present in a wide range of plant species. LEAs are proposed to play a role in various stress tolerance responses. Our study represents the first-ever survey of LEA proteins and their encoding genes in a widely distributed pine (Pinus tabuliformis) in China. Twenty-three LEA genes were identified from the P. tabuliformis belonging to seven groups. Proteins with repeated motifs are an important feature specific to LEA groups. Ten of 23 pine LEA genes were selectively expressed in specific tissues, and showed expression divergence within each group. In addition, we selected 13 genes representing each group and introduced theses genes into Escherichia coli to assess the protective function of PtaLEA under heat and salt stresses. Compared with control cells, the E. coli cells expressing PtaLEA fusion protein exhibited enhanced salt and heat resistance and viability, indicating the protein may play a protective role in cells under stress conditions. Furthermore, among these enhanced tolerance genes, a certain extent of function divergence appeared within a gene group as well as between gene groups, suggesting potential functional diversity of this gene family in conifers. PMID:26781930

  20. Functional characterization of the late embryogenesis abundant (LEA) protein gene family from Pinus tabuliformis (Pinaceae) in Escherichia coli

    PubMed Central

    Gao, Jie; Lan, Ting

    2016-01-01

    Late embryogenesis abundant (LEA) proteins are a large and highly diverse gene family present in a wide range of plant species. LEAs are proposed to play a role in various stress tolerance responses. Our study represents the first-ever survey of LEA proteins and their encoding genes in a widely distributed pine (Pinus tabuliformis) in China. Twenty–three LEA genes were identified from the P. tabuliformis belonging to seven groups. Proteins with repeated motifs are an important feature specific to LEA groups. Ten of 23 pine LEA genes were selectively expressed in specific tissues, and showed expression divergence within each group. In addition, we selected 13 genes representing each group and introduced theses genes into Escherichia coli to assess the protective function of PtaLEA under heat and salt stresses. Compared with control cells, the E. coli cells expressing PtaLEA fusion protein exhibited enhanced salt and heat resistance and viability, indicating the protein may play a protective role in cells under stress conditions. Furthermore, among these enhanced tolerance genes, a certain extent of function divergence appeared within a gene group as well as between gene groups, suggesting potential functional diversity of this gene family in conifers. PMID:26781930

  1. Abundance of plasma antioxidant proteins confers tolerance to acute hypobaric hypoxia exposure.

    PubMed

    Padhy, Gayatri; Sethy, Niroj Kumar; Ganju, Lilly; Bhargava, Kalpana

    2013-09-01

    Systematic identification of molecular signatures for hypobaric hypoxia can aid in better understanding of human adaptation to high altitude. In an attempt to identify proteins promoting hypoxia tolerance during acute exposure to high altitude, we screened and identified hypoxia tolerant and susceptible rats based on hyperventilation time to a simulated altitude of 32,000 ft (9754 m). The hypoxia tolerance was further validated by estimating 8-isoprotane levels and protein carbonyls, which revealed that hypoxia tolerant rats possessed significant lower plasma levels as compared to susceptible rats. We used a comparative plasma proteome profiling approach using 2-dimensional gel electrophoresis (2-DGE) combined with MALDI TOF/TOF for both groups, along with an hypoxic control group. This resulted in the identification of 19 differentially expressed proteins. Seven proteins (TTR, GPx-3, PON1, Rab-3D, CLC11, CRP, and Hp) were upregulated in hypoxia tolerant rats, while apolipoprotein A-I (APOA1) was upregulated in hypoxia susceptible rats. We further confirmed the consistent higher expression levels of three antioxidant proteins (PON1, TTR, and GPx-3) in hypoxia-tolerant animals using ELISA and immunoblotting. Collectively, these proteomics-based results highlight the role of antioxidant enzymes in conferring hypoxia tolerance during acute hypobaric hypoxia. The expression of these antioxidant enzymes could be used as putative biomarkers for screening altitude adaptation as well as aiding in better management of altered oxygen pathophysiologies. PMID:24067188

  2. Direct Correlation between Motile Behavior and Protein Abundance in Single Cells.

    PubMed

    Dufour, Yann S; Gillet, Sébastien; Frankel, Nicholas W; Weibel, Douglas B; Emonet, Thierry

    2016-09-01

    Understanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage. PMID:27599206

  3. Chernobyl seed project. Advances in the identification of differentially abundant proteins in a radio-contaminated environment

    PubMed Central

    Rashydov, Namik M.; Hajduch, Martin

    2015-01-01

    Plants have the ability to grow and successfully reproduce in radio-contaminated environments, which has been highlighted by nuclear accidents at Chernobyl (1986) and Fukushima (2011). The main aim of this article is to summarize the advances of the Chernobyl seed project which has the purpose to provide proteomic characterization of plants grown in the Chernobyl area. We present a summary of comparative proteomic studies on soybean and flax seeds harvested from radio-contaminated Chernobyl areas during two successive generations. Using experimental design developed for radio-contaminated areas, altered abundances of glycine betaine, seed storage proteins, and proteins associated with carbon assimilation into fatty acids were detected. Similar studies in Fukushima radio-contaminated areas might complement these data. The results from these Chernobyl experiments can be viewed in a user-friendly format at a dedicated web-based database freely available at http://www.chernobylproteomics.sav.sk. PMID:26217350

  4. Chernobyl seed project. Advances in the identification of differentially abundant proteins in a radio-contaminated environment.

    PubMed

    Rashydov, Namik M; Hajduch, Martin

    2015-01-01

    Plants have the ability to grow and successfully reproduce in radio-contaminated environments, which has been highlighted by nuclear accidents at Chernobyl (1986) and Fukushima (2011). The main aim of this article is to summarize the advances of the Chernobyl seed project which has the purpose to provide proteomic characterization of plants grown in the Chernobyl area. We present a summary of comparative proteomic studies on soybean and flax seeds harvested from radio-contaminated Chernobyl areas during two successive generations. Using experimental design developed for radio-contaminated areas, altered abundances of glycine betaine, seed storage proteins, and proteins associated with carbon assimilation into fatty acids were detected. Similar studies in Fukushima radio-contaminated areas might complement these data. The results from these Chernobyl experiments can be viewed in a user-friendly format at a dedicated web-based database freely available at http://www.chernobylproteomics.sav.sk. PMID:26217350

  5. New genetic regulators question relevance of abundant yolk protein production in C. elegans

    PubMed Central

    Rompay, Liesbeth Van; Borghgraef, Charline; Beets, Isabel; Caers, Jelle; Temmerman, Liesbet

    2015-01-01

    Vitellogenesis or maternal yolk formation is considered critical to the reproduction of egg-laying animals. In invertebrates, however, most of its regulatory genes are still unknown. Via a combined mapping and whole-genome sequencing strategy, we performed a forward genetic screen to isolate novel regulators of yolk production in the nematode model system Caenorhabditis elegans. In addition to isolating new alleles of rab-35, rab-10 and M04F3.2, we identified five mutant alleles corresponding to three novel regulatory genes potently suppressing the expression of a GFP-based yolk reporter. We confirmed that mutations in vrp-1, ceh-60 and lrp-2 disrupt endogenous yolk protein synthesis at the transcriptional and translational level. In contrast to current beliefs, our discovered set of mutants with strongly reduced yolk proteins did not show serious reproduction defects. This raises questions as to whether yolk proteins per se are needed for ultimate reproductive success. PMID:26553710

  6. Spatial mapping of protein abundances in the mouse brain by voxelation integrated with high-throughput liquid chromatography-mass spectrometry.

    PubMed

    Petyuk, Vladislav A; Qian, Wei-Jun; Chin, Mark H; Wang, Haixing; Livesay, Eric A; Monroe, Matthew E; Adkins, Joshua N; Jaitly, Navdeep; Anderson, David J; Camp, David G; Smith, Desmond J; Smith, Richard D

    2007-03-01

    Temporally and spatially resolved mapping of protein abundance patterns within the mammalian brain is of significant interest for understanding brain function and molecular etiologies of neurodegenerative diseases; however, such imaging efforts have been greatly challenged by complexity of the proteome, throughput and sensitivity of applied analytical methodologies, and accurate quantitation of protein abundances across the brain. Here, we describe a methodology for comprehensive spatial proteome mapping that addresses these challenges by employing voxelation integrated with automated microscale sample processing, high-throughput liquid chromatography (LC) system coupled with high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, and a "universal" stable isotope labeled reference sample approach for robust quantitation. We applied this methodology as a proof-of-concept trial for the analysis of protein distribution within a single coronal slice of a C57BL/6J mouse brain. For relative quantitation of the protein abundances across the slice, an 18O-isotopically labeled reference sample, derived from a whole control coronal slice from another mouse, was spiked into each voxel sample, and stable isotopic intensity ratios were used to obtain measures of relative protein abundances. In total, we generated maps of protein abundance patterns for 1028 proteins. The significant agreement of the protein distributions with previously reported data supports the validity of this methodology, which opens new opportunities for studying the spatial brain proteome and its dynamics during the course of disease progression and other important biological and associated health aspects in a discovery-driven fashion. PMID:17255552

  7. Discovery and initial verification of differentially abundant proteins between multiple sclerosis patients and controls using iTRAQ and SID-SRM.

    PubMed

    Kroksveen, Ann C; Aasebø, Elise; Vethe, Heidrun; Van Pesch, Vincent; Franciotta, Diego; Teunissen, Charlotte E; Ulvik, Rune J; Vedeler, Christian; Myhr, Kjell-Morten; Barsnes, Harald; Berven, Frode S

    2013-01-14

    In the present study, we aimed to discover cerebrospinal fluid (CSF) proteins with significant abundance difference between early multiple sclerosis patients and controls, and do an initial verification of these proteins using selected reaction monitoring (SRM). iTRAQ and Orbitrap MS were used to compare the CSF proteome of patients with clinically isolated syndrome (CIS) (n=5), patients with relapsing-remitting multiple sclerosis that had CIS at the time of lumbar puncture (n=5), and controls with other inflammatory neurological disease (n=5). Of more than 1200 identified proteins, five proteins were identified with significant abundance difference between the patients and controls. In the initial verification using SRM we analyzed a larger patient and control cohort (n=132) and also included proteins reported as differentially abundant in multiple sclerosis in the literature. We found significant abundance difference for 11 proteins after verification, of which the five proteins alpha-1-antichymotrypsin, contactin-1, apolipoprotein D, clusterin, and kallikrein-6 were significantly differentially abundant in several of the group comparisons. This initial study form the basis for further biomarker verification studies in even larger sample cohorts, to determine if these proteins have relevance as diagnostic or prognostic biomarkers for multiple sclerosis. PMID:23059536

  8. Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins.

    PubMed

    Warner, Alden H; Guo, Zhi-hao; Moshi, Sandra; Hudson, John W; Kozarova, Anna

    2016-01-01

    Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-β-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally

  9. Disordered nucleiome: Abundance of intrinsic disorder in the DNA- and RNA-binding proteins in 1121 species from Eukaryota, Bacteria and Archaea.

    PubMed

    Wang, Chen; Uversky, Vladimir N; Kurgan, Lukasz

    2016-05-01

    Intrinsically disordered proteins (IDPs) are abundant in various proteomes, where they play numerous important roles and complement biological activities of ordered proteins. Among functions assigned to IDPs are interactions with nucleic acids. However, often, such assignments are made based on the guilty-by-association principle. The validity of the extension of these correlations to all nucleic acid binding proteins has never been analyzed on a large scale across all domains of life. To fill this gap, we perform a comprehensive computational analysis of the abundance of intrinsic disorder and intrinsically disordered domains in nucleiomes (∼548 000 nucleic acid binding proteins) of 1121 species from Archaea, Bacteria and Eukaryota. Nucleiome is a whole complement of proteins involved in interactions with nucleic acids. We show that relative to other proteins in the corresponding proteomes, the DNA-binding proteins have significantly increased disorder content and are significantly enriched in disordered domains in Eukaryotes but not in Archaea and Bacteria. The RNA-binding proteins are significantly enriched in the disordered domains in Bacteria, Archaea and Eukaryota, while the overall abundance of disorder in these proteins is significantly increased in Bacteria, Archaea, animals and fungi. The high abundance of disorder in nucleiomes supports the notion that the nucleic acid binding proteins often require intrinsic disorder for their functions and regulation. PMID:27037624

  10. Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2.

    PubMed

    Smyllie, Nicola J; Pilorz, Violetta; Boyd, James; Meng, Qing-Jun; Saer, Ben; Chesham, Johanna E; Maywood, Elizabeth S; Krogager, Toke P; Spiller, David G; Boot-Handford, Raymond; White, Michael R H; Hastings, Michael H; Loudon, Andrew S I

    2016-07-25

    Transcriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5-7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock. PMID:27374340

  11. Liquid-phase-based separation systems for depletion, prefractionation and enrichment of proteins in biological fluids and matrices for in-depth proteomics analysis – An update covering the period 2011-2014

    PubMed Central

    Puangpila, Chanida; Mayadunne, Erandi; Rassi, Ziad El

    2015-01-01

    This review article expands on the previous one (S. Selvaraju and Z. El Rassi, Electrophoresis 2012, 33, 74-88) by reviewing pertinent literature in the period extending from early 2011 to present. As the previous review article, the present one is concerned with proteomic sample preparation (e.g., depletion of high abundance proteins, reduction of the protein dynamic concentration range, enrichment of a particular sub-proteome), and the subsequent chromatographic and/or electrophoretic pre-fractionation prior to peptide separation and identification by LC-MS/MS. This review article is distinguished from its second version published in Electrophoresis 2012, 33, 74-88 by expanding on capturing/enriching sub-phosphoproteomes by immobilized metal affinity chromatography and metal oxide affinity chromatography. Seventy-seven papers published in the period extending from mid 2011 to the present have been reviewed. By no means this review article is exhaustive, given the fact that its aim is to give a concise treatment of the latest developments in the field. PMID:25287967

  12. Abundant and broad expression of transcription-induced chimeras and protein products in mammalian genomes.

    PubMed

    Lu, Guanting; Wu, Jin; Zhao, Gangbin; Wang, Zhiqiang; Chen, Weihua; Mu, Shijie

    2016-02-12

    The expression of transcription-induced chimeras (TICs) was underestimated due to strategic and logical reasons. In order to thoroughly examine TICs, systematic survey of TIC events was conducted in mammalian genomes using ESTs, followed by experimental validation using RT-PCR and real-time quantitative PCR (qPCR). The expression of ∼98% TIC events in at least one tissue or cell line from both mouse and human was verified. Besides, ∼40% TICs were broadly expressed, and ∼33% of TICs showed expression levels comparable to or higher than their upstream parental genes. We further identified putative chimeric proteins in public databases and validated two using Western blotting. GO analysis revealed that proteins resided in one multi-protein complex or functioning in metabolic or signaling pathway tended to produce fused products. Taken together, we have shown substantial evidence for the underestimated TIC events; and TICs could be a novel regulated way to further increases the proteome complexity in mammalian genomes. Possible regulation mechanisms and evolution of TICs were also discussed. PMID:26718406

  13. LEA polypeptide profiling of recalcitrant and orthodox legume seeds reveals ABI3-regulated LEA protein abundance linked to desiccation tolerance

    PubMed Central

    Hundertmark, Michaela; Buitink, Julia

    2013-01-01

    In contrast to orthodox seeds that acquire desiccation tolerance during maturation, recalcitrant seeds are unable to survive drying. These desiccation-sensitive seeds constitute an interesting model for comparative analysis with phylogenetically close species that are desiccation tolerant. Considering the importance of LEA (late embryogenesis abundant) proteins as protective molecules both in drought and in desiccation tolerance, the heat-stable proteome was characterized in cotyledons of the legume Castanospermum australe and it was compared with that of the orthodox model legume Medicago truncatula. RNA sequencing identified transcripts of 16 homologues out of 17 LEA genes for which polypeptides are detected in M. truncatula seeds. It is shown that for 12 LEA genes, polypeptides were either absent or strongly reduced in C. australe cotyledons compared with M. truncatula seeds. Instead, osmotically responsive, non-seed-specific dehydrins accumulated to high levels in the recalcitrant cotyledons compared with orthodox seeds. Next, M. truncatula mutants of the ABSCISIC ACID INSENSITIVE3 (ABI3) gene were characterized. Mature Mtabi3 seeds were found to be desiccation sensitive when dried below a critical water content of 0.4g H2O g DW–1. Characterization of the LEA proteome of the Mtabi3 seeds revealed a subset of LEA proteins with severely reduced abundance that were also found to be reduced or absent in C. australe cotyledons. Transcripts of these genes were indeed shown to be ABI3 responsive. The results highlight those LEA proteins that are critical to desiccation tolerance and suggest that comparable regulatory pathways responsible for their accumulation are missing in both desiccation-sensitive genotypes, revealing new insights into the mechanistic basis of the recalcitrant trait in seeds. PMID:24043848

  14. Norvaline and Norleucine May Have Been More Abundant Protein Components during Early Stages of Cell Evolution

    NASA Astrophysics Data System (ADS)

    Alvarez-Carreño, Claudia; Becerra, Arturo; Lazcano, Antonio

    2013-10-01

    The absence of the hydrophobic norvaline and norleucine in the inventory of protein amino acids is readdressed. The well-documented intracellular accumulation of these two amino acids results from the low-substrate specificity of the branched-chain amino acid biosynthetic enzymes that act over a number of related α-ketoacids. The lack of absolute substrate specificity of leucyl-tRNA synthase leads to a mischarged norvalyl-tRNALeu that evades the translational proofreading activites and produces norvaline-containing proteins, (cf. Apostol et al. J Biol Chem 272:28980-28988, 1997). A similar situation explains the presence of minute but detectable amounts of norleucine in place of methionine. Since with few exceptions both leucine and methionine are rarely found in the catalytic sites of most enzymes, their substitution by norvaline and norleucine, respectively, would have not been strongly hindered in small structurally simple catalytic polypeptides during the early stages of biological evolution. The report that down-shifts of free oxygen lead to high levels of intracellular accumulation of pyruvate and the subsequent biosynthesis of norvaline (Soini et al. Microb Cell Factories 7:30, 2008) demonstrates the biochemical and metabolic consequences of the development of a highly oxidizing environment. The results discussed here also suggest that a broader definition of biomarkers in the search for extraterrestrial life may be required.

  15. Rho-associated protein kinase 1 (ROCK1) is increased in Alzheimer's disease and ROCK1 depletion reduces amyloid-β levels in brain.

    PubMed

    Henderson, Benjamin W; Gentry, Erik G; Rush, Travis; Troncoso, Juan C; Thambisetty, Madhav; Montine, Thomas J; Herskowitz, Jeremy H

    2016-08-01

    Alzheimer's disease (AD) is the leading cause of dementia and mitigating amyloid-β (Aβ) levels may serve as a rational therapeutic avenue to slow AD progression. Pharmacologic inhibition of the Rho-associated protein kinases (ROCK1 and ROCK2) is proposed to curb Aβ levels, and mechanisms that underlie ROCK2's effects on Aβ production are defined. How ROCK1 affects Aβ generation remains a critical barrier. Here, we report that ROCK1 protein levels were elevated in mild cognitive impairment due to AD (MCI) and AD brains compared to controls. Aβ42 oligomers marginally increased ROCK1 and ROCK2 protein levels in neurons but strongly induced phosphorylation of Lim kinase 1 (LIMK1), suggesting that Aβ42 activates ROCKs. RNAi depletion of ROCK1 or ROCK2 suppressed endogenous Aβ40 production in neurons, and Aβ40 levels were reduced in brains of ROCK1 heterozygous knock-out mice compared to wild-type littermate controls. ROCK1 knockdown decreased amyloid precursor protein (APP), and treatment with bafilomycin accumulated APP levels in neurons depleted of ROCK1. These observations suggest that reduction of ROCK1 diminishes Aβ levels by enhancing APP protein degradation. Collectively, these findings support the hypothesis that both ROCK1 and ROCK2 are therapeutic targets to combat Aβ production in AD. Mitigating amyloid-β (Aβ) levels is a rational strategy for Alzheimer's disease (AD) treatment, however, therapeutic targets with clinically available drugs are lacking. We hypothesize that Aβ accumulation in mild cognitive impairment because of AD (MCI) and AD activates the RhoA/ROCK pathway which in turn fuels production of Aβ. Escalation of this cycle over the course of many years may contribute to the buildup of amyloid pathology in MCI and/or AD. PMID:27246255

  16. A group 6 late embryogenesis abundant protein from common bean is a disordered protein with extended helical structure and oligomer-forming properties.

    PubMed

    Rivera-Najera, Lucero Y; Saab-Rincón, Gloria; Battaglia, Marina; Amero, Carlos; Pulido, Nancy O; García-Hernández, Enrique; Solórzano, Rosa M; Reyes, José L; Covarrubias, Alejandra A

    2014-11-14

    Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association. PMID:25271167

  17. A Group 6 Late Embryogenesis Abundant Protein from Common Bean Is a Disordered Protein with Extended Helical Structure and Oligomer-forming Properties*

    PubMed Central

    Rivera-Najera, Lucero Y.; Saab-Rincón, Gloria; Battaglia, Marina; Amero, Carlos; Pulido, Nancy O.; García-Hernández, Enrique; Solórzano, Rosa M.; Reyes, José L.; Covarrubias, Alejandra A.

    2014-01-01

    Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association. PMID:25271167

  18. Group 3 late embryogenesis abundant proteins from embryos of Artemia franciscana: structural properties and protective abilities during desiccation.

    PubMed

    Boswell, Leaf C; Menze, Michael A; Hand, Steven C

    2014-01-01

    Group 3 late embryogenesis abundant (LEA) proteins are highly hydrophilic, and their expression is associated with desiccation tolerance in both plants and animals. Here we show that two LEA proteins from embryos of Artemia franciscana, AfrLEA2 and AfrLEA3m, are intrinsically disordered in solution but upon desiccation gain secondary structure, as measured by circular dichroism. Trifluoroethanol and sodium dodecyl sulfate are both shown to induce α-helical structure in AfrLEA2 and AfrLEA3m. Bioinformatic predictions of secondary-structure content for both proteins correspond most closely to conformations measured in the dry state. Because some LEA proteins afford protection to desiccation-sensitive proteins during drying and subsequent rehydration, we tested for this capacity in AfrLEA2 and AfrLEA3m. The protective capacities vary, depending on the target enzyme. For the cytoplasmic enzyme lactate dehydrogenase, neither AfrLEA2 nor AfrLEA3m, with or without trehalose present, was able to afford protection better than that provided by bovine serum albumin (BSA) under the same conditions. However, for another cytoplasmic enzyme, phosphofructokinase, both AfrLEA2 and AfrLEA3m in the presence of trehalose were able to afford protection far greater than that provided by BSA with trehalose. Finally, for the mitochondrial enzyme citrate synthase, 400-μg/mL AfrLEA3m without trehalose provided significantly more protection than the same concentration of either AfrLEA2 or BSA. PMID:25244376

  19. Proteome profiling of the growth phases of Leishmania pifanoi promastigotes in axenic culture reveals differential abundance of immunostimulatory proteins.

    PubMed

    Alcolea, Pedro J; Alonso, Ana; García-Tabares, Francisco; Mena, María del Carmen; Ciordia, Sergio; Larraga, Vicente

    2016-06-01

    Leishmaniasis is a term that encompasses a compendium of neglected tropical diseases caused by dimorphic and digenetic protozoan parasites from the genus Leishmania (Kinetoplastida: Trypanosomatidae). The clinical manifestations of neotropical cutaneous leishmaniasis (NCL) caused by Leishmania pifanoi and other species of the "Leishmania mexicana complex" mainly correspond to anergic diffuse cutaneous leishmaniasis (ADCL), which is the origin of considerable morbidity. Despite the outstanding advances in the characterization of the trypanosomatid genomes and proteomes, the biology of this species has been scarcely explored. However, the close relation of L. pifanoi to the sequenced species L. mexicana and others included in the "L. mexicana complex" allowed us to perform a two-dimension electrophoresis (2DE) approach to the promastigote proteome at the differential expression level. Protein identifications were performed by matrix-assisted laser desorption-ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF). This insight has revealed similarities and differences between L. pifanoi and other species responsible for cutaneous and visceral leishmaniasis. Interestingly, certain proteins that were previously described as immunostimulatory (elongation factor 1β, trypanothione peroxidase, heat shock protein 70, enolase, GDP-forming succinyl-CoA and aldehyde dehydrogenase) are more abundant in the final growth stages of promastigotes (late-logarithmic and/or stationary phase) in the case of L. pifanoi. PMID:26992294

  20. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells.

    PubMed

    Ramírez-Carvajal, Lisbeth; Singh, Neetu; de los Santos, Teresa; Rodríguez, Luis L; Long, Charles R

    2016-01-01

    Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells. PMID:26592975

  1. Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk

    PubMed Central

    Zhao, Jianguo; Xu, Weijie; Ross, Jason W.; Walters, Eric M.; Butler, Stephen P.; Whyte, Jeff J.; Kelso, Lindsey; Fatemi, Mostafa; Vanderslice, Nicholas C.; Giroux, Keith; Spate, Lee D.; Samuel, Melissa S.; Murphy, Cliff N.; Wells, Kevin D.; Masiello, Nick C.; Prather, Randall S.; Velander, William H.

    2015-01-01

    Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to <20% of the world’s population. We used transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock. PMID:26387706

  2. Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk.

    PubMed

    Zhao, Jianguo; Xu, Weijie; Ross, Jason W; Walters, Eric M; Butler, Stephen P; Whyte, Jeff J; Kelso, Lindsey; Fatemi, Mostafa; Vanderslice, Nicholas C; Giroux, Keith; Spate, Lee D; Samuel, Melissa S; Murphy, Cliff N; Wells, Kevin D; Masiello, Nick C; Prather, Randall S; Velander, William H

    2015-01-01

    Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to <20% of the world's population. We used transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock. PMID:26387706

  3. Myosin Gene Expression and Protein Abundance in Different Castes of the Formosan Subterranean Termite (Coptotermes formosanus).

    PubMed

    Tarver, Matthew R; Florane, Christopher B; Mattison, Christopher P; Holloway, Beth A; Lax, Alan

    2012-01-01

    The Formosan subterranean termite (Coptotermes formosanus) is an important worldwide pest, each year causing millions of dollars in structural damage and control costs. Termite colonies are composed of several phenotypically distinct castes. Termites utilize these multiple castes to efficiently perform unique roles within the colony. During the molting/caste differentiation process, multiple genes are believed to be involved in the massive reorganization of the body plan. The objective of this research was to analyze the muscle gene, myosin, to further understand the role it plays in C. formosanus development. We find that comparing worker vs. solider caste myosin gene expression is up-regulated in the soldier and a myosin antibody-reactive protein suggests changes in splicing. Comparison of body regions of mature soldier and worker castes indicates a greater level of myosin transcript in the heads. The differential expression of this important muscle-related gene is anticipated considering the large amount of body plan reorganization and muscle found in the soldier caste. These results have a direct impact on our understanding of the downstream genes in the caste differentiation process and may lead to new targets for termite control. PMID:26466734

  4. Quantitation of HLA Class II Protein Incorporated into Human Immunodeficiency Type 1 Virions Purified by Anti-CD45 Immunoaffinity Depletion of Microvesicles

    PubMed Central

    Trubey, Charles M.; Chertova, Elena; Coren, Lori V.; Hilburn, Joanne M.; Hixson, Catherine V.; Nagashima, Kunio; Lifson, Jeffrey D.; Ott, David E.

    2003-01-01

    Among the many host cell-derived proteins found in human immunodeficiency virus type 1 (HIV-1), HLA class II (HLA-II) appears to be selectively incorporated onto virions and may contribute to mechanisms of indirect imunopathogenesis in HIV infection and AIDS. However, the amount of HLA-II on the surface of HIV-1 particles has not been reliably determined due to contamination of virus preparations by microvesicles containing host cell proteins, including HLA-II. Even rigorous sucrose density centrifugation is unable to completely separate HIV-1 from microvesicles. CD45, a leukocyte integral membrane protein, is found on microvesicles, yet appears to be excluded from HIV-1 particles. Exploiting this observation, we have developed a CD45-based immunoaffinity depletion method for removing CD45-containing microvesicles that yields highly purified preparations of virions. Examination of CD45-depleted HIV-1MN by high-pressure liquid chromatography, protein sequencing, and amino acid analyses determined a molar ratio of HLA-II to Gag of 0.04 to 0.05 in the purified virions, corresponding to an estimated average of 50 to 63 native HLA-II complexes (i.e., a dimer of α and β heterodimers) per virion. These values are approximately 5- to 10-fold lower than those previously determined for other virion preparations that contained microvesicles. Our observations demonstrate the utility of CD45 immunoaffinity-based approaches for producing highly purified retrovirus preparations for applications that would benefit from the use of virus that is essentially free of microvesicles. PMID:14610192

  5. Integrated analysis of transcriptomic and proteomic data of Desulfovibrio vulgaris: Zero-Inflated Poisson regression models to predict abundance of undetected proteins

    SciTech Connect

    Nie, Lei; Wu, Gang; Brockman, Fred J.; Zhang, Weiwen

    2006-05-04

    Abstract Advances in DNA microarray and proteomics technologies have enabled high-throughput measurement of mRNA expression and protein abundance. Parallel profiling of mRNA and protein on a global scale and integrative analysis of these two data types could provide additional insight into the metabolic mechanisms underlying complex biological systems. However, because protein abundance and mRNA expression are affected by many cellular and physical processes, there have been conflicting results on the correlation of these two measurements. In addition, as current proteomic methods can detect only a small fraction of proteins present in cells, no correlation study of these two data types has been done thus far at the whole-genome level. In this study, we describe a novel data-driven statistical model to integrate whole-genome microarray and proteomic data collected from Desulfovibrio vulgaris grown under three different conditions. Based on the Poisson distribution pattern of proteomic data and the fact that a large number of proteins were undetected (excess zeros), Zero-inflated Poisson models were used to define the correlation pattern of mRNA and protein abundance. The models assumed that there is a probability mass at zero representing some of the undetected proteins because of technical limitations. The models thus use abundance measurements of transcripts and proteins experimentally detected as input to generate predictions of protein abundances as output for all genes in the genome. We demonstrated the statistical models by comparatively analyzing D. vulgaris grown on lactate-based versus formate-based media. The increased expressions of Ech hydrogenase and alcohol dehydrogenase (Adh)-periplasmic Fe-only hydrogenase (Hyd) pathway for ATP synthesis were predicted for D. vulgaris grown on formate.

  6. Mononuclear phagocyte system depletion blocks interstitial tonicity-responsive enhancer binding protein/vascular endothelial growth factor C expression and induces salt-sensitive hypertension in rats.

    PubMed

    Machnik, Agnes; Dahlmann, Anke; Kopp, Christoph; Goss, Jennifer; Wagner, Hubertus; van Rooijen, Nico; Eckardt, Kai-Uwe; Müller, Dominik N; Park, Joon-Keun; Luft, Friedrich C; Kerjaschki, Dontscho; Titze, Jens

    2010-03-01

    We showed recently that mononuclear phagocyte system (MPS) cells provide a buffering mechanism for salt-sensitive hypertension by driving interstitial lymphangiogenesis, modulating interstitial Na(+) clearance, and increasing endothelial NO synthase protein expression in response to very high dietary salt via a tonicity-responsive enhancer binding protein/vascular endothelial growth factor C regulatory mechanism. We now tested whether isotonic saline and deoxycorticosterone acetate (DOCA)-salt treatment leads to a similar regulatory response in Sprague-Dawley rats. Male rats were fed a low-salt diet and received tap water (low-salt diet LSD), 1.0% saline (high-salt diet HSD), or DOCA+1.0% saline (DOCA-HSD). To test the regulatory role of interstitial MPS cells, we further depleted MPS cells with clodronate liposomes. HSD and DOCA-HSD led to Na(+) accumulation in the skin, MPS-driven tonicity-responsive enhancer binding protein/vascular endothelial growth factor C-mediated hyperplasia of interstitial lymph capillaries, and increased endothelial NO synthase protein expression in skin interstitium. Clodronate liposome MPS cell depletion blocked MPS infiltration in the skin interstitium, resulting in unchanged tonicity-responsive enhance binding protein/vascular endothelial growth factor C levels and absent hyperplasia of the lymph capillary network. Moreover, no increased skin endothelial NO synthase protein expression occurred in either clodronate liposome-treated HSD or DOCA-salt rats. Thus, absence of the MPS-cell regulatory response converted a salt-resistant blood-pressure state to a salt-sensitive state in HSD rats. Furthermore, salt-sensitive hypertension in DOCA-salt rats was aggravated. We conclude that MPS cells act as onsite controllers of interstitial volume and blood pressure homeostasis, providing a local regulatory salt-sensitive tonicity-responsive enhancer binding protein/vascular endothelial growth factor C-mediated mechanism in the skin to maintain

  7. Lipoic acid inhibits the DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) and triggers its depletion in colorectal cancer cells with concomitant autophagy induction.

    PubMed

    Göder, Anja; Nagel, Georg; Kraus, Alexander; Dörsam, Bastian; Seiwert, Nina; Kaina, Bernd; Fahrer, Jörg

    2015-08-01

    Alkylating agents are present in food and tobacco smoke, but are also used in cancer chemotherapy, inducing the DNA lesion O (6)-methylguanine. This critical adduct is repaired by O (6)-methylguanine-DNA methyltransferase (MGMT), resulting in MGMT inactivation and degradation. In the present study, we analyzed the effects of the natural disulfide compound lipoic acid (LA) on MGMT in vitro and in colorectal cancer cells. We show that LA, but not its reduced form dihydrolipoic acid, potently inhibits the activity of recombinant MGMT by interfering with its catalytic Cys-145 residue, which was partially reversible by N-acetyl cysteine. Incubation of HCT116 colorectal cancer cells with LA altered their glutathione pool and caused a decline in MGMT activity. This was mirrored by LA-induced depletion of MGMT protein, which was not attributable to changes in MGMT messenger RNA levels. Loss of MGMT protein coincided with LA-induced autophagy, a process resulting in lysosomal degradation of proteins, including presumably MGMT. LA-stimulated autophagy in a p53-independent manner as revealed by the response of isogenic HCT116 cell lines. Knockdown of the crucial autophagy component beclin-1 and chemical inhibitors blocked LA-induced autophagy, but did not abrogate LA-triggered MGMT degradation. Concomitant with MGMT depletion, LA pretreatment resulted in enhanced O (6)-methylguanine levels in DNA. It also increased the cytotoxicity of the alkylating anticancer drug temozolomide in temozolomide-resistant colorectal cancer cells. Taken together, our study showed that the natural compound LA inhibits MGMT and induces autophagy. Furthermore, LA enhanced the cytotoxic effects of temozolomide, which makes it a candidate for a supplement in cancer therapy. PMID:25998848

  8. Phytochrome-imposed oscillations in PIF3 protein abundance regulate hypocotyl growth under diurnal light/dark conditions in Arabidopsis

    PubMed Central

    Soy, Judit; Leivar, Pablo; González-Schain, Nahuel; Sentandreu, Maria; Prat, Salomé; Quail, Peter H.; Monte, Elena

    2012-01-01

    SUMMARY Arabidopsis seedlings display rhythmic growth when grown under diurnal conditions, with maximal elongation rates occurring at the end of the night under short-day photoperiods. Current evidence indicates that this behavior involves the action of the growth-promoting bHLH factors PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) at the end of the night, through a coincidence mechanism that combines their transcriptional regulation by the circadian clock with control of protein accumulation by light. To assess the possible role of PIF3 in this process, we have analyzed hypocotyl responses and marker gene expression in pif single- and higher-order mutants. The data show that PIF3 plays a prominent role as a promoter of seedling growth under diurnal light/dark conditions, in conjunction with PIF4 and PIF5. In addition, we provide evidence that PIF3 functions in this process through its intrinsic transcriptional regulatory activity, at least in part by directly targeting growth-related genes, and independently of its ability to regulate phytochrome B (phyB) levels. Furthermore, in sharp contrast to PIF4 and PIF5, our data show that the PIF3 gene is not subject to transcriptional regulation by the clock, but that PIF3 protein abundance oscillates under diurnal conditions as a result of a progressive decline in PIF3 protein degradation mediated by photoactivated phyB, and consequent accumulation of the bHLH factor during the dark period. Collectively, the data suggest that phyB-mediated, post-translational regulation allows PIF3 accumulation to peak just before dawn, at which time it accelerates hypocotyl growth, together with PIF4 and PIF5, by directly regulating the induction of growth-related genes. PMID:22409654

  9. Competition between Heterochromatic Loci Allows the Abundance of the Silencing Protein, Sir4, to Regulate de novo Assembly of Heterochromatin

    PubMed Central

    Larin, Michelle L.; Harding, Katherine; Williams, Elizabeth C.; Lianga, Noel; Doré, Carole; Pilon, Sophie; Langis, Éric; Yanofsky, Corey; Rudner, Adam D.

    2015-01-01

    Changes in the locations and boundaries of heterochromatin are critical during development, and de novo assembly of silent chromatin in budding yeast is a well-studied model for how new sites of heterochromatin assemble. De novo assembly cannot occur in the G1 phase of the cell cycle and one to two divisions are needed for complete silent chromatin assembly and transcriptional repression. Mutation of DOT1, the histone H3 lysine 79 (K79) methyltransferase, and SET1, the histone H3 lysine 4 (K4) methyltransferase, speed de novo assembly. These observations have led to the model that regulated demethylation of histones may be a mechanism for how cells control the establishment of heterochromatin. We find that the abundance of Sir4, a protein required for the assembly of silent chromatin, decreases dramatically during a G1 arrest and therefore tested if changing the levels of Sir4 would also alter the speed of de novo establishment. Halving the level of Sir4 slows heterochromatin establishment, while increasing Sir4 speeds establishment. yku70Δ and ubp10Δ cells also speed de novo assembly, and like dot1Δ cells have defects in subtelomeric silencing, suggesting that these mutants may indirectly speed de novo establishment by liberating Sir4 from telomeres. Deleting RIF1 and RIF2, which suppresses the subtelomeric silencing defects in these mutants, rescues the advanced de novo establishment in yku70Δ and ubp10Δ cells, but not in dot1Δ cells, suggesting that YKU70 and UBP10 regulate Sir4 availability by modulating subtelomeric silencing, while DOT1 functions directly to regulate establishment. Our data support a model whereby the demethylation of histone H3 K79 and changes in Sir4 abundance and availability define two rate-limiting steps that regulate de novo assembly of heterochromatin. PMID:26587833

  10. Thyrotropin-releasing hormone-induced depletion of G(q)alpha/G(11)alpha proteins from detergent-insensitive membrane domains.

    PubMed

    Pesanová, Z; Novotný, J; Cerný, J; Milligan, G; Svoboda, P

    1999-12-24

    The role of detergent-insensitive membrane domains (DIMs) in desensitisation of the G protein-coupled receptor-mediated hormone response was studied in clone E2M11 of HEK293 cells which stably express high levels of both thyrotropin-releasing hormone (TRH) receptors and G(11)alpha G protein. DIMs were prepared by flotation in equilibrium sucrose density gradients and characterised by a panel of membrane markers representing peripheral, glycosylphosphatidylinositol-bound as well as integral membrane proteins (caveolin, CD29, CD55, CD59, CD147, the alpha subunit of Na, K-ATPase) and enzyme activities (alkaline phosphatase, adenylyl cyclase). Caveolin-containing DIMs represented only a small fraction of the overall pool of G(q)alpha/G(11)alpha-rich domains. Prolonged stimulation of E2M11 cells with TRH resulted in dramatic depletion of G(q)alpha/G(11)alpha from all DIMs, which was paralleled by a concomitant G(q)alpha/G(11)alpha increase in the high-density gradient fractions containing the bulk-phase membrane constituents soluble in 1% Triton X-100. Distribution of membrane markers was unchanged under these conditions. Membrane domains thus represent a substantial structural determinant of the G protein pool relevant to desensitisation of hormone action. PMID:10611479

  11. Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation.

    PubMed

    Shah, Punit; Wang, Xiangchun; Yang, Weiming; Toghi Eshghi, Shadi; Sun, Shisheng; Hoti, Naseruddin; Chen, Lijun; Yang, Shuang; Pasay, Jered; Rubin, Abby; Zhang, Hui

    2015-10-01

    Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in

  12. Prion protein localizes at the ciliary base during neural and cardiovascular development, and its depletion affects α-tubulin post-translational modifications

    PubMed Central

    Halliez, Sophie; Martin-Lannerée, Séverine; Passet, Bruno; Hernandez-Rapp, Julia; Castille, Johan; Urien, Céline; Chat, Sophie; Laude, Hubert; Vilotte, Jean-Luc; Mouillet-Richard, Sophie; Béringue, Vincent

    2015-01-01

    Although conversion of the cellular form of the prion protein (PrPC) into a misfolded isoform is the underlying cause of prion diseases, understanding PrPC physiological functions has remained challenging. PrPC depletion or overexpression alters the proliferation and differentiation properties of various types of stem and progenitor cells in vitro by unknown mechanisms. Such involvement remains uncertain in vivo in the absence of any drastic phenotype of mice lacking PrPC. Here, we report PrPC enrichment at the base of the primary cilium in stem and progenitor cells from the central nervous system and cardiovascular system of developing mouse embryos. PrPC depletion in a neuroepithelial cell line dramatically altered key cilium-dependent processes, such as Sonic hedgehog signalling and α-tubulin post-translational modifications. These processes were also affected over a limited time window in PrPC–ablated embryos. Thus, our study reveals PrPC as a potential actor in the developmental regulation of microtubule dynamics and ciliary functions. PMID:26679898

  13. Role of Cardiac Myocytes Heart Fatty Acid Binding Protein Depletion (H-FABP) in Early Myocardial Infarction in Human Heart (Autopsy Study)

    PubMed Central

    Shabaiek, Amany; Ismael, Nour El-Hoda; Elsheikh, Samar; Amin, Hebat Allah

    2016-01-01

    BACKGROUND: Many immunohistochemical markers have been used in the postmortem detection of early myocardial infarction. AIM: In the present study we examined the role of Heart-type fatty acid binding protein (H-FABP), in the detection of early myocardial infarction. MATERIAL AND METHODS: We obtained samples from 40 human autopsy hearts with/without histopathological signs of ischemia. RESULTS: All cases of definite and probable myocardial infarction showed a well-defined area of H-FABP depletion. All of the control cases showed strong H-FABP expression, except two markedly autolysed myocardial samples that showed affected antigenicity. CONCLUSION: Thus, we suggest H-FABP as being one of the valuable tools facing the problem of postmortem detection of early myocardial infarction/ischemia, but not in autolysis.

  14. Protein ingestion does not impair exercise-induced AMPK signalling when in a glycogen-depleted state: implications for train-low compete-high.

    PubMed

    Taylor, Conor; Bartlett, Jonathan D; van de Graaf, Christian Soler; Louhelainen, Jari; Coyne, Vicki; Iqbal, Zafar; Maclaren, Don P M; Gregson, Warren; Close, Graeme L; Morton, James P

    2013-06-01

    The aim of the present study was to test the hypothesis that consuming protein does not attenuate AMPK signalling when exercise is commenced in a glycogen-depleted state. After performing a glycogen-depleting protocol the evening before, the subsequent morning ten active men performed 45 min steady-state cycling at 50 % of peak power output (PPO) followed by an exercise capacity test (1-min intervals at 80 % PPO interspersed with 1-min periods at 40 % PPO). In a repeated measures design, subjects consumed 20 g of a casein hydrolysate solution (PRO) 45 min before exercise, 10 g during and a further 20 g immediately post-exercise, or an equivalent volume of a non-calorie taste matched placebo (PLA). Resting (PRO = 134 ± 29; PLA = 136 ± 28 mmol kg(-1)) and post-exercise muscle glycogen (PRO = 43 ± 16; PLA = 47 ± 18 mmol kg(-1)) was not different (P > 0.05) between trials nor was exercise capacity (PRO = 26 ± 9; PLA = 25 ± 10 min, P > 0.05). Phosphorylation of AMPK(Thr172) increased threefold immediately post-exercise (P < 0.05) and PGC1-mRNA increased sixfold at 3 h post-exercise (P < 0.05), though there were no differences between conditions (P > 0.05). In contrast, there was a trend (P = 0.08) for a divergent response in eEF2(Thr56) phosphorylation such that 1.5 fold increases post- and 3 h post-exercise in PLA were blunted with PRO, thus indicative of greater eEF2 activation. We conclude that athletes who deliberately incorporate training phases with reduced muscle glycogen into their training programmes may consume protein before, during and after exercise without negating signalling through the AMPK cascade. PMID:23263742

  15. Single-cell analysis of the mitogen-induced calcium responses of normal and protein kinase C-depleted Swiss 3T3 cells.

    PubMed Central

    Corps, A N; Cheek, T R; Moreton, R B; Berridge, M J; Brown, K D

    1989-01-01

    Single-cell fluorescence image analysis has been used to characterize the mitogen-induced increases in intracellular free [Ca2+] ([Ca2+]i) in control and protein kinase C-depleted Swiss 3T3 cells. More than 80% of the control cells exhibited fast, transient responses to bombesin, vasopressin, or prostaglandin F2 alpha (PGF2 alpha). In contrast, the [Ca2+]i responses induced by platelet-derived growth factor (PDGF) were markedly more heterogeneous, slower, and often biphasic, with fewer cells (60-70%) responding. The peak [Ca2+]i values obtained in response to each mitogen showed substantial variation between cells. Brief pretreatment of the cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) reduced the [Ca2+]i responses to bombesin, but did not affect the responses to PDGF. Long-term pretreatment of the cells with TPA to down-modulate protein kinase C resulted in substantially prolonged [Ca2+]i responses to bombesin, vasopressin, and PGF2 alpha, but had no such effect on the responses to PDGF. We conclude that differences between the [Ca2+]i responses to bombesin and PDGF, previously reported using cell populations, reflect differences occurring in individual cells, and that the [Ca2+]i responses to bombesin, vasopressin, and PGF2 alpha (but not PDGF) are subject to feedback inhibition via protein kinase C. Images PMID:2519620

  16. Expression profiles of 12 late embryogenesis abundant protein genes from Tamarix hispida in response to abiotic stress.

    PubMed

    Gao, Caiqiu; Liu, Yali; Wang, Chao; Zhang, Kaimin; Wang, Yucheng

    2014-01-01

    Twelve embryogenesis abundant protein (LEA) genes (named ThLEA-1 to -12) were cloned from Tamarix hispida. The expression profiles of these genes in response to NaCl, PEG, and abscisic acid (ABA) in roots, stems, and leaves of T. hispida were assessed using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). These ThLEAs all showed tissue-specific expression patterns in roots, stems, and leaves under normal growth conditions. However, they shared a high similar expression patterns in the roots, stems, and leaves when exposed to NaCl and PEG stress. Furthermore, ThLEA-1, -2, -3, -4, and -11 were induced by NaCl and PEG, but ThLEA-5, -6, -8, -10, and -12 were downregulated by salt and drought stresses. Under ABA treatment, some ThLEA genes, such as ThLEA-1, -2, and -3, were only slightly differentially expressed in roots, stems, and leaves, indicating that they may be involved in the ABA-independent signaling pathway. These findings provide a basis for the elucidation of the function of LEA genes in future work. PMID:25133264

  17. Regulation of death induction and chemosensitizing action of 3-bromopyruvate in myeloid leukemia cells: energy depletion, oxidative stress, and protein kinase activity modulation.

    PubMed

    Calviño, Eva; Estañ, María Cristina; Sánchez-Martín, Carlos; Brea, Rocío; de Blas, Elena; Boyano-Adánez, María del Carmen; Rial, Eduardo; Aller, Patricio

    2014-02-01

    3-Bromopyruvate (3-BrP) is an alkylating, energy-depleting drug that is of interest in antitumor therapies, although the mechanisms underlying its cytotoxicity are ill-defined. We show here that 3-BrP causes concentration-dependent cell death of HL60 and other human myeloid leukemia cells, inducing both apoptosis and necrosis at 20-30 μM and a pure necrotic response at 60 μM. Low concentrations of 3-BrP (10-20 μM) brought about a rapid inhibition of glycolysis, which at higher concentrations was followed by the inhibition of mitochondrial respiration. The combination of these effects causes concentration-dependent ATP depletion, although this cannot explain the lethality at intermediate 3-BrP concentrations (20-30 μM). The oxidative stress caused by exposure to 3-BrP was evident as a moderate overproduction of reactive oxygen species and a concentration-dependent depletion of glutathione, which was an important determinant of 3-BrP toxicity. In addition, 3-BrP caused glutathione-dependent stimulation of p38 mitogen-activated protein kinase (MAPK), mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK), and protein kinase B (Akt)/mammalian target of rapamycin/p70S6K phosphorylation or activation, as well as rapid LKB-1/AMP kinase (AMPK) activation, which was later followed by Akt-mediated inactivation. Experiments with pharmacological inhibitors revealed that p38 MAPK activation enhances 3-BrP toxicity, which is conversely restrained by ERK and Akt activity. Finally, 3-BrP was seen to cooperate with antitumor agents like arsenic trioxide and curcumin in causing cell death, a response apparently mediated by both the generation of oxidative stress induced by 3-BrP and the attenuation of Akt and ERK activation by curcumin. In summary, 3-BrP cytotoxicity is the result of several combined regulatory mechanisms that might represent important targets to improve therapeutic efficacy. PMID:24307199

  18. Integration of multi-omics data of a genome-reduced bacterium: Prevalence of post-transcriptional regulation and its correlation with protein abundances

    PubMed Central

    Chen, Wei-Hua; van Noort, Vera; Lluch-Senar, Maria; Hennrich, Marco L.; H. Wodke, Judith A.; Yus, Eva; Alibés, Andreu; Roma, Guglielmo; Mende, Daniel R.; Pesavento, Christina; Typas, Athanasios; Gavin, Anne-Claude; Serrano, Luis; Bork, Peer

    2016-01-01

    We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated ‘-omics’ data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes. PMID:26773059

  19. Identifying protein aggregation mechanisms and quantifying aggregation rates from combined monomer depletion and continuous scattering.

    PubMed

    Barnett, Gregory V; Drenski, Michael; Razinkov, Vladimir; Reed, Wayne F; Roberts, Christopher J

    2016-10-15

    Parallel temperature initial rates (PTIR) from chromatographic separation of aggregating protein solutions are combined with continuous simultaneous multiple sample light scattering (SMSLS) to make quantitative deductions about protein aggregation kinetics and mechanisms. PTIR determines the rates at which initially monomeric proteins are converted to aggregates over a range of temperatures, under initial-rate conditions. Using SMSLS for the same set of conditions provides time courses of the absolute Rayleigh scattering ratio, IR(t), from which a potentially different measure of aggregation rates can be quantified. The present report compares these measures of aggregation rates across a range of solution conditions that result in different aggregation mechanisms for anti-streptavidin (AS) immunoglobulin gamma-1 (IgG1). The results illustrate how the two methods provide complementary information when deducing aggregation mechanisms, as well as cases where they provide new mechanistic details that were not possible to deduce in previous work. Criteria are presented for when the two techniques are expected to give equivalent results for quantitative rates, the potential limitations when solution non-idealities are large, as well as a comparison of the temperature dependence of AS-IgG1 aggregation rates with published data for other antibodies. PMID:27510552

  20. Interleukin (IL)-1 in rat parturition: IL-1 receptors 1 and 2 and accessory proteins abundance in pregnant rat uterus at term - regulation by progesterone.

    PubMed

    Ishiguro, Tomohito; Takeda, Jun; Fang, Xin; Bronson, Heather; Olson, David M

    2016-07-01

    The role of interleukin-1 (IL-1), a pro-inflammatory cytokine, in parturition is typically noted by changes in its concentrations. Studying the expression of its receptor family, IL-1 receptor (IL-1R) 1, IL-1R2, IL-1R accessory protein (IL-1RAcP), and its predominantly brain isoform, IL-1RAcPb, during late gestation in the uterus in the Long-Evans rat is another. We assessed changes in their mRNA and protein relative abundance in the uterus and compared IL-1RAcP and IL-1RAcPb mRNA abundance in uterus, cervix, ovaries, placenta, and whole blood of Long-Evans rats during late gestation or in RU486 and progesterone-treated dams using quantitative real-time PCR and western immunoblotting. IL-1R1, IL-1RAcP, and IL-1RAcPb mRNA abundance significantly increased in the uterus at delivery whereas IL-1R2 mRNA abundance significantly decreased. IL-1R1 protein increased at term and IL-1R2 protein decreased at term compared to nonpregnant uteri. IL1-RAcPb mRNA abundance was less than IL-1RAcP, but in the lower uterine segment it was the highest of all tissues examined. RU486 stimulated preterm delivery and an increase in IL-1R1 mRNA abundance whereas progesterone administration extended pregnancy and suppressed the increase in IL-1R1. These data suggest that changes in uterine sensitivity to IL-1 occur during late gestation and suggest another level of regulation for the control of delivery. The roles for IL-1RAcP and IL-1RAcPb need to be determined, but may relate to different intracellular signaling pathways. PMID:27440742

  1. Improved tolerance to salt and water stress in Drosophila melanogaster cells conferred by late embryogenesis abundant protein.

    PubMed

    Marunde, Matthew R; Samarajeewa, Dilini A; Anderson, John; Li, Shumin; Hand, Steven C; Menze, Michael A

    2013-04-01

    Mechanisms that govern anhydrobiosis involve the accumulation of highly hydrophilic macromolecules, such as late embryogenesis abundant (LEA) proteins. Group 1 LEA proteins comprised of 181 (AfLEA1.1) and 197 (AfLEA1.3) amino acids were cloned from embryos of Artemia franciscana and expressed in Drosophila melanogaster cells (Kc167). Confocal microscopy revealed a construct composed of green fluorescent protein (GFP) and AfLEA1.3 accumulates in the mitochondria (AfLEA1.3-GFP), while AfLEA1.1-GFP was found in the cytoplasm. In the presence of mixed substrates, oxygen consumption was statistically identical for permeabilized Kc167 control and Kc167-AfLEA1.3 cells. Acute titrations of permeabilized cells with NaCl up to 500 mM led to successive drops in oxygen flux, which were significantly ameliorated by 18% in Kc167-AfLEA1.3 cells compared to Kc167 controls. Mitochondria were isolated from both cell types and resuspended in a sucrose-based buffer solution. The purified mitochondria from Kc167 control cells showed significantly larger reductions in respiratory capacities after one freeze-thaw cycle (-80°C) compared to mitochondria isolated from Kc167-AfLEA1.3 cells. When cultured in the presence of a non-permeant osmolyte (50-200 mM sucrose) cells expressing AfLEA1.3 showed significantly improved viability (10-15%) during this hyperosmotic challenge as compared to Kc167 controls. Furthermore, Kc167-AfLEA1.3 cells survived desiccation by convective air drying in presence of 200 mM extracellular trehalose to lower final moisture contents than did control Kc167 cells (0.36 g H2O/g DW vs.1.02 g H2O/g DW). Thus, AfLEA1.3 exerts a protective influence on mitochondrial function and increases viability of Kc167 cells during water stress. PMID:23376561

  2. (4Fe-4S)-cluster-depleted Azotobacter vinelandii ferredoxin I: a new 3Fe iron-sulfur protein

    SciTech Connect

    Stephens, P.J.; Morgan, T.V.; Devlin, F.; Penner-Hahn, J.E.; Hodgson, K.O.; Scott, R.A.; Stout, C.D.; Burgess, B.K.

    1985-09-01

    Fe(CN)6T oxidation of the aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I, (7Fe)FdI, is a degradative reaction. Destruction of the (4Fe-4S) cluster occurs first, followed by destruction of the (3Fe-3S) cluster. At a Fe(CN)6T /(7Fe)FdI concentration ratio of 20, the product is a mixture of apoprotein and protein containing only a (3Fe-3S) cluster, (3Fe)FdI. This protein mixture, after partial purification, has been characterized by absorption, CD, magnetic CD, and EPR and Fe x-ray absorption spectroscopies. EPR and magnetic CD spectra provide strong evidence that the (3Fe-3S) cluster in (3Fe)FdI is essentially identical in structure to that in (7Fe)FdI. Analysis of the extended x-ray absorption fine structure (EXAFS) of (3Fe)FdI finds Fe scattering at an average Fe...Fe distance of approx. =2.7 A. The structure of the oxidized (3Fe-3S) cluster in solutions of oxidized (3Fe)FdI, and, by extension, of oxidized (7Fe)FdI, is thus different from that obtained by x-ray crystallography on oxidized (7Fe)FdI. Possible interpretations of this result are discussed.

  3. APOL1 kidney disease risk variants cause cytotoxicity by depleting cellular potassium and inducing stress-activated protein kinases

    PubMed Central

    Olabisi, Opeyemi A.; Zhang, Jia-Yue; VerPlank, Lynn; Zahler, Nathan; DiBartolo, Salvatore; Heneghan, John F.; Schlöndorff, Johannes S.; Suh, Jung Hee; Yan, Paul; Alper, Seth L.; Friedman, David J.; Pollak, Martin R.

    2016-01-01

    Two specific genetic variants of the apolipoprotein L1 (APOL1) gene are responsible for the high rate of kidney disease in people of recent African ancestry. Expression in cultured cells of these APOL1 risk variants, commonly referred to as G1 and G2, results in significant cytotoxicity. The underlying mechanism of this cytotoxicity is poorly understood. We hypothesized that this cytotoxicity is mediated by APOL1 risk variant-induced dysregulation of intracellular signaling relevant for cell survival. To test this hypothesis, we conditionally expressed WT human APOL1 (G0), the APOL1 G1 variant, or the APOL1 G2 variant in human embryonic kidney cells (T-REx-293) using a tetracycline-mediated (Tet-On) system. We found that expression of either G1 or G2 APOL1 variants increased apparent cell swelling and cell death compared with G0-expressing cells. These manifestations of cytotoxicity were preceded by G1 or G2 APOL1-induced net efflux of intracellular potassium as measured by X-ray fluorescence, resulting in the activation of stress-activated protein kinases (SAPKs), p38 MAPK, and JNK. Prevention of net K+ efflux inhibited activation of these SAPKs by APOL1 G1 or G2. Furthermore, inhibition of SAPK signaling and inhibition of net K+ efflux abrogated cytotoxicity associated with expression of APOL1 risk variants. These findings in cell culture raise the possibility that nephrotoxicity of APOL1 risk variants may be mediated by APOL1 risk variant-induced net loss of intracellular K+ and subsequent induction of stress-activated protein kinase pathways. PMID:26699492

  4. SOLiD-SAGE of Endophyte-Infected Red Fescue Reveals Numerous Effects on Host Transcriptome and an Abundance of Highly Expressed Fungal Secreted Proteins

    PubMed Central

    Ambrose, Karen V.; Belanger, Faith C.

    2012-01-01

    One of the most important plant-fungal symbiotic relationships is that of cool season grasses with endophytic fungi of the genera Epichloë and Neotyphodium. These associations often confer benefits, such as resistance to herbivores and improved drought tolerance, to the hosts. One benefit that appears to be unique to fine fescue grasses is disease resistance. As a first step towards understanding the basis of the endophyte-mediated disease resistance in Festuca rubra we carried out a SOLiD-SAGE quantitative transcriptome comparison of endophyte-free and Epichloë festucae-infected F. rubra. Over 200 plant genes involved in a wide variety of physiological processes were statistically significantly differentially expressed between the two samples. Many of the endophyte expressed genes were surprisingly abundant, with the most abundant fungal tag representing over 10% of the fungal mapped tags. Many of the abundant fungal tags were for secreted proteins. The second most abundantly expressed fungal gene was for a secreted antifungal protein and is of particular interest regarding the endophyte-mediated disease resistance. Similar genes in Penicillium and Aspergillus spp. have been demonstrated to have antifungal activity. Of the 10 epichloae whole genome sequences available, only one isolate of E. festucae and Neotyphodium gansuense var inebrians have an antifungal protein gene. The uniqueness of this gene in E. festucae from F. rubra, its transcript abundance, and the secreted nature of the protein, all suggest it may be involved in the disease resistance conferred to the host, which is a unique feature of the fine fescue–endophyte symbiosis. PMID:23285269

  5. Selectivity of monolithic supports under overloading conditions and their use for separation of human plasma and isolation of low abundance proteins

    PubMed Central

    Brgles, Marija; Clifton, James; Walsh, Robert; Huang, Feilei; Rucevic, Marijana; Cao, Lulu; Hixson, Douglas; Müller, Egbert

    2011-01-01

    Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These two proteins frequently interfere with detection, determination and purification of low abundance proteins that can be potential biomarkers and biomarker candidates for various diseases. Some low abundance plasma proteins such as clotting factors and inhibitors are also important therapeutic agents. In this paper, the characterization of ion-exchange monolithic supports under overloading conditions was performed by use of sample displacement chromatography (SDC). If these supports were used for separation of human plasma, the composition of bound and eluted proteins in both anion- and cation-exchange mode is dependent on column loading. Under overloading conditions, the weakly bound proteins such as HSA in anion-exchange and IgG in cation-exchange mode are displaced by stronger binding proteins, and this phenomenon was not dependent on column size. Consequently, small monolithic columns with a column volume of 100 and 200 μL are ideal supports for high-throughput screening in order to develop new methods for separation of complex mixtures, and for sample preparation in proteomic technology. PMID:21186030

  6. The relative abundance of a salivary protein, bSP30, is correlated with susceptibility to bloat in cattle herds selected for high or low bloat susceptibility.

    PubMed

    Rajan, G H; Morris, C A; Carruthers, V R; Wilkins, R J; Wheeler, T T

    1996-12-01

    Pasture bloat is a serious economic and animal welfare problem in cattle grazed on legumes in New Zealand. Analysis of salivary proteins from dairy cattle in herds bred for either low or high susceptibility to bloat has resulted in the identification of a 30 kilodalton protein, which we term bSP30, whose relative abundance is negatively correlated with bloat score (r = -0.40 +/- 0.12). From 74 animals sampled, relative abundance of bSP30 was 66 +/- 15% higher in the low-susceptibility herd than in the high-susceptibility herd. Relative abundance of bSP30 also varied significantly within individuals, according to feeding or time of day, and from day to day. A sequence homology search of 38 amino acids derived from three tryptic fragments of the protein suggests that the amino acid sequence of bSP30 has not been described previously. Amino acid analysis indicates that bSP30 is not a member of the proline-rich family of salivary proteins. The function of bSP30 is unknown but it is conceivable that it plays a role in the aetiology of bloat. PMID:9022155

  7. Correlation of mRNA expression and protein abundance affected by multiple sequence features related to translational efficiency in Desulfovibrio vulgaris: A quantitative analysis

    SciTech Connect

    Nie, Lei; Wu, Gang; Zhang, Weiwen

    2006-12-01

    The modest correlation between mRNA expression and protein abundance in large scale datasets is explained in part by experimental challenges, such as technological limitations, and in part by fundamental biological factors in the transcription and translation processes. Among various factors affecting the mRNA-protein correlation, the roles of biological factors related to translation are poorly understood. In this study, using experimental mRNA expression and protein abundance data collected from Desulfovibrio vulgaris by DNA microarray and LC-MS/MS proteomic analysis, we quantitatively examined the effects of several translational-efficiency-related sequence features on mRNA-protein correlation. Three classes of sequence features were investigated according to different translational stages: (1) initiation: Shine-Dalgarno sequences, start codon identity and start codon context; (2) elongation: codon usage and amino acid usage; and (3) termination: stop codon identity and stop codon context. Surprisingly, although it is widely accepted that translation initiation is a rate-limiting step for translation, our results showed that the mRNA-protein correlation was affected the most by the features at elongation stages, codon usage and amino acid composition (7.4-12.6% and 5.3-9.3% of the total variation of mRNA-protein correlation, respectively), followed by stop codon context and the Shine-Dalgarno sequence (2.5-4.2% and 2.3%, respectively). Taken together, all sequence features contributed to 18.4-21.8% of the total variation of mRNA-protein correlation. As the first comprehensive quantitative analysis of the mRNA-protein correlation in bacterial D. vulgaris, our results suggest that the traditional view of the relative importance of various sequence features in prokaryotic protein translation might be questionable.

  8. Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize.

    PubMed

    Wu, Xiaolin; Gong, Fangping; Yang, Le; Hu, Xiuli; Tai, Fuju; Wang, Wei

    2014-01-01

    ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5), deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs), late embryogenesis abundant (LEA) proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation. PMID:25653661

  9. Delayed Cell Cycle Progression in Selenoprotein W-depleted Cells Is Regulated by a Mitogen-activated Protein Kinase Kinase 4-p38/c-Jun NH2-terminal Kinase-p53 Pathway*

    PubMed Central

    Hawkes, Wayne Chris; Alkan, Zeynep

    2012-01-01

    Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a transient p53- and p21Cip1-dependent G1-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser-33 in p53, which is associated with decreased p53 ubiquitination and stabilization of p53. We report here that delayed cell cycle progression, Ser-33 phosphorylation, and p53 nuclear accumulation from SEPW1 depletion require mitogen-activated protein kinase kinase 4 (MKK4). Silencing MKK4 rescued G1 arrest, Ser-33 phosphorylation, and nuclear accumulation of p53 induced by SEPW1 depletion, but silencing MKK3, MKK6, or MKK7 did not. SEPW1 silencing did not change the phosphorylation state of MKK4 but increased total MKK4 protein. Silencing p38γ, p38δ, or JNK2 partially rescued G1 arrest from SEPW1 silencing, suggesting they signal downstream from MKK4. These results imply that SEPW1 silencing increases MKK4, which activates p38γ, p38δ, and JNK2 to phosphorylate p53 on Ser-33 and cause a transient G1 arrest. PMID:22730327

  10. Preovulatory Aging In Vivo and In Vitro Affects Maturation Rates, Abundance of Selected Proteins, Histone Methylation Pattern and Spindle Integrity in Murine Oocytes.

    PubMed

    Demond, Hannah; Trapphoff, Tom; Dankert, Deborah; Heiligentag, Martyna; Grümmer, Ruth; Horsthemke, Bernhard; Eichenlaub-Ritter, Ursula

    2016-01-01

    Delayed ovulation and delayed fertilization can lead to reduced developmental competence of the oocyte. In contrast to the consequences of postovulatory aging of the oocyte, hardly anything is known about the molecular processes occurring during oocyte maturation if ovulation is delayed (preovulatory aging). We investigated several aspects of oocyte maturation in two models of preovulatory aging: an in vitro follicle culture and an in vivo mouse model in which ovulation was postponed using the GnRH antagonist cetrorelix. Both models showed significantly reduced oocyte maturation rates after aging. Furthermore, in vitro preovulatory aging deregulated the protein abundance of the maternal effect genes Smarca4 and Nlrp5, decreased the levels of histone H3K9 trimethylation and caused major deterioration of chromosome alignment and spindle conformation. Protein abundance of YBX2, an important regulator of mRNA stability, storage and recruitment in the oocyte, was not affected by in vitro aging. In contrast, in vivo preovulatory aging led to reduction in Ybx2 transcript and YBX2 protein abundance. Taken together, preovulatory aging seems to affect various processes in the oocyte, which could explain the low maturation rates and the previously described failures in fertilization and embryonic development. PMID:27611906

  11. Depletion of the cellular levels of Bag-1 proteins attenuates phorbol ester-induced downregulation of I{kappa}B{alpha} and nuclear accumulation of NF-{kappa}B

    SciTech Connect

    Maier, Jana V.; Volz, Yvonne; Berger, Caroline; Schneider, Sandra; Cato, Andrew C.B.

    2010-10-22

    Research highlights: {yields}Bag-1 depletion only marginally affects the action of the glucocorticoid receptor but strongly regulates the activity of NF-{kappa}B. {yields}Bag-1 depletion attenuates phosphorylation and degradation of I{kappa}B{alpha} and nuclear accumulation of NF-{kappa}B p65 and p50. {yields}Bag-1 interacts with I{kappa}B{alpha} and partially restores I{kappa}B{alpha} and NF-{kappa}B activation in Bag-1 depleted cells. -- Abstract: Bag-1 consists in humans of four isoforms generated from the same RNA by alternative translation. Overexpression of single Bag-1 isoforms has identified Bag-1 as a negative regulator of action of many proteins including the glucocorticoid receptor (GR). Here we have analysed the ability of Bag-1 to regulate the transrepression function of the GR. Silencing Bag-1 expression only marginally affects the transrepression action of the GR but decreased the action of the transcription factor NF-{kappa}B. Furthermore phosphorylation and degradation of the inhibitor protein I{kappa}B{alpha} and nuclear accumulation of p65 and p50 NF-{kappa}B proteins in response to phorbol ester was attenuated following Bag-1 depletion in HeLa cells. Reconstitution of Bag-1 in depleted cells partially restored I{kappa}B{alpha} and NF-{kappa}B activation. Knock-down of Bag-1 expression also did not significantly alter GR-mediated transactivation but affected the basal transcription of some of the target genes. Thus Bag-1 proteins function as regulators of the action of selective transcription factors.

  12. The boron abundance of Procyon

    NASA Technical Reports Server (NTRS)

    Lemke, Michael; Lambert, David L.; Edvardsson, Bengt

    1993-01-01

    The B I 2496.8 A resonance line and HST/GHRS echelle spectra are used with model atmospheres and synthetic spectra to derive the B abundance of the F dwarfs Procyon (Alpha Canis Minoris), Theta Ursae Majoris, and Iota Pegasi. The B abundance of Theta UMa and Iota Peg is similar to that derived by Boesgaard and Heacox (1978) from the B II resonance line in spectra of A- and B-type stars. These two dwarfs show normal abundances of Li, Be, and B. Procyon, which is highly depleted in Li and Be, is depleted in B by a factor of at least 3. Comparison of the spectra of Procyon and the halo dwarf HD 140283 shows that the B abundance assigned by Duncan et al. (1992) to three halo dwarfs is not greatly overestimated as a result of contamination of the B I line by an unidentified line.

  13. Heterologous Expression of MeLEA3: A 10 kDa Late Embryogenesis Abundant Protein of Cassava, Confers Tolerance to Abiotic Stress in Escherichia coli with Recombinant Protein Showing In Vitro Chaperone Activity.

    PubMed

    Barros, Nicolle L F; da Silva, Diehgo T; Marques, Deyvid N; de Brito, Fabiano M; dos Reis, Savio P; de Souza, Claudia R B

    2015-01-01

    Late embryogenesis abundant (LEA) proteins are small molecular weight proteins involved in acquisition of tolerance to drought, salinity, high temperature, cold, and freezing stress in many plants. Previous studies revealed a cDNA sequence coding for a 10 kDa atypical LEA protein, named MeLEA3, predicted to be located into mitochondria with potential role in salt stress response of cassava (Manihot esculenta Crantz). Here we aimed to produce the recombinant MeLEA3 protein by heterologous expression in Escherichia coli and evaluate the tolerance of bacteria expressing this protein under abiotic stress. Our result revealed that the recombinant MeLEA3 protein conferred a protective function against heat and salt stress in bacterial cells. Also, the recombinant MeLEA3 protein showed in vitro chaperone activity by protection of NdeI restriction enzyme activity under heat stress. PMID:25990084

  14. A combined blood based gene expression and plasma protein abundance signature for diagnosis of epithelial ovarian cancer - a study of the OVCAD consortium

    PubMed Central

    2013-01-01

    Background The immune system is a key player in fighting cancer. Thus, we sought to identify a molecular ‘immune response signature’ indicating the presence of epithelial ovarian cancer (EOC) and to combine this with a serum protein biomarker panel to increase the specificity and sensitivity for earlier detection of EOC. Methods Comparing the expression of 32,000 genes in a leukocytes fraction from 44 EOC patients and 19 controls, three uncorrelated shrunken centroid models were selected, comprised of 7, 14, and 6 genes. A second selection step using RT-qPCR data and significance analysis of microarrays yielded 13 genes (AP2A1, B4GALT1, C1orf63, CCR2, CFP, DIS3, NEAT1, NOXA1, OSM, PAPOLG, PRIC285, ZNF419, and BC037918) which were finally used in 343 samples (90 healthy, six cystadenoma, eight low malignant potential tumor, 19 FIGO I/II, and 220 FIGO III/IV EOC patients). Using new 65 controls and 224 EOC patients (thereof 14 FIGO I/II) the abundances of six plasma proteins (MIF, prolactin, CA125, leptin, osteopondin, and IGF2) was determined and used in combination with the expression values from the 13 genes for diagnosis of EOC. Results Combined diagnostic models using either each five gene expression and plasma protein abundance values or 13 gene expression and six plasma protein abundance values can discriminate controls from patients with EOC with Receiver Operator Characteristics Area Under the Curve values of 0.998 and bootstrap .632+ validated classification errors of 3.1% and 2.8%, respectively. The sensitivities were 97.8% and 95.6%, respectively, at a set specificity of 99.6%. Conclusions The combination of gene expression and plasma protein based blood derived biomarkers in one diagnostic model increases the sensitivity and the specificity significantly. Such a diagnostic test may allow earlier diagnosis of epithelial ovarian cancer. PMID:23551967

  15. The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit.

    PubMed

    Cuevas-Velazquez, Cesar L; Saab-Rincón, Gloria; Reyes, José Luis; Covarrubias, Alejandra A

    2016-05-13

    Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics. PMID:27006402

  16. Annexin A5 is the Most Abundant Membrane-Associated Protein in Stereocilia but is Dispensable for Hair-Bundle Development and Function.

    PubMed

    Krey, Jocelyn F; Drummond, Meghan; Foster, Sarah; Porsov, Edward; Vijayakumar, Sarath; Choi, Dongseok; Friderici, Karen; Jones, Sherri M; Nuttall, Alfred L; Barr-Gillespie, Peter G

    2016-01-01

    The phospholipid- and Ca(2+)-binding protein annexin A5 (ANXA5) is the most abundant membrane-associated protein of ~P23 mouse vestibular hair bundles, the inner ear's sensory organelle. Using quantitative mass spectrometry, we estimated that ANXA5 accounts for ~15,000 copies per stereocilium, or ~2% of the total protein there. Although seven other annexin genes are expressed in mouse utricles, mass spectrometry showed that none were present at levels near ANXA5 in bundles and none were upregulated in stereocilia of Anxa5(-/-) mice. Annexins have been proposed to mediate Ca(2+)-dependent repair of membrane lesions, which could be part of the repair mechanism in hair cells after noise damage. Nevertheless, mature Anxa5(-/-) mice not only have normal hearing and balance function, but following noise exposure, they are identical to wild-type mice in their temporary or permanent changes in hearing sensitivity. We suggest that despite the unusually high levels of ANXA5 in bundles, it does not play a role in the bundle's key function, mechanotransduction, at least until after two months of age in the cochlea and six months of age in the vestibular system. These results reinforce the lack of correlation between abundance of a protein in a specific compartment or cellular structure and its functional significance. PMID:27251877

  17. Correlated Changes in the Activity, Amount of Protein, and Abundance of Transcript of NADPH:Protochlorophyllide Oxidoreductase and Chlorophyll Accumulation during Greening of Cucumber Cotyledons.

    PubMed Central

    Yoshida, K.; Chen, R. M.; Tanaka, A.; Teramoto, H.; Tanaka, R.; Timko, M. P.; Tsuji, H.

    1995-01-01

    Changes in the activity and abundance of NADPH:protochlorophyllide oxidoreductase (NPR) and the abundance of mRNA encoding it were examined during the greening of 5-d-old etiolated cucumber cotyledons under continuous illumination. To measure NPR activity in the extracts from fully greened tissues, we have developed an improved method of assay. Upon exposure of etiolated cotyledons to light, NPR activity decreased rapidly within the first 2 h of exposure. Thereafter, enzymatic activity increased transiently, reaching a submaximum level at 12 h, and decreased slowly. The level of immunodetectable NPR protein followed the same pattern of changes during 96 h of greening as observed for NPR activity. The NPR mRNA in etiolated cotyledons disappeared quickly in the 1st h of irradiation. However, the level of mRNA increased thereafter to reach 3-fold or more of the dark level at 12 h and then decreased. The changes in the activity, protein level, and mRNA level after the first rapid decreases corresponded chronologically and nearly paralleled the increase in the rate of chlorophyll accumulation. These findings suggest that the greening of cucumber cotyledons is regulated basically by the level of NPR protein without activation or repression of enzymatic activity and that NPR mRNA increased by light maintains the level of enzyme protein necessary for greening. PMID:12228591

  18. Annexin A5 is the Most Abundant Membrane-Associated Protein in Stereocilia but is Dispensable for Hair-Bundle Development and Function

    PubMed Central

    Krey, Jocelyn F.; Drummond, Meghan; Foster, Sarah; Porsov, Edward; Vijayakumar, Sarath; Choi, Dongseok; Friderici, Karen; Jones, Sherri M.; Nuttall, Alfred L.; Barr-Gillespie, Peter G.

    2016-01-01

    The phospholipid- and Ca2+-binding protein annexin A5 (ANXA5) is the most abundant membrane-associated protein of ~P23 mouse vestibular hair bundles, the inner ear’s sensory organelle. Using quantitative mass spectrometry, we estimated that ANXA5 accounts for ~15,000 copies per stereocilium, or ~2% of the total protein there. Although seven other annexin genes are expressed in mouse utricles, mass spectrometry showed that none were present at levels near ANXA5 in bundles and none were upregulated in stereocilia of Anxa5−/− mice. Annexins have been proposed to mediate Ca2+-dependent repair of membrane lesions, which could be part of the repair mechanism in hair cells after noise damage. Nevertheless, mature Anxa5−/− mice not only have normal hearing and balance function, but following noise exposure, they are identical to wild-type mice in their temporary or permanent changes in hearing sensitivity. We suggest that despite the unusually high levels of ANXA5 in bundles, it does not play a role in the bundle’s key function, mechanotransduction, at least until after two months of age in the cochlea and six months of age in the vestibular system. These results reinforce the lack of correlation between abundance of a protein in a specific compartment or cellular structure and its functional significance. PMID:27251877

  19. Effect of ultrasonic stimulation on mRNA abundance of uncoupling protein (UCP) 2 and UCP 3 in gastrocnemius muscle of rats.

    PubMed

    Kogure, Akinori; Yoshida, Toshihide; Takakura, Yasuto; Umekawa, Tsunekazu; Hioki, Chizuko; Yoshioka, Keiji; Yoshimoto, Kanji; Yoshikawa, Toshikazu

    2005-01-01

    1. The hypothesis that ultrasonic stimulation upregulates uncoupling protein (UCP) 2 and UCP3 in gastrocnemius muscle by a different mechanism of exercise was investigated in Wister rats. 2. The ultrasnonic-stimulated group was given ultrasonic stimulation to the leg (1 MHz frequency, 1 W/cm2 intensity, 10 min continuously). 3. The exercise group was given exercise training by swimming for 10 min in plastic barrels filled with warm water. 4. After 3 h, rats were killed and the gastrocnemius muscle was removed rapidly, weighed and frozen in liquid nitrogen for real-time quantitative reverse transcription-polymerase chain reaction analysis. 5. In gastrocnenius muscles of ultrasonic-stimulated rats, UCP3 mRNA abundance was significantly increased 3.6-fold and UCP2 mRNA abundance was significantly increased 2.2-fold compared with control rats. 6. In gastrocnenius muscles of exercised rats, UCP3 mRNA abundance was significantly increased 3.5-fold compared with control rats, but no change in UCP2 mRNA abundance was observed. 7. Plasma free fatty acid (FFA) levels were also significantly increased in the ultrasonic stimulation group, as well as the exercise group, compared with the control group. 8. These findings show that ultrasonic stimulation lipolyses subcutaneous fat into FFA and glycerol and upregulates UCP2 and UCP3 mRNA by a mechanism different to that of exercise. PMID:15730441

  20. Depletion of human serum albumin in embryo culture media for in vitro fertilization using monolithic columns with immobilized antibodies.

    PubMed

    Tarasova, Irina A; Lobas, Anna A; Černigoj, Urh; Solovyeva, Elizaveta M; Mahlberg, Barbara; Ivanov, Mark V; Panić-Janković, Tanja; Nagy, Zoltan; Pridatchenko, Marina L; Pungor, Andras; Nemec, Blaž; Vidic, Urška; Gašperšič, Jernej; Krajnc, Nika Lendero; Vidič, Jana; Gorshkov, Mikhail V; Mitulović, Goran

    2016-09-01

    Affinity depletion of abundant proteins such as HSA is an important stage in routine sample preparation prior to MS/MS analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low-abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low-abundance proteins becomes a challenge unless the sample is depleted from high-concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study, we performed evaluation of a novel immunoaffinity-based Convective Interaction Media analytical columns (CIMac) depletion column with specificity to HSA (CIMac-αHSA). Because of the convective flow-through channels, the polymethacrylate CIMac monoliths afford flow rate independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom-up proteomic approach followed by label-free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and cleavage/blastocyst in vitro fertilization culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100 ± 25% in albumin-enriched fractions relative to the nondepleted samples for both CIMac-αHSA column and Seppro kit. In the albumin-free fractions concentrated 5.5-fold by volume, serum albumin was identified at the levels of 5-30% and 20-30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively. PMID:27122488

  1. Robust Abundance Estimation in Animal Abundance Surveys with Imperfect Detection

    EPA Science Inventory

    Surveys of animal abundance are central to the conservation and management of living natural resources. However, detection uncertainty complicates the sampling process of many species. One sampling method employed to deal with this problem is depletion (or removal) surveys in whi...

  2. A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion.

    PubMed Central

    Pan, David A; Hardie, D Grahame

    2002-01-01

    We have identified single genes encoding homologues of the alpha, beta and gamma subunits of mammalian AMP-activated protein kinase (AMPK) in the genome of Drosophila melanogaster. Kinase activity could be detected in extracts of a Drosophila cell line using the SAMS peptide, which is a relatively specific substrate for the AMPK/SNF1 kinases in mammals and yeast. Expression of double stranded (ds) RNAs targeted at any of the putative alpha, beta or gamma subunits ablated this activity, and abolished expression of the alpha subunit. The Drosophila kinase (DmAMPK) was activated by AMP in cell-free assays (albeit to a smaller extent than mammalian AMPK), and by stresses that deplete ATP (oligomycin and hypoxia), as well as by carbohydrate deprivation, in intact cells. Using a phosphospecific antibody, we showed that activation was associated with phosphorylation of a threonine residue (Thr-184) within the 'activation loop' of the alpha subunit. We also identified a homologue of acetyl-CoA carboxylase (DmACC) in Drosophila and, using a phosphospecific antibody, showed that the site corresponding to the regulatory AMPK site on the mammalian enzyme became phosphorylated in response to oligomycin or hypoxia. By immunofluorescence microscopy of oligomycin-treated Dmel2 cells using the phosphospecific antibody, the phosphorylated DmAMPK alpha subunit was mainly detected in the nucleus. Our results show that the AMPK system is highly conserved between insects and mammals. Drosophila cells now represent an attractive system to study this pathway, because of the small, well-defined genome and the ability to ablate expression of specific gene products using interfering dsRNAs. PMID:12093363

  3. The pro-oxidant buthionine sulfoximine (BSO) reduces tumor growth of implanted Lewis lung carcinoma in mice associated with increased protein carbonyl, tubulin abundance, and aminopeptidase activity.

    PubMed

    Rodríguez-Gómez, Isabel; Carmona-Cortés, Javier; Wangensteen, Rosemary; Vargas-Tendero, Pablo; Banegas, Inmaculada; Quesada, Andrés; García-Lora, Angel M; Vargas, Félix

    2014-08-01

    This study evaluated the effects of the pro-oxidant buthionine sulfoximine (BSO) and of the interaction between BSO and TETRAC, an antagonist of αvß3 integrin, on tumor development and aminopeptidase (AP) activity in a murine model of implanted Lewis's carcinoma. Male CBA-C57 mice were untreated (controls) or treated with BSO (222 mg/100 mL in drinking water), TETRAC (10 mg/kg/day, i.p.), or BSO + TETRAC. BSO for 28 days and TETRAC were given for the last 20 days. Mice were subcutaneously inoculated with 1 × 10(6) Lewis carcinoma 3LL cells into the dorsum. Study variables were tumor weight (TW); Hb, as index of tumor-mediated angiogenesis; vascular endothelial growth factor (VEGF) protein abundance; protein carbonyl content; α-tubulin abundance; and GluAp, AlaAp, and AspAp activities. BSO produced a major decrease in TW (203 ± 18 mg) with respect to controls (365 ± 26) and a reduction in Hb content. The TETRAC group also showed marked reductions in TW (129 ± 15) and Hb concentration associated with a reduced VEGF content. The BSO + TETRAC group showed a major TW reduction (125 ± 13); although, the difference with the TETRAC group was not significant. BSO treatment increased protein carbonyl and tubulin abundance in comparison to controls. The activity of all APs was increased in the three experimental groups and was strongly and negatively correlated with TW. In conclusion, administration of BSO reduced the TW, which inversely correlated with protein carbonyl content, suggesting a loss of microtubule polymerization. The finding of a negative correlation between TW and AP activity opens up new perspectives for the study of APs as tumor growth modulators. PMID:24816945

  4. Brain α2-adrenoceptors in monoamine-depleted rats: increased receptor density, G coupling proteins, receptor turnover and receptor mRNA

    PubMed Central

    Ribas, Catalina; Miralles, Antonio; Busquets, Xavier; García-Sevilla, Jesús A

    2001-01-01

    This study was designed to assess the molecular and cellular events involved in the up-regulation (and receptor supersensitivity) of brain α2-adrenoceptors as a result of chronic depletion of noradrenaline (and other monoamines) by reserpine. Chronic reserpine (0.25 mg kg−1 s.c., every 48 h for 6 – 14 days) increased significantly the density (Bmax values) of cortical α2-adrenoceptor agonist sites (34 – 48% for [3H]-UK14304, 22 – 32% for [3H]-clonidine) but not that of antagonist sites (11 – 18% for [3H]-RX821002). Competition of [3H]-RX821002 binding by (−)-adrenaline further indicated that chronic reserpine was associated with up-regulation of the high-affinity state of α2-adrenoceptors. In cortical membranes of reserpine-treated rats (0.25 mg kg−1 s.c., every 48 h for 20 days), the immunoreactivities of various G proteins (Gαi1/2, Gαi3, Gαo and Gαs) were increased (25 – 34%). Because the high-affinity conformation of the α2-adrenoceptor is most probably related to the complex with Gαi2 proteins, these results suggested an increase in signal transduction through α2-adrenoceptors (and other monoamine receptors) induced by chronic reserpine. After α2-adrenoceptor alkylation, the analysis of receptor recovery (Bmax for [3H]-UK14304) indicated that the increased density of cortical α2-adrenoceptors in reserpine-treated rats was probably due to a higher appearance rate constant of the receptor (Δr=57%) and not to a decreased disappearance rate constant (Δk=7%). Northern- and dot-blot analyses of RNA extracted from the cerebral cortex of saline- and reserpine-treated rats (0.25 mg kg−1, s.c., every 48 h for 20 days) revealed that reserpine markedly increased the expression of α2a-adrenoceptor mRNA in the brain (125%). This transcriptional activation of the receptor gene expression appears to be the cellular mechanism by which reserpine induces up-regulation in the density of brain α2-adrenoceptors

  5. Does an infrasonic acoustic shock wave resonance of the manganese 3+ loaded/copper depleted prion protein initiate the pathogenesis of TSE?

    PubMed

    Purdey, Mark

    2003-06-01

    Intensive exposures to natural and artificial sources of infrasonic acoustic shock (tectonic disturbances, supersonic aeroplanes, etc.) have been observed in ecosystems supporting mammalian populations that are blighted by clusters of traditional and new variant strains of transmissible spongiform encephalopathy (TSE). But TSEs will only emerge in those 'infrasound-rich' environments which are simultaneously influenced by eco-factors that induce a high manganese (Mn)/low copper (Cu)-zinc (Zn) ratio in brains of local mammalian populations. Since cellular prion protein (PrPc) is a cupro-protein expressed throughout the circadian mediated pathways of the body, it is proposed that PrP's Cu component performs a role in the conduction and distribution of endogenous electromagnetic energy; energy that has been transduced from incoming ultraviolet, acoustic, geomagnetic radiations. TSE pathogenesis is initiated once Mn substitutes at the vacant Cu domain on PrPc and forms a nonpathogenic, protease resistant, 'sleeping' prion. A second stage of pathogenesis comes into play once a low frequency wave of infrasonic shock metamorphoses the piezoelectric atomic structure of the Mn 3+ component of the prion, thereby 'priming' the sleeping prion into its fully fledged, pathogenic TSE isoform - where the paramagnetic status of the Mn 3+ atom is transformed into a stable ferrimagnetic lattice work, due to the strong electron-phonon coupling resulting from the dynamic 'Jahn-Teller' type distortions of the oxygen octahedra specific to the trivalent Mn species. The so called 'infectivity' of the prion is a misnomer and should be correctly defined as the contagious field inducing capacity of the ferrimagnetic Mn 3+ component of the prion; which remains pathogenic at all temperatures below the 'curie point'. A progressive domino-like 'metal to ligand to metal' ferrimagnetic corruption of the conduits of electromagnetic superexchange is initiated. The TSE diseased brain can be likened to

  6. DEPLETED URANIUM TECHNICAL WORK

    EPA Science Inventory

    The Depleted Uranium Technical Work is designed to convey available information and knowledge about depleted uranium to EPA Remedial Project Managers, On-Scene Coordinators, contractors, and other Agency managers involved with the remediation of sites contaminated with this mater...

  7. Bee bread increases honeybee haemolymph protein and promote better survival despite of causing higher Nosema ceranae abundance in honeybees.

    PubMed

    Basualdo, Marina; Barragán, Sergio; Antúnez, Karina

    2014-08-01

    Adequate protein nutrition supports healthy honeybees and reduces the susceptibility to disease. However little is known concerning the effect of the diet on Nosema ceranae development, an obligate intracellular parasite that disturbs the protein metabolism of honeybees (Apis mellifera). Here we tested the effect of natural (bee bread) and non-natural protein diets (substitute) on haemolymph proteins titers of honeybee and N. ceranae spore production. The natural diet induced higher levels of protein and parasite development, but the survival of bees was also higher than with non-natural diets. The data showed that the administration of an artificially high nutritious diet in terms of crude protein content is not sufficient to promote healthy bees; rather the protein ingested should be efficiently assimilated. The overall results support the idea that the physiological condition of the bees is linked to protein levels in the haemolymph, which affects the tolerance to parasite; consequently the negative impact of the parasite on host fitness is not associated only with the level of infection. PMID:24992539

  8. KvLEA, a New Isolated Late Embryogenesis Abundant Protein Gene from Kosteletzkya virginica Responding to Multiabiotic Stresses

    PubMed Central

    Tang, Xiaoli; Wang, Hongyan; Chu, Liye; Shao, Hongbo

    2016-01-01

    The LEA proteins are a kind of hydrophilic proteins, playing main functions in desiccation tolerance. However, their importance as a kind of stress proteins in abiotic stress is being clarified little by little. In this study we isolated, cloned, and identified the first KvLEA gene in Kosteletzkya virginica. Bioinformatic analysis showed that the protein encoded by this gene had common properties of LEA proteins and the multiple sequences alignment and phylogenetic analysis further showed that this protein had high homology with two Arabidopsis LEA proteins. Gene expression analysis revealed that this gene had a higher expression in root and it was induced obviously by salt stress. Moreover, the transcripts of KvLEA were also induced by other abiotic stresses including drought, high temperature, chilling, and ABA treatment. Among these abiotic stresses, ABA treatment brought about the biggest changes to this gene. Collectively, our research discovered a novel LEA gene and uncovered its involvement in multiabiotic stresses in K. virginica. This research not only enriched studies on LEA gene in plant but also would accelerate more studies on K. virginica in the future. PMID:27123459

  9. KvLEA, a New Isolated Late Embryogenesis Abundant Protein Gene from Kosteletzkya virginica Responding to Multiabiotic Stresses.

    PubMed

    Tang, Xiaoli; Wang, Hongyan; Chu, Liye; Shao, Hongbo

    2016-01-01

    The LEA proteins are a kind of hydrophilic proteins, playing main functions in desiccation tolerance. However, their importance as a kind of stress proteins in abiotic stress is being clarified little by little. In this study we isolated, cloned, and identified the first KvLEA gene in Kosteletzkya virginica. Bioinformatic analysis showed that the protein encoded by this gene had common properties of LEA proteins and the multiple sequences alignment and phylogenetic analysis further showed that this protein had high homology with two Arabidopsis LEA proteins. Gene expression analysis revealed that this gene had a higher expression in root and it was induced obviously by salt stress. Moreover, the transcripts of KvLEA were also induced by other abiotic stresses including drought, high temperature, chilling, and ABA treatment. Among these abiotic stresses, ABA treatment brought about the biggest changes to this gene. Collectively, our research discovered a novel LEA gene and uncovered its involvement in multiabiotic stresses in K. virginica. This research not only enriched studies on LEA gene in plant but also would accelerate more studies on K. virginica in the future. PMID:27123459

  10. PHYSCOMITRELLA PATENS ARABINOGALACTAN PROTEINS CONTAIN ABUNDANT TERMINAL 3-O-METHYL-L-RHAMMOSYL RESIDUES NOT FOUND IN ANGIOSPERMS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A biochemical investigation of arabinogalactan proteins (AGPs) in Physcomitrella patens was undertaken with particular emphasis on the glycan chains. Following homogenization and differential centrifugation of moss gametophytes, AGPs were obtained by Arrive phenylglycoside-induced precipitation from...

  11. Correlation between mRNA and protein abundance in Desulfovibrio vulgaris: A multiple regression to identify sources of variations

    SciTech Connect

    Nie, Lei; Wu, G; Zhang, Weiwen

    2006-01-13

    Using whole-genome microarray and LC-MC/MS proteomic data collected from Desulfovibrio vulgaris grown under three different conditions, we systematically investigate the relationship between mRNA and protein abundunce by a multiple regression approach.

  12. Core-shell molecularly imprinted polymer nanoparticles with assistant recognition polymer chains for effective recognition and enrichment of natural low-abundance protein.

    PubMed

    Liu, Dejing; Yang, Qian; Jin, Susu; Song, Yingying; Gao, Junfei; Wang, Ying; Mi, Huaifeng

    2014-02-01

    Core-shell molecular imprinting of nanomaterials overcomes difficulties with template transfer and achieves higher binding capacities for macromolecular imprinting, which are more important to the imprinting of natural low-abundance proteins from cell extracts. In the present study, a novel strategy of preparing core-shell nanostructured molecularly imprinted polymers (MIPs) was developed that combined the core-shell approach with assistant recognition polymer chains (ARPCs). Vinyl-modified silica nanoparticles were used as support and ARPCs were used as additional functional monomers. Immunoglobulin heavy chain binding protein (BiP) from the endoplasmic reticulum (ER) was chosen as the model protein. The cloned template protein BiP was selectively assembled with ARPCs from their library, which contained numerous limited-length polymer chains with randomly distributed recognition and immobilization sites. The resulting complex was copolymerized onto the surface of vinyl-modified silica nanoparticles under low concentrations of the monomers. After template removal, core-shell-structured nanoparticles with a thin imprinted polymer layer were produced. The particles demonstrated considerably high adsorption capacity, fast adsorption kinetics and selective binding affinities toward the template BiP. Furthermore, the synthesized MIP nanoparticles successfully isolated cloned protein BiP from protein mixtures and highly enriched BiP from an ER extract containing thousands of kinds of proteins. The enrichment reached 115-fold and the binding capacity was 5.4 μg g(-1), which were higher than those achieved by using traditional MIP microspheres. The advantageous properties of MIP nanoparticles hold promise for further practical applications in biology, such as protein analysis and purification. PMID:24140608

  13. The highly abundant chlorophyll-protein complex of iron-deficient Synechococcus sp. PCC7942 (CP43') is encoded by the isiA gene.

    PubMed Central

    Burnap, R L; Troyan, T; Sherman, L A

    1993-01-01

    A chlorophyll (Chl)-protein complex designated CPVI-4 becomes the major pigment-protein complex in Synechococcus sp. PCC7942 cells grown under conditions of iron limitation. Work by Laudenbach et al. (J Bacteriol [1988] 170: 5018-5026) has identified an iron-repressible operon, designated isiAB, containing the flavodoxin gene and a gene predicted to encode a Chl-binding protein resembling CP43 of photosystem II. To test the hypothesis that the CP43-like protein is a component of the CPVI-4 complex, we have inactivated the isiAB operon in Synechococcus sp. PCC7942 using directed insertional mutagenesis. Mutant cells grown under conditions of iron limitation exhibit pronounced changes in their spectroscopic and photosynthetic properties relative to similarly grown wild-type cells. Notably, the strong 77 K fluorescence emission at 685 nm, which dominates the spectrum of iron-deficient wild-type cells, is dramatically reduced in the mutant. The loss of this emission appears to unmask the otherwise obscured photosystem II emissions at 685 and 695 nm. Most importantly, mildly denaturing gel electrophoresis shows that mutant cells no longer express the CPVI-4 complex, indicating that the isiA gene encodes a component of this abundant Chl-protein complex. PMID:8022940

  14. Identification of Putative Genes Involved in Bisphenol A Degradation Using Differential Protein Abundance Analysis of Sphingobium sp. BiD32.

    PubMed

    Zhou, Nicolette A; Kjeldal, Henrik; Gough, Heidi L; Nielsen, Jeppe L

    2015-10-20

    Discharge of the endocrine disrupting compound bisphenol A (BPA) with wastewater treatment plant (WWTP) effluents into surface waters results in deleterious effects on aquatic life. Sphingobium sp. BiD32 was previously isolated from activated sludge based on its ability to degrade BPA. This study investigated BPA metabolism by Sphingobium sp. BiD32 using label-free quantitative proteomics. The genome of Sphingobium sp. BiD32 was sequenced to provide a species-specific platform for optimal protein identification. The bacterial proteomes of Sphingobium sp. BiD32 in the presence and absence of BPA were identified and quantified. A total of 2155 proteins were identified; 1174 of these proteins were quantified, and 184 of these proteins had a statistically significant change in abundance in response to the presence/absence of BPA (p ≤ 0.05). Proteins encoded by genes previously identified to be responsible for protocatechuate degradation were upregulated in the presence of BPA. The analysis of the metabolites from BPA degradation by Sphingobium sp. BiD32 detected a hydroxylated metabolite. A novel p-hydroxybenzoate hydroxylase enzyme detected by proteomics was implicated in the metabolic pathway associated with the detected metabolite. This enzyme is hypothesized to be involved in BPA degradation by Sphingobium sp. BiD32, and may serve as a future genetic marker for BPA degradation. PMID:26390302

  15. Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Strategies for assignments.

    PubMed

    Oldfield, E; Norton, R S; Allerhand, A

    1975-08-25

    Natural abundance 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes) of aqueous native proteins yield numerous narrow single carbon resonances of nonprotonated aromatic carbons. Techniques for the assignment of these resonances are presented. Each technique is applied to one or more of the following proteins: ferricytochrome c from horse heart and Candida krusei, ferrocytochrome c and cyanoferricytochrome c from horse heart, lysozyme from hen egg white, cyanoferrimyoglobins from horse and sperm whale skeletal muscle, and carbon monoxide myoglobin from horse. In all of the protein spectra we have examined, methine aromatic carbons give rise to broad bands. Studies of the narrow resonances of nonprotonated aromatic carbons of proteins are facilitated by removal of these broad bands by means of the convolution-difference method, preferably from spectra recorded under conditions of noise-modulated off-resonance proton decoupling. We present a summary of the chemical shift ranges for the various types of nonprotonated aromatic carbons of amino acid residues and hemes of diamagnetic proteins, based on our results for hen egg white lysozyme, horse heart ferrocytochrome c, horse carbon monoxide myoglobin, and carbon monoxide hemoglobins from various species... PMID:169240

  16. Modification of expansin protein abundance in tomato fruit alters softening and cell wall polymer metabolism during ripening

    PubMed Central

    Brummell, DA; Harpster, MH; Civello, PM; Palys, JM; Bennett, AB; Dunsmuir, P

    1999-01-01

    The role of the ripening-specific expansin Exp1 protein in fruit softening and cell wall metabolism was investigated by suppression and overexpression of Exp1 in transgenic tomato plants. Fruit in which Exp1 protein accumulation was suppressed to 3% that of wild-type levels were firmer than controls throughout ripening. Suppression of Exp1 protein also substantially inhibited polyuronide depolymerization late in ripening but did not prevent the breakdown of structurally important hemicelluloses, a major contributor to softening. In contrast, fruit overexpressing high levels of recombinant Exp1 protein were much softer than controls, even in mature green fruit before ripening commenced. This softening was correlated with the precocious and extensive depolymerization of structural hemicelluloses, whereas polyuronide depolymerization was not altered. These data are consistent with there being at least three components to fruit softening and textural changes. One component is a relaxation of the wall directly mediated by Exp1, which indirectly limits part of a second component due to polyuronide depolymerization late in ripening, perhaps by controlling access of a pectinase to its substrate. The third component is caused by depolymerization of hemicelluloses, which occurs independently of or requires only very small amounts of Exp1 protein. PMID:10559444

  17. Comparison of the Effects of PRKAR1A and PRKAR2B Depletion on Signaling Pathways, Cell Growth, and Cell Cycle Control of Adrenocortical Cells

    PubMed Central

    Basso, F.; Rocchetti, F.; Rodriguez, S.; Nesterova, M.; Cormier, F.; Stratakis, C.; Ragazzon, B.; Bertherat, J.; Rizk-Rabin, M.

    2016-01-01

    The cyclic AMP/protein kinase A signaling cascade is one of the main pathways involved in the pathogenesis of adrenocortical tumors. The PKA R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. Inactivating mutations of PRKAR1A are associated with Carney complex and a subset of sporadic tumors and the abundance of R2B protein is low in a subset of secreting adrenocortical adenomas. We previously showed that PRKAR1A and PRKAR2B inactivation have anti-apoptotic effects on the adrenocortical carcinoma cell line H295R. The aim of this study was to compare the effects of PRKAR1A and PRKAR2B depletion on cell proliferation, apoptosis, cell signaling pathways, and cell cycle regulation. We found that PRKAR2B depletion is compensated by an upregulation in the abundance of R1A protein, whereas PRKAR1A depletion has no effect on the production of R2B. The depletion of either PRKAR1A or PRKAR2B promotes the expression of Bcl-xL and resistance to apoptosis; and is associated with a high percentage of cells in S and G2 phase, activates PKA and MEK/ERK pathways, and impairs the expression of IkB leading to activate the NF-κB pathway. Nonetheless, we observed differences in the regulation of cyclins. The depletion of PRKAR1A leads to the accumulation of cyclin D1 and p27kip, whereas the depletion of PRKAR2B promotes the accumulation of cyclin A, B, cdk1, cdc2, and p21Cip. In conclusion, although the depletion of PRKAR1A and PRKAR2B in adrenocortical cells has similar effects on cell proliferation and apoptosis; loss of these PKA subunits differentially affects cyclin expression. PMID:25268545

  18. Dataset of liver proteins of eu- and hypothyroid rats affected in abundance by any of three factors: in vivo exposure to hexabromocyclododecane (HBCD), thyroid status, gender differences.

    PubMed

    Miller, I; Renaut, J; Cambier, S; Murk, A J; Gutleb, A C; Serchi, T

    2016-09-01

    Male Wistar rats with different thyroid status (eu-, hypothyroid) were exposed to 0, 3 or 30 mg/kg body weight of the flame retardant HBCD for 7 days and obtained data compared with a previous study in females, "Hexabromocyclododecane (HBCD) induced changes in the liver proteome of eu- and hypothyroid female rats" (Miller et al., 2016) [1]. Specifically, proteomic investigation of liver protein patterns obtained by 2D-DIGE was performed and differences between animals groups recorded, based on the factors exposure, thyroid status and gender. All proteins with significantly changed abundance in any of these comparisons were identified by mass spectrometry. General, hormone and proteomic data of both the present and the previous studies are discussed in Miller et al. (2016) [1] and in "Gender specific differences in the liver proteome of rats exposed to hexabromocyclododecane (HBCD)" Miller et al. (2016) [2]. PMID:27579339

  19. The abundant class III chitinase homolog in young developing banana fruits behaves as a transient vegetative storage protein and most probably serves as an important supply of amino acids for the synthesis of ripening-associated proteins.

    PubMed

    Peumans, Willy J; Proost, Paul; Swennen, Rony L; Van Damme, Els J M

    2002-10-01

    Analyses of the protein content and composition revealed dramatic changes in gene expression during in situ banana (Musa spp.) fruit formation/ripening. The total banana protein content rapidly increases during the first 60 to 70 d, but remains constant for the rest of fruit formation/ripening. During the phase of rapid protein accumulation, an inactive homolog of class III chitinases accounts for up to 40% (w/v) of the total protein. Concomitant with the arrest of net protein accumulation, the chitinase-related protein (CRP) progressively decreases and several novel proteins appear in the electropherograms. Hence, CRP behaves as a fruit-specific vegetative storage protein that accumulates during early fruit formation and serves as a source of amino acids for the synthesis of ripening-associated proteins. Analyses of individual proteins revealed that a thaumatin-like protein, a beta-1,3-glucanase, a class I chitinase, and a mannose-binding lectin are the most abundant ripening-associated proteins. Because during the ripening of prematurely harvested bananas, similar changes take place as in the in situ ripening bananas, CRP present in immature fruits is a sufficient source of amino acids for a quasi-normal synthesis of ripening-associated proteins. However, it is evident that the conversion of CRP in ripening-associated proteins takes place at an accelerated rate, especially when climacteric ripening is induced by ethylene. The present report also includes a discussion of the accumulation of the major banana allergens and the identification of suitable promoters for the production of vaccines in transgenic bananas. PMID:12376669

  20. Overexpression of Drosophila juvenile hormone esterase binding protein results in anti-JH effects and reduced pheromone abundance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The titer of juvenile hormone (JH), which has wide ranging physiological effects in insects, is regulated in part by JH esterase (JHE). We show that overexpression in Drosophila melanogaster of the JHE binding protein, DmP29 results in a series of apparent anti-JH effects. We hypothesize that DmP29 ...

  1. Identification of an abundant 56 kDa protein implicated in food allergy as granule-bound starch synthase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rice, the staple food of South and East Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16 kDa, 26 kDa, 33 kDa and 56 kDa have been identified as allergens. Re...

  2. Analysis of crude protein and allergen abundance in peanuts (Arachis hypogaea cv. Walter) from three growing regions in Australia.

    PubMed

    Walczyk, Nicole E; Smith, Penelope M C; Tovey, Euan; Wright, Graeme C; Fleischfresser, Dayle B; Roberts, Thomas H

    2013-04-17

    The effects of plant growth conditions on concentrations of proteins, including allergens, in peanut ( Arachis hypogaea L.) kernels are largely unknown. Peanuts (cv. Walter) were grown at five sites (Taabinga, Redvale, Childers, Bundaberg, and Kairi) covering three commercial growing regions in Queensland, Australia. Differences in temperature, rainfall, and solar radiation during the growing season were evaluated. Kernel yield varied from 2.3 t/ha (Kairi) to 3.9 t/ha (Childers), probably due to differences in solar radiation. Crude protein appeared to vary only between Kairi and Childers, whereas Ara h 1 and 2 concentrations were similar in all locations. 2D-DIGE revealed significant differences in spot volumes for only two minor protein spots from peanuts grown in the five locations. Western blotting using peanut-allergic serum revealed no qualitative differences in recognition of antigens. It was concluded that peanuts grown in different growing regions in Queensland, Australia, had similar protein compositions and therefore were unlikely to show differences in allergenicity. PMID:23495786

  3. A definition of depletion of fish stocks

    USGS Publications Warehouse

    Van Oosten, John

    1949-01-01

    Attention was focused on the need of a common and better understanding of the term depletion as applied to the fisheries in order to eliminate if possible the existing inexactness of thought on the subject. Depletion has been confused at various times with at least ten different ideas associated with it but which, as has has heen pointed out, are not synonymous at all. In defining depletion we must recognize that the term represents a condition and must not he confounded with the cause (overfishing) that leads to this condition or with the symptoms that identify it. Depletion was defined as a reduction, through overfishing, in the level of abundance of the exploitable segment of a stock that prevents the realization of the maximum productive capacity.

  4. RcLEA, a late embryogenesis abundant protein gene isolated from Rosa chinensis, confers tolerance to Escherichia coli and Arabidopsis thaliana and stabilizes enzyme activity under diverse stresses.

    PubMed

    Zhang, Xuan; Lu, Songchong; Jiang, Changhua; Wang, Yaofeng; Lv, Bo; Shen, Jiabin; Ming, Feng

    2014-07-01

    The late embryogenesis abundant (LEA) protein family is a large protein family that is closely associated with resistance to abiotic stresses in many organisms, such as plants, bacteria and animals. In this study, we isolated a LEA gene, RcLEA, which was cytoplasm-localized, from Rosa chinensis. RcLEA was found to be induced by high temperature through RT-PCR. Overexpression of RcLEA in Escherichia coli improved its growth performance compared with the control under high temperature, low temperature, NaCl and oxidative stress conditions. RcLEA was also overexpressed in Arabidopsis thaliana. The transgenic Arabidopsis showed better growth after high and low temperature treatment and exhibited less peroxide according to 3, 3-diaminobenzidine staining. However, RcLEA did not improve the tolerance to NaCl or osmotic stress in Arabidopsis. In vitro analysis showed that RcLEA was able to prevent the freeze-thaw-induced inactivation or heat-induced aggregation of various substrates, such as lactate dehydrogenase and citrate synthase. It also protected the proteome of E. coli from denaturation when the proteins were heat-shocked or subjected to acidic conditions. Furthermore, bimolecular fluorescence complementation assays suggested that RcLEA proteins function in a complex manner by making the form of homodimers. PMID:24760474

  5. Retinoic acid-induced differentiation of human neuroblastoma SH-SY5Y cells is associated with changes in the abundance of G proteins.

    PubMed

    Ammer, H; Schulz, R

    1994-04-01

    Western blot analysis, using subtype-specific anti-G protein antibodies, revealed the presence of the following G protein subunits in human neuroblastoma SH-SY5Y cells: Gs alpha, Gi alpha 1, Gi alpha 2, Go alpha, Gz alpha, and G beta. Differentiation of the cells by all-trans-retinoic acid (RA) treatment (10 mumol/L; 6 days) caused substantial alterations in the abundance of distinct G protein subunits. Concomitant with an enhanced expression of mu-opioid binding sites, the levels of the inhibitory G proteins Gi alpha 1 and Gi alpha 2 were found to be significantly increased. This coordinate up-regulation is accompanied by functional changes in mu-opioid receptor-stimulated low-Km GTPase, mu-receptor-mediated adenylate cyclase inhibition, and receptor-independent guanosine 5'-(beta gamma-imido)triphosphate [Gpp(NH)p; 10 nmol/L]-mediated attenuation of adenylate cyclase activity. In contrast, increased levels of inhibitory G proteins had no effect on muscarinic cholinergic receptor-mediated adenylate cyclase inhibition. With respect to stimulatory receptor systems, a reciprocal regulation was observed for prostaglandin E1 (PGE1) receptors and Gs alpha, the G protein subunit activating adenylate cyclase. RA treatment of SH-SY5Y cells increases both the number of PGE1 binding sites and PGE1-stimulated adenylate cyclase activity, but significantly reduced amounts of Gs alpha were found. This down-regulation is paralleled by a decrease in the stimulatory activity of Gs alpha as assessed in S49 cyc- reconstitution assays.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8133263

  6. Optimal Allocation of Sampling Effort in Depletion Surveys

    EPA Science Inventory

    We consider the problem of designing a depletion or removal survey as part of estimating animal abundance for populations with imperfect capture or detection rates. In a depletion survey, animals are captured from a given area, counted, and withheld from the population. This proc...

  7. The freshwater Amazonian stingray, Potamotrygon motoro, up-regulates glutamine synthetase activity and protein abundance, and accumulates glutamine when exposed to brackish (15 per thousand) water.

    PubMed

    Ip, Y K; Loong, A M; Ching, B; Tham, G H Y; Wong, W P; Chew, S F

    2009-12-01

    This study aimed to examine whether the stenohaline freshwater stingray, Potamotrygon motoro, which lacks a functional ornithine-urea cycle, would up-regulate glutamine synthetase (GS) activity and protein abundance, and accumulate glutamine during a progressive transfer from freshwater to brackish (15 per thousand) water with daily feeding. Our results revealed that, similar to other freshwater teleosts, P. motoro performed hyperosmotic regulation, with very low urea concentrations in plasma and tissues, in freshwater. In 15 per thousand water, it was non-ureotelic and non-ureoosmotic, acting mainly as an osmoconformer with its plasma osmolality, [Na+] and [Cl-] comparable to those of the external medium. There were significant increases in the content of several free amino acids (FAAs), including glutamate, glutamine and glycine, in muscle and liver, but not in plasma, indicating that FAAs could contribute in part to cell volume regulation. Furthermore, exposure of P. motoro to 15 per thousand water led to up-regulation of GS activity and protein abundance in both liver and muscle. Thus, our results indicate for the first time that, despite the inability to synthesize urea and the lack of functional carbamoyl phosphate synthetase III (CPS III) which uses glutamine as a substrate, P. motoro retained the capacity to up-regulate the activity and protein expression of GS in response to salinity stress. Potamotrygon motoro was not nitrogen (N) limited when exposed to 15 per thousand water with feeding, and there were no significant changes in the amination and deamination activities of hepatic glutamate dehydrogenase. In contrast, P. motoro became N limited when exposed to 10 per thousand water with fasting and could not survive well in 15 per thousand water without food. PMID:19915125

  8. Abundant Type III Lipid Transfer Proteins in Arabidopsis Tapetum Are Secreted to the Locule and Become a Constituent of the Pollen Exine1[W][OPEN

    PubMed Central

    Huang, Ming-Der; Chen, Tung-Ling L.; Huang, Anthony H.C.

    2013-01-01

    Lipid transfer proteins (LTPs) are small secretory proteins in plants with defined lipid-binding structures for possible lipid exocytosis. Special groups of LTPs unique to the anther tapetum are abundant, but their functions are unclear. We studied a special group of LTPs, type III LTPs, in Arabidopsis (Arabidopsis thaliana). Their transcripts were restricted to the anther tapetum, with levels peaking at the developmental stage of maximal pollen-wall exine synthesis. We constructed an LTP-Green Fluorescent Protein (LTP-GFP) plasmid, transformed it into wild-type plants, and monitored LTP-GFP in developing anthers with confocal laser scanning microscopy. LTP-GFP appeared in the tapetum and was secreted via the endoplasmic reticulum-trans-Golgi network machinery into the locule. It then moved to the microspore surface and remained as a component of exine. Immuno-transmission electron microscopy of native LTP in anthers confirmed the LTP-GFP observations. The in vivo association of LTP-GFP and exine in anthers was not observed with non-type III or structurally modified type III LTPs or in transformed exine-defective mutant plants. RNA interference knockdown of individual type III LTPs produced no observable mutant phenotypes. RNA interference knockdown of two type III LTPs produced microscopy-observable morphologic changes in the intine underneath the exine (presumably as a consequence of changes in the exine not observed by transmission electron microscopy) and pollen susceptible to dehydration damage. Overall, we reveal a novel transfer pathway of LTPs in which LTPs bound or nonbound to exine precursors are secreted from the tapetum to become microspore exine constituents; this pathway explains the need for plentiful LTPs to incorporate into the abundant exine. PMID:24096413

  9. DNA polymorphisms and transcript abundance of PRKAG2 and phosphorylated AMP-activated protein kinase in the rumen are associated with gain and feed intake in beef steers.

    PubMed

    Lindholm-Perry, A K; Kuehn, L A; Oliver, W T; Kern, R J; Cushman, R A; Miles, J R; McNeel, A K; Freetly, H C

    2014-08-01

    Beef steers with variation in feed efficiency phenotypes were evaluated previously on a high-density SNP panel. Ten markers from rs110125325-rs41652818 on bovine chromosome 4 were associated with average daily gain (ADG). To identify the gene(s) in this 1.2-Mb region responsible for variation in ADG, genotyping with 157 additional markers was performed. Several markers (n = 41) were nominally associated with ADG, and three of these, including the only marker to withstand Bonferroni correction, were located within the protein kinase, AMP-activated, gamma 2 non-catalytic subunit (PRKAG2) gene. An additional population of cross-bred steers (n = 406) was genotyped for validation. One marker located within the PRKAG2 loci approached a significant association with gain. To evaluate PRKAG2 for differences in transcript abundance, we measured expression in the liver, muscle, rumen and intestine from steers (n = 32) with extreme feed efficiency phenotypes collected over two seasons. No differences in PRKAG2 transcript abundance were detected in small intestine, liver or muscle. Correlation between gene expression level of PRKAG2 in rumen and average daily feed intake (ADFI) was detected in both seasons (P < 0.05); however, the direction differed by season. Lastly, we evaluated AMP-activated protein kinase (AMPK), of which PRKAG2 is a subunit, for differences among ADG and ADFI and found that the phosphorylated form of AMPK was associated with ADFI in the rumen. These data suggest that PRKAG2 and its mature protein, AMPK, are involved in feed efficiency traits in beef steers. This is the first evidence to suggest that rumen AMPK may be contributing to ADFI in cattle. PMID:24730749

  10. Abundant type III lipid transfer proteins in Arabidopsis tapetum are secreted to the locule and become a constituent of the pollen exine.

    PubMed

    Huang, Ming-Der; Chen, Tung-Ling L; Huang, Anthony H C

    2013-11-01

    Lipid transfer proteins (LTPs) are small secretory proteins in plants with defined lipid-binding structures for possible lipid exocytosis. Special groups of LTPs unique to the anther tapetum are abundant, but their functions are unclear. We studied a special group of LTPs, type III LTPs, in Arabidopsis (Arabidopsis thaliana). Their transcripts were restricted to the anther tapetum, with levels peaking at the developmental stage of maximal pollen-wall exine synthesis. We constructed an LTP-Green Fluorescent Protein (LTP-GFP) plasmid, transformed it into wild-type plants, and monitored LTP-GFP in developing anthers with confocal laser scanning microscopy. LTP-GFP appeared in the tapetum and was secreted via the endoplasmic reticulum-trans-Golgi network machinery into the locule. It then moved to the microspore surface and remained as a component of exine. Immuno-transmission electron microscopy of native LTP in anthers confirmed the LTP-GFP observations. The in vivo association of LTP-GFP and exine in anthers was not observed with non-type III or structurally modified type III LTPs or in transformed exine-defective mutant plants. RNA interference knockdown of individual type III LTPs produced no observable mutant phenotypes. RNA interference knockdown of two type III LTPs produced microscopy-observable morphologic changes in the intine underneath the exine (presumably as a consequence of changes in the exine not observed by transmission electron microscopy) and pollen susceptible to dehydration damage. Overall, we reveal a novel transfer pathway of LTPs in which LTPs bound or nonbound to exine precursors are secreted from the tapetum to become microspore exine constituents; this pathway explains the need for plentiful LTPs to incorporate into the abundant exine. PMID:24096413

  11. Treatment of green tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins, depletion of antioxidant enzymes and phosphorylation of MAPK proteins in SKH-1 hairless mouse skin.

    PubMed

    Vayalil, Praveen K; Elmets, Craig A; Katiyar, Santosh K

    2003-05-01

    The use of botanical supplements has received immense interest in recent years to protect human skin from adverse biological effects of solar ultraviolet (UV) radiation. The polyphenols from green tea are one of them and have been shown to prevent photocarcinogenesis in animal models but their mechanism of photoprotection is not well understood. To determine the mechanism of photoprotection in in vivo mouse model, topical treatment of polyphenols from green tea (GTP) or its most chemopreventive constituent (-)-epigallocatechin-3-gallate (EGCG) (1 mg/cm(2) skin area) in hydrophilic ointment USP before single (180 mJ/cm(2)) or multiple UVB exposures (180 mJ/cm(2), daily for 10 days) resulted in significant prevention of UVB-induced depletion of antioxidant enzymes such as glutathione peroxidase (78-100%, P < 0.005-0.001), catalase (51-92%, P < 0.001) and glutathione level (87-100%, P < 0.005). Treatment of EGCG or GTP also inhibited UVB-induced oxidative stress when measured in terms of lipid peroxidation (76-95%, P < 0.001), and protein oxidation (67-75%, P > 0.001). Further, to delineate the inhibition of UVB-induced oxidative stress with cell signaling pathways, treatment of EGCG to mouse skin resulted in marked inhibition of a single UVB irradiation-induced phosphorylation of ERK1/2 (16-95%), JNK (46-100%) and p38 (100%) proteins of MAPK family in a time-dependent manner. Identical photoprotective effects of EGCG or GTP were also observed against multiple UVB irradiation-induced phosphorylation of the proteins of MAPK family in vivo mouse skin. Photoprotective efficacy of GTP given in drinking water (d.w.) (0.2%, w/v) was also determined and compared with that of topical treatment of EGCG and GTP. Treatment of GTP in d.w. also significantly prevented single or multiple UVB irradiation-induced depletion of antioxidant enzymes (44-61%, P < 0.01-0.001), oxidative stress (33-71%, P < 0.01) and phosphorylation of ERK1/2, JNK and p38 proteins of MAPK family but the

  12. Dimerization and DNA-binding of ASR1, a small hydrophilic protein abundant in plant tissues suffering from water loss

    SciTech Connect

    Maskin, Laura; Frankel, Nicolas; Gudesblat, Gustavo; Demergasso, Maria J.; Pietrasanta, Lia I.; Iusem, Norberto D. . E-mail: norbius@fbmc.fcen.uba.ar

    2007-01-26

    The Asr gene family is present in Spermatophyta. Its members are generally activated under water stress. We present evidence that tomato ASR1, one of the proteins of the family, accumulates in seed during late stages of embryogenesis, a physiological process characterized by water loss. In vitro, electrophoretic assays show a homo-dimeric structure for ASR1 and highlight strong non-covalent interactions between monomers prone to self-assemble. Direct visualization of single molecules by atomic force microscopy (AFM) confirms that ASR1 forms homodimers and that uncovers both monomers and dimers bind double stranded DNA.

  13. An abundantly expressed mucin-like protein from Toxocara canis infective larvae: the precursor of the larval surface coat glycoproteins.

    PubMed Central

    Gems, D; Maizels, R M

    1996-01-01

    Evasion of host immunity by Toxocara canis infective larvae is mediated by the nematode surface coat, which is shed in response to binding by host antibody molecules or effector cells. The major constituent of the coat is the TES-120 glycoprotein series. We have isolated a 730-bp cDNA from the gene encoding the apoprotein precursor of TES-120. The mRNA is absent from T. canis adults but hyperabundant in larvae, making up approximately 10% of total mRNA, and is trans-spliced with the nematode 5' leader sequence SL1. It encodes a 15.8-kDa protein (after signal peptide removal) containing a typical mucin domain: 86 amino acid residues, 72.1% of which are Ser or Thr, organized into an array of heptameric repeats, interspersed with proline residues. At the C-terminal end of the putative protein are two 36-amino acid repeats containing six Cys residues, in a motif that can also be identified in several genes in Caenorhabditis elegans. Although TES-120 displays size and charge heterogeneity, there is a single copy gene and a homogeneous size of mRNA. The association of overexpression of some membrane-associated mucins with immunosuppression and tumor metastasis suggests a possible model for the role of the surface coat in immune evasion by parasitic nematodes. Images Fig. 1 Fig. 4 PMID:8643687

  14. Protein covalent immobilization via its scarce thiol versus abundant amine groups: Effect on orientation, cell binding domain exposure and conformational lability.

    PubMed

    Ba, O M; Hindie, M; Marmey, P; Gallet, O; Anselme, K; Ponche, A; Duncan, A C

    2015-10-01

    Quantity, orientation, conformation and covalent linkage of naturally cell adhesive proteins adsorbed or covalently linked to a surface, are known to influence the preservation of their subsequent long term cell adhesion properties and bioactivity. In the present work, we explore two different strategies for the covalent linking of plasma fibronectin (pFN) - used as a cell adhesive model protein, onto a polystyrene (PS) surface. One is aimed at tethering the protein to the surface in a semi-oriented fashion (via one of the 4 free thiol reactive groups on the protein) with a heterofunctional coupling agent (SSMPB method). The other aims to immobilize the protein in a more random fashion by reaction between the abundant pendant primary amine bearing amino acids of the pFN and activated carboxylic surface functions obtained after glutaric anhydride surface treatment (GA method). The overall goal will be to verify the hypothesis of a correlation between covalent immobilization of a model cell adhesive protein to a PS surface in a semi-oriented configuration (versus randomly oriented) with promotion of enhanced exposure of the protein's cell binding domain. This in turn would lead to enhanced cell adhesion. Ideally the goal is to elaborate substrates exhibiting a long term stable protein monolayer with preserved cell adhesive properties and bioactivity for biomaterial and/or cell adhesion commercial plate applications. However, the initial restrictive objective of this paper is to first quantitatively and qualitatively investigate the reversibly (merely adsorbed) versus covalently irreversibly bound protein to the surface after the immobilization procedure. Although immobilized surface amounts were similar (close to the monolayer range) for all immobilization approaches, covalent grafting showed improved retention and stronger "tethering" of the pFN protein to the surface (roughly 40%) after SDS rinsing compared to that for mere adsorption (0%) suggesting an added value

  15. The Deoxynucleoside Triphosphate Triphosphohydrolase Activity of SAMHD1 Protein Contributes to the Mitochondrial DNA Depletion Associated with Genetic Deficiency of Deoxyguanosine Kinase*

    PubMed Central

    Franzolin, Elisa; Salata, Cristiano; Bianchi, Vera; Rampazzo, Chiara

    2015-01-01

    The dNTP triphosphohydrolase SAMHD1 is a nuclear antiviral host restriction factor limiting HIV-1 infection in macrophages and a major regulator of dNTP concentrations in human cells. In normal human fibroblasts its expression increases during quiescence, contributing to the small dNTP pool sizes of these cells. Down-regulation of SAMHD1 by siRNA expands all four dNTP pools, with dGTP undergoing the largest relative increase. The deoxyguanosine released by SAMHD1 from dGTP can be phosphorylated inside mitochondria by deoxyguanosine kinase (dGK) or degraded in the cytosol by purine nucleoside phosphorylase. Genetic mutations of dGK cause mitochondrial (mt) DNA depletion in noncycling cells and hepato-cerebral mtDNA depletion syndrome in humans. We studied if SAMHD1 and dGK interact in the regulation of the dGTP pool during quiescence employing dGK-mutated skin fibroblasts derived from three unrelated patients. In the presence of SAMHD1 quiescent mutant fibroblasts manifested mt dNTP pool imbalance and mtDNA depletion. When SAMHD1 was silenced by siRNA transfection the composition of the mt dNTP pool approached that of the controls, and mtDNA copy number increased, compensating the depletion to various degrees in the different mutant fibroblasts. Chemical inhibition of purine nucleoside phosphorylase did not improve deoxyguanosine recycling by dGK in WT cells. We conclude that the activity of SAMHD1 contributes to the pathological phenotype of dGK deficiency. Our results prove the importance of SAMHD1 in the regulation of all dNTP pools and suggest that dGK inside mitochondria has the function of recycling the deoxyguanosine derived from endogenous dGTP degraded by SAMHD1 in the nucleus. PMID:26342080

  16. Regulation of mRNA Abundance by Polypyrimidine Tract-Binding Protein-Controlled Alternate 5′ Splice Site Choice

    PubMed Central

    Hamid, Fursham M.; Makeyev, Eugene V.

    2014-01-01

    Alternative splicing (AS) provides a potent mechanism for increasing protein diversity and modulating gene expression levels. How alternate splice sites are selected by the splicing machinery and how AS is integrated into gene regulation networks remain important questions of eukaryotic biology. Here we report that polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I) controls alternate 5′ and 3′ splice site (5′ss and 3′ss) usage in a large set of mammalian transcripts. A top scoring event identified by our analysis was the choice between competing upstream and downstream 5′ss (u5′ss and d5′ss) in the exon 18 of the Hps1 gene. Hps1 is essential for proper biogenesis of lysosome-related organelles and loss of its function leads to a disease called type 1 Hermansky-Pudlak Syndrome (HPS). We show that Ptbp1 promotes preferential utilization of the u5′ss giving rise to stable mRNAs encoding a full-length Hps1 protein, whereas bias towards d5′ss triggered by Ptbp1 down-regulation generates transcripts susceptible to nonsense-mediated decay (NMD). We further demonstrate that Ptbp1 binds to pyrimidine-rich sequences between the u5′ss and d5′ss and activates the former site rather than repressing the latter. Consistent with this mechanism, u5′ss is intrinsically weaker than d5′ss, with a similar tendency observed for other genes with Ptbp1-induced u5′ss bias. Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5′ss utilization but with a considerably lower efficiency than Ptbp1. This may account for the tight correlation between Hps1 with Ptbp1 expression levels observed across mammalian tissues. More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism. PMID:25375251

  17. The pepper late embryogenesis abundant protein CaLEA1 acts in regulating abscisic acid signaling, drought and salt stress response.

    PubMed

    Lim, Chae Woo; Lim, Sohee; Baek, Woonhee; Lee, Sung Chul

    2015-08-01

    As sessile organisms, plants are constantly challenged by environmental stresses, including drought and high salinity. Among the various abiotic stresses, osmotic stress is one of the most important factors for growth and significantly reduces crop productivity in agriculture. Here, we report a function of the CaLEA1 protein in the defense responses of plants to osmotic stress. Our analyses showed that the CaLEA1 gene was strongly induced in pepper leaves exposed to drought and increased salinity. Furthermore, we determined that the CaLEA1 protein has a late embryogenesis abundant (LEA)_3 homolog domain highly conserved among other known group 5 LEA proteins and is localized in the processing body. We generated CaLEA1-silenced peppers and CaLEA1-overexpressing (OX) transgenic Arabidopsis plants to evaluate their responses to dehydration and high salinity. Virus-induced gene silencing of CaLEA1 in pepper plants conferred enhanced sensitivity to drought and salt stresses, which was accompanied by high levels of lipid peroxidation in dehydrated and NaCl-treated leaves. CaLEA1-OX plants exhibited enhanced sensitivity to abscisic acid (ABA) during seed germination and in the seedling stage; furthermore, these plants were more tolerant to drought and salt stress than the wild-type plants because of enhanced stomatal closure and increased expression of stress-responsive genes. Collectively, our data suggest that CaLEA1 positively regulates drought and salinity tolerance through ABA-mediated cell signaling. PMID:25302464

  18. The AP2 clathrin adaptor protein complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans

    PubMed Central

    Garafalo, Steven D.; Luth, Eric S.; Moss, Benjamin J.; Monteiro, Michael I.; Malkin, Emily; Juo, Peter

    2015-01-01

    Regulation of glutamate receptor (GluR) abundance at synapses by clathrin-mediated endocytosis can control synaptic strength and plasticity. We take advantage of viable, null mutations in subunits of the clathrin adaptor protein 2 (AP2) complex in Caenorhabditis elegans to characterize the in vivo role of AP2 in GluR trafficking. In contrast to our predictions for an endocytic adaptor, we found that levels of the GluR GLR-1 are decreased at synapses in the ventral nerve cord (VNC) of animals with mutations in the AP2 subunits APM-2/μ2, APA-2/α, or APS-2/σ2. Rescue experiments indicate that APM-2/μ2 functions in glr-1–expressing interneurons and the mature nervous system to promote GLR-1 levels in the VNC. Genetic analyses suggest that APM-2/μ2 acts upstream of GLR-1 endocytosis in the VNC. Consistent with this, GLR-1 accumulates in cell bodies of apm-2 mutants. However, GLR-1 does not appear to accumulate at the plasma membrane of the cell body as expected, but instead accumulates in intracellular compartments including Syntaxin-13– and RAB-14–labeled endosomes. This study reveals a novel role for the AP2 clathrin adaptor in promoting the abundance of GluRs at synapses in vivo, and implicates AP2 in the regulation of GluR trafficking at an early step in the secretory pathway. PMID:25788288

  19. Knockout of the abundant Trichomonas vaginalis hydrogenosomal membrane protein TvHMP23 increases hydrogenosome size but induces no compensatory up-regulation of paralogous copies.

    PubMed

    Brás, Xavier Pereira; Zimorski, Verena; Bolte, Kathrin; Maier, Uwe-G; Martin, William F; Gould, Sven B

    2013-05-01

    The Trichomonas vaginalis genome encodes up to 60000 genes, many of which stem from genome duplication events. Paralogous copies thus accompany most T. vaginalis genes, a phenomenon that limits genetic manipulation. We characterized one of the parasite's most abundant hydrogenosomal membrane proteins, TvHMP23, which is phylogenetically distinct from canonical metabolite carriers, and which localizes to the inner hydrogenosomal membrane as shown through sub-organellar fractionation and protease protection assays. Knockout of Tvhmp23 through insertion of the selectable neomycin marker led to a size increase of hydrogenosomes, the first knockout-induced phenotypes reported for Trichomonas, but no growth impairment. The transcriptional response of its four paralogous copies then analyzed revealed that they are not up-regulated, and hence do not compensate for the Tvhmp23 knockout. PMID:23499435

  20. Water Depletion Threatens Agriculture

    NASA Astrophysics Data System (ADS)

    Brauman, K. A.; Richter, B. D.; Postel, S.; Floerke, M.; Malsy, M.

    2014-12-01

    Irrigated agriculture is the human activity that has by far the largest impact on water, constituting 85% of global water consumption and 67% of global water withdrawals. Much of this water use occurs in places where water depletion, the ratio of water consumption to water availability, exceeds 75% for at least one month of the year. Although only 17% of global watershed area experiences depletion at this level or more, nearly 30% of total cropland and 60% of irrigated cropland are found in these depleted watersheds. Staple crops are particularly at risk, with 75% of global irrigated wheat production and 65% of irrigated maize production found in watersheds that are at least seasonally depleted. Of importance to textile production, 75% of cotton production occurs in the same watersheds. For crop production in depleted watersheds, we find that one half to two-thirds of production occurs in watersheds that have not just seasonal but annual water shortages, suggesting that re-distributing water supply over the course of the year cannot be an effective solution to shortage. We explore the degree to which irrigated production in depleted watersheds reflects limitations in supply, a byproduct of the need for irrigation in perennially or seasonally dry landscapes, and identify heavy irrigation consumption that leads to watershed depletion in more humid climates. For watersheds that are not depleted, we evaluate the potential impact of an increase in irrigated production. Finally, we evaluate the benefits of irrigated agriculture in depleted and non-depleted watersheds, quantifying the fraction of irrigated production going to food production, animal feed, and biofuels.

  1. The solar abundance of beryllium

    NASA Technical Reports Server (NTRS)

    Ross, J. E.; Aller, L. H.

    1974-01-01

    The solar abundance of beryllium is deduced from high-resolution Kitt Peak observations of the 3130.43- and 3131.08-A lines of Be II interpreted by the method of spectrum synthesis. The results are in good agreement with those previously obtained by Grevesse (1968) and by Hauge and Engvold (1968) and indicate that in the photospheric layers, beryllium is depleted below the chondritic value by a factor of about two. It is found that the beryllium abundance is equal to logN(Be)/N(H) + 12 = 1.08 plus or minus 0.05.

  2. Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein

    PubMed Central

    Gao, Zhuangqiang; Hou, Li; Xu, Mingdi; Tang, Dianping

    2014-01-01

    Methods based on enzyme labels have been developed for colorimetric immunoassays, but most involve poor sensitivity and are unsuitable for routine use. Herein, we design an enhanced colorimetric immunoassay for prostate-specific antigen (PSA) coupling with an enzyme-cascade-amplification strategy (ECAS-CIA). In the presence of target PSA, the labeled alkaline phosphatase on secondary antibody catalyzes the formation of palladium nanostructures, which catalyze 3,3′,5,5′-tetramethylbenzidine-H2O2 system to produce the colored products, thus resulting in the signal cascade amplification. Results indicated that the ECAS-CIA presents good responses toward PSA, and allows detection of PSA at a concentration as low as 0.05 ng mL−1. Intra- and inter-assay coefficients of variation are below 9.5% and 10.7%, respectively. Additionally, the methodology is validated for analysis of clinical serum specimens with consistent results obtained by PSA ELISA kit. Importantly, the ECAS-CIA opens a new horizon for protein diagnostics and biosecurity. PMID:24509941

  3. Influence of pregnancy in mid-to-late gestation on circulating metabolites, visceral organ mass, and abundance of proteins relating to energy metabolism in mature beef cows.

    PubMed

    Wood, K M; Awda, B J; Fitzsimmons, C; Miller, S P; McBride, B W; Swanson, K C

    2013-12-01

    In mid-to-late gestation, nutrient demand increases to meet the growth requirements of the conceptus and cows may alter metabolism in response to energy demands of pregnancy. By better understanding the metabolic role of pregnancy, there may be opportunities to better understand maintenance energy costs and improve overall feed efficiency. Eighteen mature Simmental/Angus crossbred cows, pregnant (PREG; n = 9) and nonpregnant (OPEN; n = 9), were used to investigate the effect of pregnancy on BW change, carcass traits, visceral organ mass, and circulating serum metabolites. Cows were blocked by day of expected parturition such that each block was slaughtered 4 to 5 wk before parturition. Cows were individually fed for ad libitum intake using Calan gates for 89 to 105 d. Cows were weighed, ultrasounded for rib (over the 12th and 13th rib) and rump fat, and a serum sample obtained at d 1, 56, and 3 to 5 d before slaughter. At slaughter, organs were removed, trimmed of fat, and weighed. Serum was analyzed for β-hydroxybutyrate (BHBA), NEFA, glucose, urea, total cholesterol, and triiodothyronine (T3). Tissue samples from liver, kidney, sternomandibularis muscle, ruminal papillae, pancreas, and small intestinal mucosa were collected at slaughter and snap frozen in liquid N. Western blots were conducted to quantify abundance of: proliferating cell nuclear antigen (PCNA), ATP synthase, ubiquitin, and Na(+)/K+ ATPase for all tissues; PPARγ, PPARγ coactivator 1α (PGC1-α), 5'-adenosine monophosphate-activated protein kinase (AMPK) and phosphorylated-AMPK (pAMPK) for liver, muscle, and rumen; phosphoenolpyruvate carboxykinase (PEPCK) for liver and kidney; and uncoupling protein 2 (UCP2) for liver. Data were analyzed using PROC MIXED in SAS as a replicated randomized complete block. Liver weights (actual, relative to BW, relative to HCW) were heavier (P ≤ 0.02) in OPEN. Rumen mass and kidney fat weight, both relative to BW, were also greater (P ≤ 0.04) in OPEN. On d 56

  4. Comparison of the effects of PRKAR1A and PRKAR2B depletion on signaling pathways, cell growth, and cell cycle control of adrenocortical cells.

    PubMed

    Basso, F; Rocchetti, F; Rodriguez, S; Nesterova, M; Cormier, F; Stratakis, C A; Ragazzon, B; Bertherat, J; Rizk-Rabin, M

    2014-11-01

    The cyclic AMP/protein kinase A signaling cascade is one of the main pathways involved in the pathogenesis of adrenocortical tumors. The PKA R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. Inactivating mutations of PRKAR1A are associated with Carney complex and a subset of sporadic tumors and the abundance of R2B protein is low in a subset of secreting adrenocortical adenomas. We previously showed that PRKAR1A and PRKAR2B inactivation have anti-apoptotic effects on the adrenocortical carcinoma cell line H295R. The aim of this study was to compare the effects of PRKAR1A and PRKAR2B depletion on cell proliferation, apoptosis, cell signaling pathways, and cell cycle regulation. We found that PRKAR2B depletion is compensated by an upregulation of R1A protein, whereas PRKAR1A depletion has no effect on the production of R2B. The depletion of either PRKAR1A or PRKAR2B promotes the expression of Bcl-xL and resistance to apoptosis; and is associated with a high percentage of cells in S and G2 phase, activates PKA and MEK/ERK pathways, and impairs the expression of IkB leading to activate the NF-κB pathway. However, we observed differences in the regulation of cyclins. The depletion of PRKAR1A leads to the accumulation of cyclin D1 and p27kip, whereas the depletion of PRKAR2B promotes the accumulation of cyclin A, B, cdk1, cdc2, and p21Cip. In conclusion, although the depletion of PRKAR1A and PRKAR2B in adrenocortical cells has similar effects on cell proliferation and apoptosis; loss of these PKA subunits differentially affects cyclin expression. PMID:25268545

  5. CpLEA5, the Late Embryogenesis Abundant Protein Gene from Chimonanthus praecox, Possesses Low Temperature and Osmotic Resistances in Prokaryote and Eukaryotes.

    PubMed

    Liu, Yiling; Xie, Lixia; Liang, Xilong; Zhang, Shihong

    2015-01-01

    Plants synthesize and accumulate a series of stress-resistance proteins to protect normal physiological activities under adverse conditions. Chimonanthus praecox which blooms in freezing weather accumulates late embryogenesis abundant proteins (LEAs) in flowers, but C. praecox LEAs are little reported. Here, we report a group of five LEA genes of C. praecox (CpLEA5, KT727031). Prokaryotic-expressed CpLEA5 was employed in Escherichia coli to investigate bioactivities and membrane permeability at low-temperature. In comparison with the vacant strains, CpLEA5-containing strains survived in a 20% higher rate; and the degree of cell membrane damage in CpLEA5-containing strains was 55% of that of the vacant strains according to a conductivity test, revealing the low-temperature resistance of CpLEA5 in bacteria. CpLEA5 was also expressed in Pichia pastoris. Interestingly, besides low-temperature resistance, CpLEA5 conferred high resistance to salt and alkali in CpLEA5 overexpressing yeast. The CpLEA5 gene was transferred into Arabidopsis thaliana to also demonstrate CpLEA5 actions in plants. As expected, the transgenic lines were more resistant against low-temperature and drought while compared with the wild type. Taken together, CpLEA5-conferred resistances to several conditions in prokaryote and eukaryotes could have great value as a genetic technology to enhance osmotic stress and low-temperature tolerance. PMID:26569231

  6. A late embryogenesis abundant protein HVA1 regulated by an inducible promoter enhances root growth and abiotic stress tolerance in rice without yield penalty.

    PubMed

    Chen, Yi-Shih; Lo, Shuen-Fang; Sun, Peng-Kai; Lu, Chung-An; Ho, Tuan-Hua D; Yu, Su-May

    2015-01-01

    Regulation of root architecture is essential for maintaining plant growth under adverse environment. A synthetic abscisic acid (ABA)/stress-inducible promoter was designed to control the expression of a late embryogenesis abundant protein (HVA1) in transgenic rice. The background of HVA1 is low but highly inducible by ABA, salt, dehydration and cold. HVA1 was highly accumulated in root apical meristem (RAM) and lateral root primordia (LRP) after ABA/stress treatments, leading to enhanced root system expansion. Water-use efficiency (WUE) and biomass also increased in transgenic rice, likely due to the maintenance of normal cell functions and metabolic activities conferred by HVA1 which is capable of stabilizing proteins, under osmotic stress. HVA1 promotes lateral root (LR) initiation, elongation and emergence and primary root (PR) elongation via an auxin-dependent process, particularly by intensifying asymmetrical accumulation of auxin in LRP founder cells and RAM, even under ABA/stress-suppressive conditions. We demonstrate a successful application of an inducible promoter in regulating the spatial and temporal expression of HVA1 for improving root architecture and multiple stress tolerance without yield penalty. PMID:25200982

  7. CpLEA5, the Late Embryogenesis Abundant Protein Gene from Chimonanthus praecox, Possesses Low Temperature and Osmotic Resistances in Prokaryote and Eukaryotes

    PubMed Central

    Liu, Yiling; Xie, Lixia; Liang, Xilong; Zhang, Shihong

    2015-01-01

    Plants synthesize and accumulate a series of stress-resistance proteins to protect normal physiological activities under adverse conditions. Chimonanthus praecox which blooms in freezing weather accumulates late embryogenesis abundant proteins (LEAs) in flowers, but C. praecox LEAs are little reported. Here, we report a group of five LEA genes of C. praecox (CpLEA5, KT727031). Prokaryotic-expressed CpLEA5 was employed in Escherichia coli to investigate bioactivities and membrane permeability at low-temperature. In comparison with the vacant strains, CpLEA5-containing strains survived in a 20% higher rate; and the degree of cell membrane damage in CpLEA5-containing strains was 55% of that of the vacant strains according to a conductivity test, revealing the low-temperature resistance of CpLEA5 in bacteria. CpLEA5 was also expressed in Pichia pastoris. Interestingly, besides low-temperature resistance, CpLEA5 conferred high resistance to salt and alkali in CpLEA5 overexpressing yeast. The CpLEA5 gene was transferred into Arabidopsis thaliana to also demonstrate CpLEA5 actions in plants. As expected, the transgenic lines were more resistant against low-temperature and drought while compared with the wild type. Taken together, CpLEA5-conferred resistances to several conditions in prokaryote and eukaryotes could have great value as a genetic technology to enhance osmotic stress and low-temperature tolerance. PMID:26569231

  8. Depletion of complement has distinct effects on the primary and secondary antibody responses to a conjugate of pneumococcal serotype 14 capsular polysaccharide and a T-cell-dependent protein carrier.

    PubMed

    Test, Samuel T; Mitsuyoshi, Joyce K; Hu, Yong

    2005-01-01

    Complement activation plays a critical role in the immune response to T-cell-dependent and T-cell-independent antigens. However, the effect of conjugation of T-cell-dependent protein carriers to T-cell-independent type 2 antigens on the requirement for complement in the humoral immune response to such antigens remains unknown. We studied the role of complement activation on the antibody response of BALB/c mice immunized with the T-cell-independent type 2 antigen serotype 14 pneumococcal capsular polysaccharide (PPS14), either in unmodified form or conjugated to ovalbumin (OVA). In mice immunized with either PPS14 or PPS14-OVA, depletion of endogenous complement at the time of primary immunization by treatment with cobra venom factor (CVF) diminished serum anti-PPS14 concentrations after primary immunization but enhanced antibody responses after secondary immunization. The secondary immunoglobulin G (IgG) anti-PPS14 antibody response after immunization with PPS14-OVA was especially enhanced by complement depletion, was observed at doses as low as 0.2 mug of antigen, and was maximal when CVF was administered within 2 days of immunization. The avidity and opsonophagocytic functions of IgG anti-PPS14 antibodies were comparable in mice immunized with PPS14-OVA with or without complement depletion. Serum anti-PPS14 antibody concentrations were near normal, and the enhancing effects of CVF treatment on the secondary anti-PPS14 antibody response were also apparent in splenectomized mice immunized with PPS14-OVA. These results demonstrate that complement activation can have distinct effects on the primary and secondary antibody responses to a T-cell-independent type 2 antigen, either unmodified or conjugated to a T-cell-dependent protein carrier. These differences should be taken into consideration when using complement to modulate the immune response to vaccines. PMID:15618164

  9. High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding*

    PubMed Central

    Cabrera, Paula V.; Pang, Mabel; Marshall, Jamie L.; Kung, Raymond; Nelson, Stanley F.; Stalnaker, Stephanie H.; Wells, Lance; Crosbie-Watson, Rachelle H.; Baum, Linda G.

    2012-01-01

    Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function. PMID:22570487

  10. Mitochondrial genome depletion in human liver cells abolishes bile acid-induced apoptosis: role of the Akt/mTOR survival pathway and Bcl-2 family proteins.

    PubMed

    Marin, Jose J G; Hernandez, Alicia; Revuelta, Isabel E; Gonzalez-Sanchez, Ester; Gonzalez-Buitrago, Jose M; Perez, Maria J

    2013-08-01

    Acute accumulation of bile acids in hepatocytes may cause cell death. However, during long-term exposure due to prolonged cholestasis, hepatocytes may develop a certain degree of chemoresistance to these compounds. Because mitochondrial adaptation to persistent oxidative stress may be involved in this process, here we have investigated the effects of complete mitochondrial genome depletion on the response to bile acid-induced hepatocellular injury. A subline (Rho) of human hepatoma SK-Hep-1 cells totally depleted of mitochondrial DNA (mtDNA) was obtained, and bile acid-induced concentration-dependent activation of apoptosis/necrosis and survival signaling pathways was studied. In the absence of changes in intracellular ATP content, Rho cells were highly resistant to bile acid-induced apoptosis and partially resistant to bile acid-induced necrosis. In Rho cells, both basal and bile acid-induced generation of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, was decreased. Bile acid-induced proapoptotic signals were also decreased, as evidenced by a reduction in the expression ratios Bax-α/Bcl-2, Bcl-xS/Bcl-2, and Bcl-xS/Bcl-xL. This was mainly due to a downregulation of Bax-α and Bcl-xS. Moreover, in these cells the Akt/mTOR pathway was constitutively activated in a ROS-independent manner and remained similarly activated in the presence of bile acid treatment. In contrast, ERK1/2 activation was constitutively reduced and was not activated by incubation with bile acids. In conclusion, these results suggest that impaired mitochondrial function associated with mtDNA alterations, which may occur in liver cells during prolonged cholestasis, may activate mechanisms of cell survival accounting for an enhanced resistance of hepatocytes to bile acid-induced apoptosis. PMID:23597504

  11. Identification of Insulin-Like Growth Factor-I Receptor (IGF-IR) Gene Promoter-Binding Proteins in Estrogen Receptor (ER)-Positive and ER-Depleted Breast Cancer Cells

    PubMed Central

    Sarfstein, Rive; Belfiore, Antonino; Werner, Haim

    2010-01-01

    The insulin-like growth factor I receptor (IGF-IR) has been implicated in the etiology of breast cancer. Overexpression of the IGF-IR gene is a typical feature of most primary breast cancers, whereas low IGF-IR levels are seen at advanced stages. Hence, evaluation of IGF-IR levels might be important for assessing prognosis. In the present study, we employed a proteomic approach based on DNA affinity chromatography followed either by mass spectroscopy (MS) or Western blot analysis to identify transcription factors that may associate with the IGF-IR promoter in estrogen receptor (ER)-positive and ER-depleted breast cancer cells. A biotinylated IGF-IR promoter fragment was bound to streptavidin magnetic beads and incubated with nuclear extracts of breast cancer cells. IGF-IR promoter-binding proteins were eluted with high salt and analyzed by MS and Western blots. Among the proteins that were found to bind to the IGF-IR promoter we identified zinc finger transcription factors Sp1 and KLF6, ER-α, p53, c-jun, and poly (ADP-ribosylation) polymerase. Furthermore, chromatin immune-precipitation (ChIP) analysis confirmed the direct in vivo binding of some of these transcription factors to IGF-IR promoter DNA. The functional relevance of binding data was assessed by cotransfection experiments with specific expression vectors along with an IGF-IR promoter reporter. In summary, we identified nuclear proteins that are potentially responsible for the differential expression of the IGF-IR gene in ER-positive and ER-depleted breast cancer cells. PMID:24281069

  12. Analysis of genetic diversity of the heat shock protein 70 gene on the basis of abundant sequence polymorphisms in chicken breeds.

    PubMed

    Gan, J K; Jiang, L Y; Kong, L N; Zhang, X Q; Luo, Q B

    2015-01-01

    This study was designed to detect the sequence variation of the chicken heat shock protein 70 (HSP70) gene. A total of 102 individuals from 8 native Chinese breeds together with Dwarf White Chicken and Red Junglefowl were used to detect sequence variations. The coding regions of the chicken HSP70 gene from 102 individuals were cloned and sequenced. Thirty-six variations were identified, which included 34 single nucleotide polymorphisms and 2 indel mutations. Fifty-seven haplotypes were observed, of which, 43 were breed-specific and 14 were shared. There were 7 Red Junglefowl-specific haplotypes, while Haidong and Silkie only had 2 specific haplotypes. Eleven and 3 haplotypes were shared between and within species, respectively. The variation in nucleotide diversity (Pi) and average number of nucleotide differences (K) among species were consistent. The total Pi of HSP70 was 0.0016, and the total K was 4.1998. The Pi value of Red Junglefowl was the highest (0.0018) and K was 4.8000, while the Pi of Silkie was the lowest (0.0010) and K was 2.5000. These results demonstrated that variation in chicken HSP70 was abundant between and within species. PMID:25867297

  13. FcLDP1, a Gene Encoding a Late Embryogenesis Abundant (LEA) Domain Protein, Responds to Brassinosteroids and Abscisic Acid during the Development of Fruits in Fragaria chiloensis.

    PubMed

    Espinoza, Analía; Contreras, Rodrigo; Zúñiga, Gustavo E; Herrera, Raúl; Moya-León, María Alejandra; Norambuena, Lorena; Handford, Michael

    2016-01-01

    White Chilean strawberries (Fragaria chiloensis) are non-climacteric fruits, with an exotic color and aroma. In order to discover genes involved in the development of these fruits, we identified a fragment of a gene encoding a late embryogenesis abundant domain protein, FcLDP1, that was expressed in early stages of fruit development, particularly in receptacles. Hormones play key roles in regulating the development of non-climacteric fruits. We show that the brassinosteroid content of the white strawberry varies during development. Additionally, FcLDP1 as well as the closest ortholog in the woodland strawberry, F. vesca (FvLDP1) possess multiple brassinosteroid, as well as abscisic acid (ABA) response motifs in the promoter region, consistent with the response of transiently expressed FcLDP1 promoter-GFP fusions to these hormones, and the rise in FcLDP1 transcript levels in white strawberry fruits treated with brassinosteroids or ABA. These findings suggest that both hormones regulate FcLDP1 expression during the development of white strawberries. PMID:27379111

  14. FcLDP1, a Gene Encoding a Late Embryogenesis Abundant (LEA) Domain Protein, Responds to Brassinosteroids and Abscisic Acid during the Development of Fruits in Fragaria chiloensis

    PubMed Central

    Espinoza, Analía; Contreras, Rodrigo; Zúñiga, Gustavo E.; Herrera, Raúl; Moya-León, María Alejandra; Norambuena, Lorena; Handford, Michael

    2016-01-01

    White Chilean strawberries (Fragaria chiloensis) are non-climacteric fruits, with an exotic color and aroma. In order to discover genes involved in the development of these fruits, we identified a fragment of a gene encoding a late embryogenesis abundant domain protein, FcLDP1, that was expressed in early stages of fruit development, particularly in receptacles. Hormones play key roles in regulating the development of non-climacteric fruits. We show that the brassinosteroid content of the white strawberry varies during development. Additionally, FcLDP1 as well as the closest ortholog in the woodland strawberry, F. vesca (FvLDP1) possess multiple brassinosteroid, as well as abscisic acid (ABA) response motifs in the promoter region, consistent with the response of transiently expressed FcLDP1 promoter-GFP fusions to these hormones, and the rise in FcLDP1 transcript levels in white strawberry fruits treated with brassinosteroids or ABA. These findings suggest that both hormones regulate FcLDP1 expression during the development of white strawberries. PMID:27379111

  15. Elemental abundance determinations for meteors by spectroscopy.

    NASA Technical Reports Server (NTRS)

    Harvey, G. A.

    1973-01-01

    Relative elemental abundance determinations for meteors by spectroscopy are discussed. Relative abundances of spectroscopically accessible elements of four major shower meteors and one sporadic meteor are presented. A sporadic meteor with dominant sodium radiation and an iron-deficient sporadic meteor are analyzed. Empirical and theoretical tests for self-absorption in optical meteor plasmas have been conducted. Both ionization and incomplete dissociation are found to severely deplete certain neutral atoms from meteor plasmas.

  16. THE LOCAL EXPRESSION AND ABUNDANCE OF INSULIN-LIKE GROWTH FACTOR (IGF) BINDING PROTEINS IN SKELETAL MUSCLE ARE REGULATED BY AGE AND GENDER BUT NOT LOCAL IGF-I "IN VIVO"

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We wished to determine whether sustained IGF-I production in skeletal muscle increases local IGF binding protein (IGFBP) abundance, thereby mitigating the long-term stimulation of muscle growth by IGF-I. Muscle growth of transgenic mice that overexpress IGF-I in muscle (SIS2) and of wild-type (Wt) m...

  17. Increased Reactive Oxygen Species Production and Lower Abundance of Complex I Subunits and Carnitine Palmitoyltransferase 1B Protein Despite Normal Mitochondrial Respiration in Insulin-Resistant Human Skeletal Muscle

    PubMed Central

    Lefort, Natalie; Glancy, Brian; Bowen, Benjamin; Willis, Wayne T.; Bailowitz, Zachary; De Filippis, Elena A.; Brophy, Colleen; Meyer, Christian; Højlund, Kurt; Yi, Zhengping; Mandarino, Lawrence J.

    2010-01-01

    OBJECTIVE The contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle. RESEARCH DESIGN AND METHODS Mitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method. RESULTS NADH- and FADH2-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-dl-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B). CONCLUSIONS We provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance. PMID:20682693

  18. Induction of ketosis in rats fed low-carbohydrate, high-fat diets depends on the relative abundance of dietary fat and protein.

    PubMed

    Bielohuby, Maximilian; Menhofer, Dominik; Kirchner, Henriette; Stoehr, Barbara J M; Müller, Timo D; Stock, Peggy; Hempel, Madlen; Stemmer, Kerstin; Pfluger, Paul T; Kienzle, Ellen; Christ, Bruno; Tschöp, Matthias H; Bidlingmaier, Martin

    2011-01-01

    Low-carbohydrate/high-fat diets (LC-HFDs) in rodent models have been implicated with both weight loss and as a therapeutic approach to treat neurological diseases. LC-HFDs are known to induce ketosis; however, systematic studies analyzing the impact of the macronutrient composition on ketosis induction and weight loss success are lacking. Male Wistar rats were pair-fed for 4 wk either a standard chow diet or one of three different LC-HFDs, which only differed in the relative abundance of fat and protein (percentages of fat/protein in dry matter: LC-75/10; LC-65/20; LC-55/30). We subsequently measured body composition by nuclear magnetic resonance (NMR), analyzed blood chemistry and urine acetone content, evaluated gene expression changes of key ketogenic and gluconeogenic genes, and measured energy expenditure (EE) and locomotor activity (LA) during the first 4 days and after 3 wk on the respective diets. Compared with chow, rats fed with LC-75/10, LC-65/20, and LC-55/30 gained significantly less body weight. Reductions in body weight were mainly due to lower lean body mass and paralleled by significantly increased fat mass. Levels of β-hydroxybutyate were significantly elevated feeding LC-75/10 and LC-65/20 but decreased in parallel to reductions in dietary fat. Acetone was about 16-fold higher with LC-75/10 only (P < 0.001). In contrast, rats fed with LC-55/30 were not ketotic. Serum fibroblast growth factor-21, hepatic mRNA expression of hydroxymethylglutaryl-CoA-lyase, peroxisome proliferator-activated receptor-γ coactivator-1α, and peroxisome proliferator-activated receptor-γ coactivator-1β were increased with LC-75/10 only. Expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase was downregulated by 50-70% in LC-HF groups. Furthermore, EE and LA were significantly decreased in all groups fed with LC-HFDs after 3 wk on the diets. In rats, the absence of dietary carbohydrates per se does not induce ketosis. LC-HFDs must be high in fat

  19. Late Embryogenesis Abundant (LEA) Constitutes a Large and Diverse Family of Proteins Involved in Development and Abiotic Stress Responses in Sweet Orange (Citrus sinensis L. Osb.)

    PubMed Central

    Pedrosa, Andresa Muniz; Martins, Cristina de Paula Santos; Gonçalves, Luana Pereira; Costa, Marcio Gilberto Cardoso

    2015-01-01

    Late Embryogenesis Abundant (LEA) proteins are an ubiquitous group of polypeptides that were first described to accumulate during plant seed dehydration, at the later stages of embryogenesis. Since then they have also been recorded in vegetative plant tissues experiencing water limitation and in anhydrobiotic bacteria and invertebrates and, thereby, correlated with the acquisition of desiccation tolerance. This study provides the first comprehensive study about the LEA gene family in sweet orange (Citrus sinensis L. Osb.), the most important and widely grown fruit crop around the world. A surprisingly high number (72) of genes encoding C. sinensis LEAs (CsLEAs) were identified and classified into seven groups (LEA_1, LEA_2, LEA_3 and LEA_4, LEA_5, DEHYDRIN and SMP) based on their predicted amino acid sequences and also on their phylogenetic relationships with the complete set of Arabidopsis thaliana LEA proteins (AtLEAs). Approximately 60% of the CsLEAs identified in this study belongs to the unusual LEA_2 group of more hydrophobic LEA proteins, while the other LEA groups contained a relatively small number of members typically hydrophilic. A correlation between gene structure and motif composition was observed within each LEA group. Investigation of their chromosomal localizations revealed that the CsLEAs were non-randomly distributed across all nine chromosomes and that 33% of all CsLEAs are segmentally or tandemly duplicated genes. Analysis of the upstream sequences required for transcription revealed the presence of various stress-responsive cis-acting regulatory elements in the promoter regions of CsLEAs, including ABRE, DRE/CRT, MYBS and LTRE. Expression analysis using both RNA-seq data and quantitative real-time RT-PCR (qPCR) revealed that the CsLEA genes are widely expressed in various tissues, and that many genes containing the ABRE promoter sequence are induced by drought, salt and PEG. These results provide a useful reference for further exploration of

  20. Late Embryogenesis Abundant (LEA) Constitutes a Large and Diverse Family of Proteins Involved in Development and Abiotic Stress Responses in Sweet Orange (Citrus sinensis L. Osb.).

    PubMed

    Pedrosa, Andresa Muniz; Martins, Cristina de Paula Santos; Gonçalves, Luana Pereira; Costa, Marcio Gilberto Cardoso

    2015-01-01

    Late Embryogenesis Abundant (LEA) proteins are an ubiquitous group of polypeptides that were first described to accumulate during plant seed dehydration, at the later stages of embryogenesis. Since then they have also been recorded in vegetative plant tissues experiencing water limitation and in anhydrobiotic bacteria and invertebrates and, thereby, correlated with the acquisition of desiccation tolerance. This study provides the first comprehensive study about the LEA gene family in sweet orange (Citrus sinensis L. Osb.), the most important and widely grown fruit crop around the world. A surprisingly high number (72) of genes encoding C. sinensis LEAs (CsLEAs) were identified and classified into seven groups (LEA_1, LEA_2, LEA_3 and LEA_4, LEA_5, DEHYDRIN and SMP) based on their predicted amino acid sequences and also on their phylogenetic relationships with the complete set of Arabidopsis thaliana LEA proteins (AtLEAs). Approximately 60% of the CsLEAs identified in this study belongs to the unusual LEA_2 group of more hydrophobic LEA proteins, while the other LEA groups contained a relatively small number of members typically hydrophilic. A correlation between gene structure and motif composition was observed within each LEA group. Investigation of their chromosomal localizations revealed that the CsLEAs were non-randomly distributed across all nine chromosomes and that 33% of all CsLEAs are segmentally or tandemly duplicated genes. Analysis of the upstream sequences required for transcription revealed the presence of various stress-responsive cis-acting regulatory elements in the promoter regions of CsLEAs, including ABRE, DRE/CRT, MYBS and LTRE. Expression analysis using both RNA-seq data and quantitative real-time RT-PCR (qPCR) revealed that the CsLEA genes are widely expressed in various tissues, and that many genes containing the ABRE promoter sequence are induced by drought, salt and PEG. These results provide a useful reference for further exploration of

  1. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 genome editing enhances antiviral response in porcine cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Type I interferons (IFN) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF7), the master regulator of IFN transcription. The role of 4EBPs in the negat...

  2. A mode of action of glucosinolate-derived isothiocyanates: Detoxification depletes glutathione and cysteine levels with ramifications on protein metabolism in Spodoptera littoralis.

    PubMed

    Jeschke, Verena; Gershenzon, Jonathan; Vassão, Daniel Giddings

    2016-04-01

    Glucosinolates are activated plant defenses common in the order Brassicales that release isothiocyanates (ITCs) and other hydrolysis products upon tissue damage. The reactive ITCs are toxic to insects resulting in reduced growth, delayed development and occasionally mortality. Generalist lepidopteran larvae often detoxify ingested ITCs via conjugation to glutathione (GSH) and survive on low glucosinolate diets, but it is not known how this process influences other aspects of metabolism. We investigated the impact of the aliphatic 4-methylsulfinylbutyl-ITC (4msob-ITC, sulforaphane) on the metabolism of Spodoptera littoralis larvae, which suffer a significant growth decline on 4msob-ITC-containing diets while excreting ITC-glutathione conjugates and their derivatives in the frass. The most striking effects were a decrease of GSH in midgut tissue and hemolymph due to losses by conjugation to ITC during detoxification, and a decline of the GSH biosynthetic precursor cysteine. Protein content was likewise reduced by ITC treatment suggesting that protein is actively catabolized in an attempt to supply cysteine for GSH biosynthesis. The negative growth and protein effects were relieved by dietary supplementation with cystine. Other consequences of protein breakdown included deamination of amino acids with increased excretion of uric acid and elevated lipid content. Thus metabolic detoxification of ITCs provokes a cascade of negative effects on insects that result in reduced fitness. PMID:26855197

  3. Cholesterol depletion induces autophagy

    SciTech Connect

    Cheng, Jinglei; Ohsaki, Yuki; Tauchi-Sato, Kumi; Fujita, Akikazu; Fujimoto, Toyoshi . E-mail: tfujimot@med.nagoya-u.ac.jp

    2006-12-08

    Autophagy is a mechanism to digest cells' own components, and its importance in many physiological and pathological processes is being recognized. But the molecular mechanism that regulates autophagy is not understood in detail. In the present study, we found that cholesterol depletion induces macroautophagy. The cellular cholesterol in human fibroblasts was depleted either acutely using 5 mM methyl-{beta}-cyclodextrin or 10-20 {mu}g/ml nystatin for 1 h, or metabolically by 20 {mu}M mevastatin and 200 {mu}M mevalonolactone along with 10% lipoprotein-deficient serum for 2-3 days. By any of these protocols, marked increase of LC3-II was detected by immunoblotting and by immunofluorescence microscopy, and the increase was more extensive than that caused by amino acid starvation, i.e., incubation in Hanks' solution for several hours. The induction of autophagic vacuoles by cholesterol depletion was also observed in other cell types, and the LC3-positive membranes were often seen as long tubules, >50 {mu}m in length. The increase of LC3-II by methyl-{beta}-cyclodextrin was suppressed by phosphatidylinositol 3-kinase inhibitors and was accompanied by dephosphorylation of mammalian target of rapamycin. By electron microscopy, autophagic vacuoles induced by cholesterol depletion were indistinguishable from those seen after amino acid starvation. These results demonstrate that a decrease in cholesterol activates autophagy by a phosphatidylinositol 3-kinase-dependent mechanism.

  4. Depleted uranium disposal options.

    SciTech Connect

    Biwer, B. M.; Ranek, N. L.; Goldberg, M.; Avci, H. I.

    2000-04-01

    Depleted uranium hexafluoride (UF{sub 6}) has been produced in the United States since the 1940s as part of both the military program and the civilian nuclear energy program. The U.S. Department of Energy (DOE) is the agency responsible for managing most of the depleted UF{sub 6} that has been produced in the United States. The total quantity of depleted UF{sub 6} that DOE has to or will have to manage is approximately 700,000 Mg. Studies have been conducted to evaluate the various alternatives for managing this material. This paper evaluates and summarizes the alternative of disposal as low-level waste (LLW). Results of the analysis indicate that UF{sub 6} needs to be converted to a more stable form, such as U{sub 3}O{sub 8}, before disposal as LLW. Estimates of the environmental impacts of disposal in a dry environment are within the currently applicable standards and regulations. Of the currently operating LLW disposal facilities, available information indicates that either of two DOE facilities--the Hanford Site or the Nevada Test Site--or a commercial facility--Envirocare of Utah--would be able to dispose of up to the entire DOE inventory of depleted UF{sub 6}.

  5. Revisiting Antarctic Ozone Depletion

    NASA Astrophysics Data System (ADS)

    Grooß, Jens-Uwe; Tritscher, Ines; Müller, Rolf

    2015-04-01

    Antarctic ozone depletion is known for almost three decades and it has been well settled that it is caused by chlorine catalysed ozone depletion inside the polar vortex. However, there are still some details, which need to be clarified. In particular, there is a current debate on the relative importance of liquid aerosol and crystalline NAT and ice particles for chlorine activation. Particles have a threefold impact on polar chlorine chemistry, temporary removal of HNO3 from the gas-phase (uptake), permanent removal of HNO3 from the atmosphere (denitrification), and chlorine activation through heterogeneous reactions. We have performed simulations with the Chemical Lagrangian Model of the Stratosphere (CLaMS) employing a recently developed algorithm for saturation-dependent NAT nucleation for the Antarctic winters 2011 and 2012. The simulation results are compared with different satellite observations. With the help of these simulations, we investigate the role of the different processes responsible for chlorine activation and ozone depletion. Especially the sensitivity with respect to the particle type has been investigated. If temperatures are artificially forced to only allow cold binary liquid aerosol, the simulation still shows significant chlorine activation and ozone depletion. The results of the 3-D Chemical Transport Model CLaMS simulations differ from purely Lagrangian longtime trajectory box model simulations which indicates the importance of mixing processes.

  6. Cloning of human Ca2+ homoeostasis endoplasmic reticulum protein (CHERP): regulated expression of antisense cDNA depletes CHERP, inhibits intracellular Ca2+ mobilization and decreases cell proliferation.

    PubMed Central

    Laplante, J M; O'Rourke, F; Lu, X; Fein, A; Olsen, A; Feinstein, M B

    2000-01-01

    A monoclonal antibody which blocks InsP(3)-induced Ca(2+) release from isolated endoplasmic reticulum was used to isolate a novel 4.0 kb cDNA from a human erythroleukaemia (HEL) cell cDNA expression library. A corresponding mRNA transcript of approx. 4.2 kb was present in all human cell lines and tissues examined, but cardiac and skeletal muscle had an additional transcript of 6.4 kb. The identification in GenBank(R) of homologous expressed sequence tags from many tissues and organisms suggests that the gene is ubiquitously expressed in higher eukaryotes. The gene was mapped to human chromosome 19p13.1. The cDNA predicts a 100 kDa protein, designated Ca(2+) homoeostasis endoplasmic reticulum protein (CHERP), with two putative transmembrane domains, multiple consensus phosphorylation sites, a polyglutamine tract of 12 repeats and regions of imperfect tryptophan and histadine octa- and nona-peptide repeats. In vitro translation of the full-length cDNA produced proteins of M(r) 128000 and 100000, corresponding to protein bands detected by Western blotting of many cell types. CHERP was co-localized in HEL cells with the InsP(3) receptor by two-colour immunofluorescence. Transfection of HEL cells with antisense cDNA led to an 80% decline in CHERP within 5 days of antisense induction, with markedly decreased intracellular Ca(2+) mobilization by thrombin, decreased DNA synthesis and growth arrest, indicating that the protein has an important function in Ca(2+) homoeostasis, growth and proliferation. PMID:10794731

  7. Red light-regulated growth. I. Changes in the abundance of indoleacetic acid and a 22-kilodalton auxin-binding protein in the maize mesocotyl

    NASA Technical Reports Server (NTRS)

    Jones, A. M.; Cochran, D. S.; Lamerson, P. M.; Evans, M. L.; Cohen, J. D.

    1991-01-01

    We examined the changes in the levels of indoleacetic acid (IAA), IAA esters, and a 22-kilodalton subunit auxin-binding protein (ABP1) in apical mesocotyl tissue of maize (Zea mays L.) during continuous red light (R) irradiation. These changes were compared with the kinetics of R-induced growth inhibition in the same tissue. Upon the onset of continuous irradiation, growth decreased in a continuous manner following a brief lag period. The decrease in growth continued for 5 hours, then remained constant at 25% of the dark rate. The abundance of ABP1 and the level of free IAA both decreased in the mesocotyl. Only the kinetics of the decrease in IAA within the apical mesocotyl correlated with the initial change in growth, although growth continued to decrease even after IAA content reached its final level, 50% of the dark control. This decrease in IAA within the mesocotyl probably occurs primarily by a change in its transport within the shoot since auxin applied as a pulse move basipetally in R-irradiated tissue at the same rate but with half the area as dark control tissue. In situ localization of auxin in etiolated maize shoots revealed that R-irradiated shoots contained less auxin in the epidermis than the dark controls. Irradiated mesocotyl grew 50% less than the dark controls even when incubated in an optimal level of auxin. However, irradiated and dark tissue contained essentially the same amount of radioactivity after incubation in [14C]IAA indicating that the light treatment does not affect the uptake into the tissue through the cut end, although it is possible that a small subset of cells within the mesocotyl is affected. These observations support the hypothesis that R causes a decrease in the level of auxin in epidermal cells of the mesocotyl, consequently constraining the growth of the entire mesocotyl.

  8. A proteomics platform combining depletion, multi-lectin affinity chromatography (M-LAC), and isoelectric focusing to study the breast cancer proteome.

    PubMed

    Zeng, Zhi; Hincapie, Marina; Pitteri, Sharon J; Hanash, Samir; Schalkwijk, Joost; Hogan, Jason M; Wang, Hong; Hancock, William S

    2011-06-15

    The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation, and determination of treatment strategy for the disease. In this study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as to simultaneously detect glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC-MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation, and LC-MS analysis has been applied to discover breast cancer-associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component, and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies. PMID:21513341

  9. A Proteomics Platform Combining Depletion, Multi-lectin Affinity Chromatography (M-LAC) and Isoelectric Focusing to Study the Breast Cancer Proteome

    PubMed Central

    Zeng, Zhi; Hincapie, Marina; Pitteri, Sharon J.; Hanash, Samir; Schalkwijk, Joost; Hogan, Jason M.; Wang, Hon; Hancock, William S.

    2011-01-01

    The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation and determination of treatment strategy for the disease. In this study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as simultaneously detecting glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC-MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation and LC-MS analysis has been applied to discover breast cancer associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies. PMID:21513341

  10. Ozone depletion by hydrofluorocarbons

    NASA Astrophysics Data System (ADS)

    Hurwitz, Margaret M.; Fleming, Eric L.; Newman, Paul A.; Li, Feng; Mlawer, Eli; Cady-Pereira, Karen; Bailey, Roshelle

    2015-10-01

    Atmospheric concentrations of hydrofluorocarbons (HFCs) are projected to increase considerably in the coming decades. Chemistry climate model simulations forced by current projections show that HFCs will impact the global atmosphere increasingly through 2050. As strong radiative forcers, HFCs increase tropospheric and stratospheric temperatures, thereby enhancing ozone-destroying catalytic cycles and modifying the atmospheric circulation. These changes lead to a weak depletion of stratospheric ozone. Simulations with the NASA Goddard Space Flight Center 2-D model show that HFC-125 is the most important contributor to HFC-related atmospheric change in 2050; its effects are comparable to the combined impacts of HFC-23, HFC-32, HFC-134a, and HFC-143a. Incorporating the interactions between chemistry, radiation, and dynamics, ozone depletion potentials (ODPs) for HFCs range from 0.39 × 10-3 to 30.0 × 10-3, approximately 100 times larger than previous ODP estimates which were based solely on chemical effects.

  11. Chronic Glutathione Depletion Confers Protection against Alcohol-induced Steatosis: Implication for Redox Activation of AMP-activated Protein Kinase Pathway

    PubMed Central

    Chen, Ying; Singh, Surendra; Matsumoto, Akiko; Manna, Soumen K.; Abdelmegeed, Mohamed A.; Golla, Srujana; Murphy, Robert C.; Dong, Hongbin; Song, Byoung-Joon; Gonzalez, Frank J.; Thompson, David C.; Vasiliou, Vasilis

    2016-01-01

    The pathogenesis of alcoholic liver disease (ALD) is not well established. However, oxidative stress and associated decreases in levels of glutathione (GSH) are known to play a central role in ALD. The present study examines the effect of GSH deficiency on alcohol-induced liver steatosis in Gclm knockout (KO) mice that constitutively have ≈15% normal hepatic levels of GSH. Following chronic (6 week) feeding with an ethanol-containing liquid diet, the Gclm KO mice were unexpectedly found to be protected against steatosis despite showing increased oxidative stress (as reflected in elevated levels of CYP2E1 and protein carbonyls). Gclm KO mice also exhibit constitutive activation of liver AMP-activated protein kinase (AMPK) pathway and nuclear factor-erythroid 2–related factor 2 target genes, and show enhanced ethanol clearance, altered hepatic lipid profiles in favor of increased levels of polyunsaturated fatty acids and concordant changes in expression of genes associated with lipogenesis and fatty acid oxidation. In summary, our data implicate a novel mechanism protecting against liver steatosis via an oxidative stress adaptive response that activates the AMPK pathway. We propose redox activation of the AMPK may represent a new therapeutic strategy for preventing ALD. PMID:27403993

  12. Chronic Glutathione Depletion Confers Protection against Alcohol-induced Steatosis: Implication for Redox Activation of AMP-activated Protein Kinase Pathway.

    PubMed

    Chen, Ying; Singh, Surendra; Matsumoto, Akiko; Manna, Soumen K; Abdelmegeed, Mohamed A; Golla, Srujana; Murphy, Robert C; Dong, Hongbin; Song, Byoung-Joon; Gonzalez, Frank J; Thompson, David C; Vasiliou, Vasilis

    2016-01-01

    The pathogenesis of alcoholic liver disease (ALD) is not well established. However, oxidative stress and associated decreases in levels of glutathione (GSH) are known to play a central role in ALD. The present study examines the effect of GSH deficiency on alcohol-induced liver steatosis in Gclm knockout (KO) mice that constitutively have ≈15% normal hepatic levels of GSH. Following chronic (6 week) feeding with an ethanol-containing liquid diet, the Gclm KO mice were unexpectedly found to be protected against steatosis despite showing increased oxidative stress (as reflected in elevated levels of CYP2E1 and protein carbonyls). Gclm KO mice also exhibit constitutive activation of liver AMP-activated protein kinase (AMPK) pathway and nuclear factor-erythroid 2-related factor 2 target genes, and show enhanced ethanol clearance, altered hepatic lipid profiles in favor of increased levels of polyunsaturated fatty acids and concordant changes in expression of genes associated with lipogenesis and fatty acid oxidation. In summary, our data implicate a novel mechanism protecting against liver steatosis via an oxidative stress adaptive response that activates the AMPK pathway. We propose redox activation of the AMPK may represent a new therapeutic strategy for preventing ALD. PMID:27403993

  13. Demonstration of jackhammer incorporating depleted uranium

    SciTech Connect

    Fischer, L E; Hoard, R W; Carter, D L; Saculla, M D; Wilson, G V

    2000-04-01

    The United States Government currently has an abundance of depleted uranium (DU). This surplus of about 1 billion pounds is the result of an enrichment process using gaseous diffusion to produce enriched and depleted uranium. The enriched uranium has been used primarily for either nuclear weapons for the military or nuclear fuel for the commercial power industry. Most of the depleted uranium remains at the enrichment process plants in the form of depleted uranium hexafluoride (DUF{sub 6}). The Department of Energy (DOE) recently began a study to identify possible commercial applications for the surplus material. One of these potential applications is to use the DU in high-density strikers/hammers in pneumatically driven tools, such as jack hammers and piledrivers to improve their impulse performance. The use of DU could potentially increase tunneling velocity and excavation into target materials with improved efficiency. This report describes the efforts undertaken to analyze the particulars of using DU in two specific striking applications: the jackhammer and chipper tool.

  14. Elemental Depletions in the Magellanic Clouds and the Evolution of Depletions with Metallicity

    NASA Astrophysics Data System (ADS)

    Tchernyshyov, Kirill; Meixner, Margaret; Seale, Jonathan; Fox, Andrew; Friedman, Scott D.; Dwek, Eli; Galliano, Frédéric

    2015-10-01

    We present a study of the composition of gas and dust in the Large and Small Magellanic Clouds (LMC and SMC) using UV absorption spectroscopy. We measure P ii and Fe ii along 84 spatially distributed sightlines toward the MCs using archival Far Ultraviolet Spectroscopic Explorer observations. For 16 of those sightlines, we also measure Si ii, Cr ii, and Zn ii from new Hubble Space Telescope Cosmic Origins Spectrograph observations. We analyze these spectra using a new spectral line analysis technique based on a semi-parametric Voigt profile model. We have combined these measurements with H i and H2 column densities and reference stellar abundances from the literature to derive gas-phase abundances, depletions, and gas-to-dust ratios (GDRs). Of our 84 P and 16 Zn measurements, 80 and 13, respectively, are depleted by more than 0.1 dex, suggesting that P and Zn abundances are not accurate metallicity indicators at and above the metallicity of the SMC. Si, Cr, and Fe are systematically less depleted in the SMC than in the Milky Way (MW) or LMC. The minimum Si depletion in the SMC is consistent with zero. We find GDR ranges of 190-565 in the LMC and 480-2100 in the SMC, which is broadly consistent with GDRs from the literature. These ranges represent actual location to location variation and are evidence of dust destruction and/or growth in the diffuse neutral phase of the interstellar medium. Where they overlap in metallicity, the gas-phase abundances of the MW, LMC, and SMC and damped Lyα systems evolve similarly with metallicity.

  15. The local expression and abundance of insulin-like growth factor (IGF) binding proteins in skeletal muscle are regulated by age and gender but not local IGF-I in vivo.

    PubMed

    Oliver, William T; Rosenberger, Judy; Lopez, Rusmely; Gomez, Adam; Cummings, Kathleen K; Fiorotto, Marta L

    2005-12-01

    We wished to determine whether sustained IGF-I production in skeletal muscle increases local IGF binding protein (IGFBP) abundance, thereby mitigating the long-term stimulation of muscle growth by IGF-I. Muscle growth of transgenic mice that overexpress IGF-I in muscle (SIS2) and of wild-type (Wt) mice was compared. At 3, 5, 10, and 20 wk of age, hind-limb muscle weights and IGFBP-3, -4, -5, and -6 mRNA and protein abundances were quantified. Additional mice were injected with IGF-I or LR3-IGF-I, and phosphorylation of the type 1 IGF receptor (IGF-1R) was compared. Muscle mass was 20% greater in SIS2 compared with Wt mice by 10 wk of age (P < 0.01), and this difference was maintained to 20 wk. IGFBP mRNA and protein abundances were unaffected by genotype. IGFBP-4 and -5 protein abundances increased with age, whereas for IGFBP-3 and -6, there was a sexual dimorphic response (P < 0.01); after 5 wk of age, IGFBP-3 decreased in males but increased in females, whereas IGFBP-6 decreased in females and remained unchanged in males. These protein expression patterns resulted from differences at both the transcriptional and posttranscriptional levels. LR3-IGF-I stimulated IGF-1R phosphorylation to a greater extent than IGF-I at both 5 and 10 wk of age (P < 0.01), regardless of gender or genotype (P > 0.21). Thus, variations in local IGF-I levels do not appear to regulate muscle IGFBP expression. The age- and gender-specific differences in muscle IGFBP expression are not sufficient to alter the response of the muscle to the IGFs but may impact the IGF-independent effects of these IGFBPs. PMID:16166219

  16. Neutrophil depletion in the early inflammatory phase delayed cutaneous wound healing in older rats: improvements due to the use of un-denatured camel whey protein

    PubMed Central

    2014-01-01

    Background While it is known that advanced age alters the recruitment of neutrophils during wound healing, thereby delaying the wound healing process, little is known about prolonged wound healing in advanced ages. Thus, we investigated the correlation of neutrophil recruitment with healing events, and the impact of whey protein (WP) on neutrophil activation. Methods The animals were allocated into wounded young group, wounded older group and wounded older rats with daily treatment of WP at a dose of 100 mg/kg of body weight. Results Our results pointed to a marked deficiency in the number of neutrophils in the wounds of older rats, which was accompanied with impairment of the healing process. In the group of older rats, phagocytic activity, as tested by fluorescence microscopy, declined throughout the first 24 hours after wounding. Both the neutrophil number and the phagocytic activity recovered in older rats which received WP supplementation. Interestingly, WP was found to significantly up-regulate the MIP-1α and CINC-1 mRNA expression in old rats. On the other hand, the wound size in older rats was significantly higher than that in younger ones. Blood angiogenesis was also significantly delayed in the older group as opposed to the young rats. WP, however, was found to return these indices to normal levels in the older rats. Proliferation and epidermal migration of the keratinocytes and the collagen deposition were also returned to the normal rates. Conclusions This data confirms the critical role of neutrophil recruitment in the early inflammatory phase of wound healing in older rats. In addition, WP protein was used to improve neutrophil function in older rats, healing events returned to a more normal profile. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2100966986117779. PMID:24593823

  17. Pvlea-18, a Member of a New Late-Embryogenesis-Abundant Protein Family That Accumulates during Water Stress and in the Growing Regions of Well-Irrigated Bean Seedlings1

    PubMed Central

    Colmenero-Flores, José M.; Moreno, Liz P.; Smith, Claudia E.; Covarrubias, Alejandra A.

    1999-01-01

    Pvlea-18 is a novel stress gene whose transcript is present in the dry embryo and the endosperm from bean (Phaseolus vulgaris) seeds. It accumulates in vegetative tissues in response to water deficit and abscisic acid application (J.M. Colmenero-Flores, F. Campos, A. Garciarrubio, A.A. Covarrubias [1997] Plant Mol Biol 35: 393–405). We show that the Pvlea-18 gene encodes a 14-kD protein that accumulates during late embryogenesis. Related proteins have been detected in both monocots and dicots, indicating that PvLEA-18 is a member of a new family of LEA (Late Embryogenesis Abundant) proteins. We also show that the PvLEA-18 transcript and protein accumulate not only in different organs of the bean seedlings during water stress but also in well-irrigated seedlings. This accumulation occurs in seedling regions with more negative values of water and osmotic potentials, such as the growing region of the hypocotyl. This phenomenon has not previously been described for LEA proteins. Immunohistochemical localization showed that the PvLEA-18 protein is present in the nucleus and cytoplasm of all cell types, with a higher accumulation in the epidermis and vascular cylinder tissues, particularly in protoxylem cells and root meristematic tissues. We found a similar localization but a higher abundance in water-stressed seedlings. PMID:10318687

  18. Lithium Depletion in the Beta Pictoris Moving Group

    NASA Astrophysics Data System (ADS)

    Yee, Jennifer C.; Jensen, E. L.; Reaser, B. E.

    2006-12-01

    We present a study of lithium depletion in twelve late-type pre-main-sequence stars in the coeval Beta Pictoris Moving Group (BPMG). The age of this group ( 12 Myr) is well constrained because all of the stars in the sample have Hipparcos distances. We have determined Li abundances for these K and M stars using equivalent width measurements of the 6707.8 Angstrom Li I line from new high-resolution, high-S/N echelle spectra, and we compare these abundances to models of pre-main-sequence Li depletion by Baraffe et al. (1998), D'Antona & Mazzitelli (1997, 1998), and Siess, Dufour, & Forestini (2000). Significantly more lithium depletion is observed in the sample than is predicted for a group of this age. In particular, the discrepancy between the predicted and the observed lithium abundances increases with decreasing effective temperature, suggesting a problem with theories describing pre-main-sequence lithium depletion. Our data indicate that M stars deplete lithium more rapidly than predicted, which could make M-type post-T-Tauri stars difficult to identify. In addition, we compare our results to the work of Song, Bessell, & Zuckerman (2002) on HIP 112312. In contrast to that work, we did not observe the lithium depletion boundary of the BPMG; none of the three M4.5 stars in the sample showed evidence of lithium (log N(Li) < -0.5), indicating a lithium depletion boundary later than M4.5, further underscoring the gap between age estimates from lithium depletion and those from theoretical evolutionary tracks. We gratefully acknowledge the support of the National Science Foundation through grant AST-0307830.

  19. Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast.

    PubMed

    Xu, Yong-Jie

    2016-04-01

    DNA replication checkpoint is a highly conserved cellular signaling pathway critical for maintaining genome integrity in eukaryotes. It is activated when DNA replication is perturbed. In Schizosaccharomyces pombe, perturbed replication forks activate the sensor kinase Rad3 (ATR/Mec1), which works cooperatively with mediator Mrc1 and the 9-1-1 checkpoint clamp to phosphorylate the effector kinase Cds1 (CHK2/Rad53). Phosphorylation of Cds1 promotes autoactivation of the kinase. Activated Cds1 diffuses away from the forks and stimulates most of the checkpoint responses under replication stress. Although this signaling pathway has been well understood in fission yeast, how the signaling is initiated and thus regulated remains incompletely understood. Previous studies have shown that deletion of lem2(+) sensitizes cells to the inhibitor of ribonucleotide reductase, hydroxyurea. However, the underlying mechanism is still not well understood. This study shows that in the presence of hydroxyurea, Lem2 facilitates Rad3-mediated checkpoint signaling for Cds1 activation. Without Lem2, all known Rad3-dependent phosphorylations critical for replication checkpoint signaling are seriously compromised, which likely causes the aberrant mitosis and drug sensitivity observed in this mutant. Interestingly, the mutant is not very sensitive to DNA damage and the DNA damage checkpoint remains largely intact, suggesting that the main function of Lem2 is to facilitate checkpoint signaling in response to replication stress. Since Lem2 is an inner nuclear membrane protein, these results also suggest that the replication checkpoint may be spatially regulated inside the nucleus, a previously unknown mechanism. PMID:26746798

  20. Depleted uranium management alternatives

    SciTech Connect

    Hertzler, T.J.; Nishimoto, D.D.

    1994-08-01

    This report evaluates two management alternatives for Department of Energy depleted uranium: continued storage as uranium hexafluoride, and conversion to uranium metal and fabrication to shielding for spent nuclear fuel containers. The results will be used to compare the costs with other alternatives, such as disposal. Cost estimates for the continued storage alternative are based on a life-cycle of 27 years through the year 2020. Cost estimates for the recycle alternative are based on existing conversion process costs and Capital costs for fabricating the containers. Additionally, the recycle alternative accounts for costs associated with intermediate product resale and secondary waste disposal for materials generated during the conversion process.

  1. Tank depletion flow controller

    DOEpatents

    Georgeson, Melvin A.

    1976-10-26

    A flow control system includes two bubbler tubes installed at different levels within a tank containing such as radioactive liquid. As the tank is depleted, a differential pressure transmitter monitors pressure differences imparted by the two bubbler tubes at a remote, shielded location during uniform time intervals. At the end of each uniform interval, balance pots containing a dense liquid are valved together to equalize the pressures. The resulting sawtooth-shaped signal generated by the differential pressure transmitter is compared with a second sawtooth signal representing the desired flow rate during each time interval. Variations in the two signals are employed by a control instrument to regulate flow rate.

  2. Platelet depletion and severity of streptococcal endocarditis

    PubMed Central

    Dall, Lawrence; Miller, Todd; Herndon, Betty; Diez, Ireneo; Dew, Michelle

    1998-01-01

    OBJECTIVE: To evaluate the importance of thrombocytopenia in streptococcal endocarditis using an animal model. DESIGN: A model of human septic endocarditis was established in rats (polyethylene catheters across the aortic valve and administration of Streptococcus sanguis, 5×107 colony forming units [cfu] intravenous). Thrombocytopenia at four levels was produced by antiplatelet serum. Secondary methods of producing thrombocytopenia were also evaluated. At sacrifice (96 h after platelet depletion and 72 h after infection), vegetations were removed, weighed, diluted, plated and counted. Potential mechanisms of the dose-response relationship between vegetation density and platelet count were evaluated. SETTING: Controlled research laboratory experiments. POPULATION STUDIED: Animal models of streptococcal endocarditis. MAIN RESULTS: The bacterial density of the aortic valve vegetations significantly increased as the platelet count decreased (P=0.0007). In severely thrombocytopenic animals (two-dose antiplatelet serum), data suggest increased vegetation embolism. Platelet depletion, which was minimal with chemical methods, was produced most effectively by antithrombocyte serum. Platelet surfaces in endocarditis were found to express elevated CD62p proteins (72.7% endocarditis, 34.7% control). Platelet protein fractions were evaluated in vitro by both streptocidal (P=0.19) and phagocytosis-stimulating assays. Platelet presence in mature aortic valve vegetations averaged only about 2%. CONCLUSIONS: In platelet depletion experiments using a rat model, a dose-response relationship of peripheral circulating platelet depletion to aortic valve vegetation density was found. The mechanism relating thrombocytopenia to endocarditis severity remains unresolved. PMID:22346555

  3. How Depleted is the MORB mantle?

    NASA Astrophysics Data System (ADS)

    Hofmann, A. W.; Hart, S. R.

    2015-12-01

    Knowledge of the degree of mantle depletion of highly incompatible elements is critically important for assessing Earth's internal heat production and Urey number. Current views of the degree of MORB source depletion are dominated by Salters and Stracke (2004), and Workman and Hart (2005). The first is based on an assessment of average MORB compositions, whereas the second considers trace element data of oceanic peridotites. Both require an independent determination of one absolute concentration, Lu (Salters & Stracke), or Nd (Workman & Hart). Both use parent-daughter ratios Lu/Hf, Sm/Nd, and Rb/Sr calculated from MORB isotopes combined with continental-crust extraction models, as well as "canonical" trace element ratios, to boot-strap the full range of trace element abundances. We show that the single most important factor in determining the ultimate degree of incompatible element depletion in the MORB source lies in the assumptions about the timing of continent extraction, exemplified by continuous extraction versus simple two-stage models. Continued crust extraction generates additional, recent mantle depletion, without affecting the isotopic composition of the residual mantle significantly. Previous emphasis on chemical compositions of MORB and/or peridotites has tended to obscure this. We will explore the effect of different continent extraction models on the degree of U, Th, and K depletion in the MORB source. Given the uncertainties of the two most popular models, the uncertainties of U and Th in DMM are at least ±50%, and this impacts the constraints on the terrestrial Urey ratio. Salters, F.J.M. and Stracke, A., 2004, Geochem. Geophys. Geosyst. 5, Q05004. Workman, R.K. and Hart, S.R., 2005, EPSL 231, 53-72.

  4. RuBisCO depletion improved proteome coverage of cold responsive S-nitrosylated targets in Brassica juncea

    PubMed Central

    Sehrawat, Ankita; Abat, Jasmeet K.; Deswal, Renu

    2013-01-01

    Although in the last few years good number of S-nitrosylated proteins are identified but information on endogenous targets is still limiting. Therefore, an attempt is made to decipher NO signaling in cold treated Brassica juncea seedlings. Treatment of seedlings with substrate, cofactor and inhibitor of Nitric-oxide synthase and nitrate reductase (NR), indicated NR mediated NO biosynthesis in cold. Analysis of the in vivo thiols showed depletion of low molecular weight thiols and enhancement of available protein thiols, suggesting redox changes. To have a detailed view, S-nitrosylation analysis was done using biotin switch technique (BST) and avidin-affinity chromatography. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is S-nitrosylated and therefore, is identified as target repeatedly due to its abundance. It also competes out low abundant proteins which are important NO signaling components. Therefore, RuBisCO was removed (over 80%) using immunoaffinity purification. Purified S-nitrosylated RuBisCO depleted proteins were resolved on 2-D gel as 110 spots, including 13 new, which were absent in the crude S-nitrosoproteome. These were identified by nLC-MS/MS as thioredoxin, fructose biphosphate aldolase class I, myrosinase, salt responsive proteins, peptidyl-prolyl cis-trans isomerase and malate dehydrogenase. Cold showed differential S-nitrosylation of 15 spots, enhanced superoxide dismutase activity (via S-nitrosylation) and promoted the detoxification of superoxide radicals. Increased S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase sedoheptulose-biphosphatase, and fructose biphosphate aldolase, indicated regulation of Calvin cycle by S-nitrosylation. The results showed that RuBisCO depletion improved proteome coverage and provided clues for NO signaling in cold. PMID:24032038

  5. RuBisCO depletion improved proteome coverage of cold responsive S-nitrosylated targets in Brassica juncea.

    PubMed

    Sehrawat, Ankita; Abat, Jasmeet K; Deswal, Renu

    2013-01-01

    Although in the last few years good number of S-nitrosylated proteins are identified but information on endogenous targets is still limiting. Therefore, an attempt is made to decipher NO signaling in cold treated Brassica juncea seedlings. Treatment of seedlings with substrate, cofactor and inhibitor of Nitric-oxide synthase and nitrate reductase (NR), indicated NR mediated NO biosynthesis in cold. Analysis of the in vivo thiols showed depletion of low molecular weight thiols and enhancement of available protein thiols, suggesting redox changes. To have a detailed view, S-nitrosylation analysis was done using biotin switch technique (BST) and avidin-affinity chromatography. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is S-nitrosylated and therefore, is identified as target repeatedly due to its abundance. It also competes out low abundant proteins which are important NO signaling components. Therefore, RuBisCO was removed (over 80%) using immunoaffinity purification. Purified S-nitrosylated RuBisCO depleted proteins were resolved on 2-D gel as 110 spots, including 13 new, which were absent in the crude S-nitrosoproteome. These were identified by nLC-MS/MS as thioredoxin, fructose biphosphate aldolase class I, myrosinase, salt responsive proteins, peptidyl-prolyl cis-trans isomerase and malate dehydrogenase. Cold showed differential S-nitrosylation of 15 spots, enhanced superoxide dismutase activity (via S-nitrosylation) and promoted the detoxification of superoxide radicals. Increased S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase sedoheptulose-biphosphatase, and fructose biphosphate aldolase, indicated regulation of Calvin cycle by S-nitrosylation. The results showed that RuBisCO depletion improved proteome coverage and provided clues for NO signaling in cold. PMID:24032038

  6. Improved method for identification of low abundance proteins using 2D-gel electrophoresis, MALDI-TOF and TOF/TOF

    EPA Science Inventory

    Introduction: Differential protein expression studies have been routinely performed in our laboratory to determine the health effects of environmentally-important chemicals. In this abstract, improvements in the in-gel protein digestion, MALDI plate spotting and data acquisition...

  7. Proteomic identification of germline proteins in Caenorhabditis elegans

    PubMed Central

    Turner, B Elizabeth; Basecke, Sophia M; Bazan, Grace C; Dodge, Eric S; Haire, Cassy M; Heussman, Dylan J; Johnson, Chelsey L; Mukai, Chelsea K; Naccarati, Adrianna M; Norton, Sunny-June; Sato, Jennifer R; Talavera, Chihara O; Wade, Michael V; Hillers, Kenneth J

    2015-01-01

    Sexual reproduction involves fusion of 2 haploid gametes to form diploid offspring with genetic contributions from both parents. Gamete formation represents a unique developmental program involving the action of numerous germline-specific proteins. In an attempt to identify novel proteins involved in reproduction and embryonic development, we have carried out a proteomic characterization of the process in Caenorhabditis elegans. To identify candidate proteins, we used 2D gel electrophoresis (2DGE) to compare protein abundance in nucleus-enriched extracts from wild-type C. elegans, and in extracts from mutant worms with greatly reduced gonads (glp-4(bn2) worms reared at 25°C); 84 proteins whose abundance correlated with germline presence were identified. To validate candidates, we used feeding RNAi to deplete candidate proteins, and looked for reduction in fertility and/or germline cytological defects. Of 20 candidates so screened for involvement in fertility, depletion of 13 (65%) caused a significant reduction in fertility, and 6 (30%) resulted in sterility (<5 % of wild-type fertility). Five of the 13 proteins with demonstrated roles in fertility have not previously been implicated in germline function. The high frequency of defects observed after RNAi depletion of candidate proteins suggests that this approach is effective at identifying germline proteins, thus contributing to our understanding of this complex organ. PMID:26435885

  8. Endoplasmic-Reticulum Calcium Depletion and Disease

    PubMed Central

    Mekahli, Djalila; Bultynck, Geert; Parys, Jan B.; De Smedt, Humbert; Missiaen, Ludwig

    2011-01-01

    The endoplasmic reticulum (ER) as an intracellular Ca2+ store not only sets up cytosolic Ca2+ signals, but, among other functions, also assembles and folds newly synthesized proteins. Alterations in ER homeostasis, including severe Ca2+ depletion, are an upstream event in the pathophysiology of many diseases. On the one hand, insufficient release of activator Ca2+ may no longer sustain essential cell functions. On the other hand, loss of luminal Ca2+ causes ER stress and activates an unfolded protein response, which, depending on the duration and severity of the stress, can reestablish normal ER function or lead to cell death. We will review these various diseases by mainly focusing on the mechanisms that cause ER Ca2+ depletion. PMID:21441595

  9. Ozone Depletion by Hydrofluorocarbons

    NASA Astrophysics Data System (ADS)

    Hurwitz, M.; Fleming, E. L.; Newman, P. A.; Li, F.; Mlawer, E. J.; Cady-Pereira, K. E.; Bailey, R.

    2015-12-01

    Hydrofluorocarbons (HFCs) are second-generation replacements for the chlorofluorocarbons (CFCs), halons and other substances that caused the 'ozone hole'. Atmospheric concentrations of HFCs are projected to increase dramatically in the coming decades. Coupled chemistry-climate simulations forced by these projections show that HFCs will impact the global atmosphere in 2050. As strong radiative forcers, HFCs modulate atmospheric temperature, thereby changing ozone-destroying catalytic cycles and enhancing the stratospheric circulation. These changes lead to a weak depletion of stratospheric ozone. Sensitivity simulations with the NASA Goddard Space Flight Center (GSFC) 2D model show that HFC-125 is the most important contributor to atmospheric change in 2050, as compared with HFC-23, HFC-32, HFC-134a and HFC-143a. Incorporating the interactions between chemistry, radiation and dynamics, for a likely 2050 climate, ozone depletion potentials (ODPs) for HFCs range from 4.3x10-4 to 3.5x10-2; previously HFCs were assumed to have negligible ODPs since these species lack chlorine or bromine atoms. The ozone impacts of HFCs are further investigated with the Goddard Earth Observing System Chemistry-Climate Model (GEOSCCM). The GEOSCCM is a three-dimensional, fully coupled ocean-atmosphere model with interactive stratospheric chemistry. Sensitivity simulations in which CO2, CFC-11 and HCFC-22 are enhanced individually are used as proxies for the atmospheric response to the HFC concentrations expected by the mid-21st century. Sensitivity simulations provide quantitative estimates of the impacts of these greenhouse gases on global total ozone, and can be used to assess their effects on the recovery of Antarctic ozone.

  10. Surface abundances of OC supergiants

    NASA Astrophysics Data System (ADS)

    Martins, F.; Foschino, S.; Bouret, J.-C.; Barbá, R.; Howarth, I.

    2016-04-01

    Context. Some O and B stars show unusually strong or weak lines of carbon and/or nitrogen. These objects are classified as OBN or OBC stars. It has recently been shown that nitrogen enrichment and carbon depletion are the most likely explanations for the existence of the ON class. Aims: We investigate OC stars (all being supergiants) to check that surface abundances are responsible for the observed anomalous line strengths. Methods: We perform a spectroscopic analysis of three OC supergiants using atmosphere models. A fourth star was previously studied by us. Our sample thus comprises all OC stars known to date in the Galaxy. We determine the stellar parameters and He, C, N, and O surface abundances. Results: We show that all stars have effective temperatures and surface gravities fully consistent with morphologically normal O supergiants. However, OC stars show little, if any, nitrogen enrichment and carbon surface abundances consistent with the initial composition. OC supergiants are thus barely chemically evolved, unlike morphologically normal O supergiants. Based on observations obtained at the ESO/La Silla Observatory under programs 081.D-2008, 083.D-0589, 089.D-0975.

  11. Modelling CO depletion in starless cores

    NASA Astrophysics Data System (ADS)

    Rawlings, Jonathan M. C.

    In recent years there has been a dramatic increase in the observational detection of molecular depletion in star-forming dark clouds. In many cases the data is of very high quality and what was once considered a rather hypothetical process is now almost universally accepted as a (the) major cause of the presence of emission 'holes' in molecular maps of dense cores. However, the interpretation of the data can be severely undermined by uncertainties in the physics and chemistry. This is particularly true in the case of general molecular studies of active star-forming regions. For these objects there exist strong degeneracies between various chemical effects (gas-phase time-dependencies, desorption processes and efficiencies, ionization rates etc.) and poorly constrained physics (most particularly in the assumed kinematics and evolutionary history). Whilst these problems result in unacceptable ambiguities in the case of evolved sources, we can make significant progress for young, near-static cores and using molecular species with simple chemistries. A considerable set of constraints on the free parameters is now provided by the extensive sub-millimetre continuum and infra-red absorption studies of starless cores. These observations give us good descriptions of the temperature and density profiles in these sources. Moreover CO is to a large extent chemically inert, so that any decline of abundance at high densities can (primarily) be interpreted as being a consequence of freeze-out. Thus there are only two major free parameters:- the net depletion/desorption rate and the chemical age of the source. In this study CO abundance profiles are calculated as a function of time for cores whose temperature and density profiles have already been determined. The results are corrected for excitation effects and converted to synthetic maps, assuming typical single-dish beam parameters and source characteristics. A strong correlation with existing depletion maps is found and strong

  12. CO depletion in ATLASGAL-selected high-mass clumps

    NASA Astrophysics Data System (ADS)

    Giannetti, A.; Wyrowski, F.; Brand, J.; Csengeri, T.; Fontani, F.; Walmsley, C. M.; Nguyen Luong, Q.; Beuther, H.; Schuller, F.; Güsten, R.; Menten, K. M.

    2016-05-01

    In the low-mass regime, it is found that the gas-phase abundances of C-bearing molecules in cold starless cores rapidly decrease with increasing density. Here the molecules tend to stick to the grains, forming ice mantles. We study CO depletion in the TOP100 sample of the ATLASGAL survey, and investigate its correlation with evolutionary stage and with the physical parameters of the sources. We use low-J emission lines of CO isotopologues and the dust continuum emission to infer the depletion factor fD. RATRAN one-dimensional models were also used to determine fD and to investigate the presence of depletion above a density threshold. The isotopic ratios and optical depth were derived with a Bayesian approach. We find a significant number of clumps with a large CO depletion, up to ˜20. Larger values are found for colder clumps, thus for earlier evolutionary phases. For massive clumps in the earliest stages of evolution we estimate the radius of the region where CO depletion is important to be a few tenths of a pc. CO depletion in high-mass clumps seems to behave as in the low-mass regime, with less evolved clumps showing larger values for the depletion than their more evolved counterparts, and increasing for denser sources.

  13. Coronae of Stars with Supersolar Elemental Abundances

    NASA Technical Reports Server (NTRS)

    Peretz, Uria; Behar, Ehud; Drake, Stephen A.

    2015-01-01

    Coronal elemental abundances are known to deviate from the photospheric values of their parent star, with the degree of deviation depending on the first ionization potential (FIP). This study focuses on the coronal composition of stars with supersolar photospheric abundances. We present the coronal abundances of six such stars: 11 LMi, iota Hor, HR 7291, tau Boo, and alpha Cen A and B. These stars all have high-statistics X-ray spectra, three of which are presented for the first time. The abundances we measured were obtained using the line-resolved spectra of the Reflection Grating Spectrometer (RGS) in conjunction with the higher throughput EPIC-pn camera spectra onboard the XMM-Newton observatory. A collisionally ionized plasma model with two or three temperature components is found to represent the spectra well. All elements are found to be consistently depleted in the coronae compared to their respective photospheres. For 11 LMi and tau Boo no FIP effect is present, while iota Hor, HR 7291, and alpha Cen A and B show a clear FIP trend. These conclusions hold whether the comparison is made with solar abundances or the individual stellar abundances. Unlike the solar corona, where low-FIP elements are enriched, in these stars the FIP effect is consistently due to a depletion of high-FIP elements with respect to actual photospheric abundances. A comparison with solar (instead of stellar) abundances yields the same fractionation trend as on the Sun. In both cases, a similar FIP bias is inferred, but different fractionation mechanisms need to be invoked.

  14. Coronae of stars with supersolar elemental abundances

    NASA Astrophysics Data System (ADS)

    Peretz, Uria; Behar, Ehud; Drake, Stephen A.

    2015-05-01

    Coronal elemental abundances are known to deviate from the photospheric values of their parent star, with the degree of deviation depending on the first ionization potential (FIP). This study focuses on the coronal composition of stars with supersolar photospheric abundances. We present the coronal abundances of six such stars: 11 LMi, ι Hor, HR 7291, τ Boo, and α Cen A and B. These stars all have high-statistics X-ray spectra, three of which are presented for the first time. The abundances we measured were obtained using the line-resolved spectra of the Reflection Grating Spectrometer (RGS) in conjunction with the higher throughput EPIC-pn camera spectra onboard the XMM-Newton observatory. A collisionally ionized plasma model with two or three temperature components is found to represent the spectra well. All elements are found to be consistently depleted in the coronae compared to their respective photospheres. For 11 LMi and τ Boo no FIP effect is present, while ι Hor, HR 7291, and α Cen A and B show a clear FIP trend. These conclusions hold whether the comparison is made with solar abundances or the individual stellar abundances. Unlike the solar corona, where low-FIP elements are enriched, in these stars the FIP effect is consistently due to a depletion of high-FIP elements with respect to actual photospheric abundances. A comparison with solar (instead of stellar) abundances yields the same fractionation trend as on the Sun. In both cases, a similar FIP bias is inferred, but different fractionation mechanisms need to be invoked.

  15. 12. VIEW OF DEPLETED URANIUM INGOT AND MOLDS. DEPLETED URANIUM ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. VIEW OF DEPLETED URANIUM INGOT AND MOLDS. DEPLETED URANIUM CASTING OPERATIONS CEASED IN 1988. (11/14/57) - Rocky Flats Plant, Non-Nuclear Production Facility, South of Cottonwood Avenue, west of Seventh Avenue & east of Building 460, Golden, Jefferson County, CO

  16. Iron depletion suppresses mTORC1-directed signalling in intestinal Caco-2 cells via induction of REDD1.

    PubMed

    Watson, Ailsa; Lipina, Christopher; McArdle, Harry J; Taylor, Peter M; Hundal, Harinder S

    2016-05-01

    Iron is an indispensable micronutrient that regulates many aspects of cell function, including growth and proliferation. These processes are critically dependent upon signalling via the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Herein, we test whether iron depletion induced by cell incubation with the iron chelator, deferoxamine (DFO), mediates its effects on cell growth through mTORC1-directed signalling and protein synthesis. We have used Caco-2 cells, a well-established in vitro model of human intestinal epithelia. Iron depletion increased expression of iron-regulated proteins (TfR, transferrin receptor and DMT1, divalent metal transporter, as predicted, but it also promoted a marked reduction in growth and proliferation of Caco-2 cells. This was strongly associated with suppressed mTORC1 signalling, as judged by reduced phosphorylation of mTOR substrates, S6K1 and 4E-BP1, and diminished protein synthesis. The reduction in mTORC1 signalling was tightly coupled with increased expression and accumulation of REDD1 (regulated in DNA damage and development 1) and reduced phosphorylation of Akt and TSC2. The increase in REDD1 abundance was rapidly reversed upon iron repletion of cells but was also attenuated by inhibitors of gene transcription, protein phosphatase 2A (PP2A) and by REDD1 siRNA--strategies that also antagonised the loss in mTORC1 signalling associated with iron depletion. Our findings implicate REDD1 and PP2A as crucial regulators of mTORC1 activity in iron-depleted cells and indicate that their modulation may help mitigate atrophy of the intestinal mucosa that may occur in response to iron deficiency. PMID:26827808

  17. Iron depletion suppresses mTORC1-directed signalling in intestinal Caco-2 cells via induction of REDD1

    PubMed Central

    Watson, Ailsa; Lipina, Christopher; McArdle, Harry J.; Taylor, Peter M.; Hundal, Harinder S.

    2016-01-01

    Iron is an indispensable micronutrient that regulates many aspects of cell function, including growth and proliferation. These processes are critically dependent upon signalling via the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Herein, we test whether iron depletion induced by cell incubation with the iron chelator, deferoxamine (DFO), mediates its effects on cell growth through mTORC1-directed signalling and protein synthesis. We have used Caco-2 cells, a well-established in vitro model of human intestinal epithelia. Iron depletion increased expression of iron-regulated proteins (TfR, transferrin receptor and DMT1, divalent metal transporter, as predicted, but it also promoted a marked reduction in growth and proliferation of Caco-2 cells. This was strongly associated with suppressed mTORC1 signalling, as judged by reduced phosphorylation of mTOR substrates, S6K1 and 4E-BP1, and diminished protein synthesis. The reduction in mTORC1 signalling was tightly coupled with increased expression and accumulation of REDD1 (regulated in DNA damage and development 1) and reduced phosphorylation of Akt and TSC2. The increase in REDD1 abundance was rapidly reversed upon iron repletion of cells but was also attenuated by inhibitors of gene transcription, protein phosphatase 2A (PP2A) and by REDD1 siRNA — strategies that also antagonised the loss in mTORC1 signalling associated with iron depletion. Our findings implicate REDD1 and PP2A as crucial regulators of mTORC1 activity in iron-depleted cells and indicate that their modulation may help mitigate atrophy of the intestinal mucosa that may occur in response to iron deficiency. PMID:26827808

  18. Dynamical implications of Jupiter's tropospheric ammonia abundance

    NASA Astrophysics Data System (ADS)

    Showman, Adam P.; de Pater, Imke

    2005-03-01

    Groundbased radio observations indicate that Jupiter's ammonia is globally depleted from 0.6 bars to at least 4-6 bars relative to the deep abundance of ˜3 times solar, a fact that has so far defied explanation. The observations also indicate that (i) the depletion is greater in belts than zones, and (ii) the greatest depletion occurs within Jupiter's local 5-μm hot spots, which have recently been detected at radio wavelengths. Here, we first show that both the global depletion and its belt-zone variation can be explained by a simple model for the interaction of moist convection with Jupiter's cloud-layer circulation. If the global depletion is dynamical in origin, then important endmember models for the belt-zone circulation can be ruled out. Next, we show that the radio observations of Jupiter's 5-μm hot spots imply that the equatorial wave inferred to cause hot spots induces vertical parcel oscillation of a factor of ˜2 in pressure near the 2-bar level, which places important constraints on hot-spot dynamics. Finally, using spatially resolved radio maps, we demonstrate that low-latitude features exceeding ˜4000 km diameter, such as the equatorial plumes and large vortices, are also depleted in ammonia from 0.6 bars to at least 2 bars relative to the deep abundance of 3 times solar. If any low-latitude features exist that contain 3-times-solar ammonia up to the 0.6-bar ammonia condensation level, they must have diameters less than ˜4000 km.

  19. Depleted Uranium in Repositories

    SciTech Connect

    Haire, M.J.; Croff, A.G.

    1997-12-31

    For uranium to be useful in most fission nuclear reactors, it must be enriched (i.e. the concentration of the fissile isotope 235U must be increased). Therefore, depleted uranium (DU)-uranium which has less than naturally occurring concentrations of 235U-is a co-product of the enrichment process. Four to six tons of DU exist for every ton of fresh light water reactor fuel. There were 407,006 MgU 407,000 metric tons (t) of DU stored on U.S. Department of Energy (DOE) sites as of July 1993. If this DU were to be declared surplus, converted to a stable oxide form, and emplaced in a near surface disposal facility, the costs are estimated to be several billion dollars. However, the U.S. Nuclear Regulatory Commission has stated that near surface disposal of large quantities of DU tails is not appropriate. Thus, there is the possibility that disposition via disposal will be in a deep geological repository. One alternative that may significantly reduce the cost of DU disposition is to use it beneficially. In fact, DOE has begun the Beneficial Uses of DU Project to identify large scale uses of DU and to encourage its reuse. Several beneficial uses, many of which involve applications in the repository per se or in managing the wastes to go into the repository, are discussed in this report.

  20. Preparation of biocompatible molecularly imprinted shell on superparamagnetic iron oxide nanoparticles for selective depletion of bovine hemoglobin in biological sample.

    PubMed

    Hao, Yi; Gao, Ruixia; Liu, Dechun; Zhang, Bianbian; Tang, Yuhai; Guo, Zengjun

    2016-05-15

    Bovine hemoglobin (BHb), as one of the high-abundance proteins, could seriously mask and hamper the analysis of low-abundance proteins in serum. To selectively deplete BHb, we design a simple and effective strategy for preparation of biocompatible molecularly imprinted shell on superparamagnetic iron oxide nanoparticles through surface imprinting technique combined with template immobilization strategy. Firstly, template proteins are immobilized on the directly aldehyde-functionalized magnetic nanoparticles through imine bonds. Then, with gelatin as functional monomer, a polymeric network molded around the immobilized template proteins is obtained. Finally, the specific cavities for BHb are fabricated after removing the template proteins. The effects of imprinting conditions were investigated and the optimal imprinting conditions are found to be 40mg of BHb, 150mg of gelatin, and 8h of polymerization time. The resultant materials exhibit good dispersion, high crystallinity, and satisfactory superparamagnetic property with a high saturation magnetization (33.43emug(-1)). The adsorption experiments show that the imprinted nanomaterials have high adsorption capacity of 93.1mgg(-1), fast equilibrium time of 35min, and satisfactory selectivity for target protein. Meanwhile, the obtained polymers could be used without obvious deterioration after six adsorption-desorption cycles. In addition, the resultant polymers are successfully applied in the selective isolation BHb from bovine blood sample, which could provide an alternative solution for the preparatory work of proteomics. PMID:26939073

  1. An "Andesitic" Component in Shergottites with Restored LREE Abundances?

    NASA Technical Reports Server (NTRS)

    Nyquist, L. E.; Shih, C.-Y.; Wiesmann, H.; Barrat, J. A.

    2002-01-01

    The shergottite Martian meteorites present a variety of oft-confusing petrologic features. In particular, represented among this subgroup are basalts with very depleted LREE abundances, as well as those with nearly chondritic overall REE abundances. The LREE-depleted basalts appear to more closely record the REE and isotopic features of their mantle source legions. Those basalts with more nearly chondritic REE abundances appear to contain an extra component often referred to as a "crustal" component. The addition of the crustal component tends to restore the overall REE abundance pattern towards chondritic relative abundances. Here we suggest that the crustal component could derive from andesitic rocks observed remotely to occur on the Martian surface, and which were analysed at the Pathfinder site.

  2. An "Andestic" Component in Shergottites with Restored LREE Abundances?

    NASA Technical Reports Server (NTRS)

    Nyquist, L. E.; Shih, C.-Y.; Wiesmann, H.; Barrat, J. A.

    2002-01-01

    The shergottite Martian meteorites present a variety of oft-confusing petrologic features. In particular, represented among this subgroup are basalts with very depleted LREE abundances, as well as those with nearly chondritic overall REE abundances. The LREE-depleted basalts appear to more closely record the REE and isotopic features of their mantle source regions. Those basalts with more nearly chondritic REE abundances appear to contain an extra component often referred to as a "crustal" component. The addition of the crustal component tends to restore the overall REE abundance pattern towards chondritic relative abundances. Here we suggest that the crustal component could derive from "andesitic" rocks observed remotely to occur on the Martian surface, and which were analysed at the Pathfinder site.

  3. Ghrelin protects against depleted uranium-induced apoptosis of MC3T3-E1 cells through oxidative stress-mediated p38-mitogen-activated protein kinase pathway.

    PubMed

    Hao, Yuhui; Liu, Cong; Huang, Jiawei; Gu, Ying; Li, Hong; Yang, Zhangyou; Liu, Jing; Wang, Weidong; Li, Rong

    2016-01-01

    Depleted uranium (DU) mainly accumulates in the bone over the long term. Osteoblast cells are responsible for the formation of bone, and they are sensitive to DU damage. However, studies investigating methods of reducing DU damage in osteoblasts are rarely reported. Ghrelin is a stomach hormone that stimulates growth hormones released from the hypothalamic-pituitary axis, and it is believed to play an important physiological role in bone metabolism. This study evaluates the impact of ghrelin on DU-induced apoptosis of the osteoblast MC3T3-E1 and investigates its underlying mechanisms. The results show that ghrelin relieved the intracellular oxidative stress induced by DU, eliminated reactive oxygen species (ROS) and reduced lipid peroxidation by increasing intracellular GSH levels; in addition, ghrelin effectively suppressed apoptosis, enhanced mitochondrial membrane potential, and inhibited cytochrome c release and caspase-3 activation after DU exposure. Moreover, ghrelin significantly reduced the expression of DU-induced phosphorylated p38-mitogen-activated protein kinase (MAPK). A specific inhibitor (SB203580) or specific siRNA of p38-MAPK could significantly suppress DU-induced apoptosis and related signals, whereas ROS production was not affected. In addition, ghrelin receptor inhibition could reduce the anti-apoptosis effect of ghrelin on DU and reverse the effect of ghrelin on intracellular ROS and p38-MAPK after DU exposure. These results suggest that ghrelin can suppress DU-induced apoptosis of MC3T3-E1 cells, reduce DU-induced oxidative stress by interacting with its receptor, and inhibit downstream p38-MAPK activation, thereby suppressing the mitochondrial-dependent apoptosis pathway. PMID:26529667

  4. The Toxicity of Depleted Uranium

    PubMed Central

    Briner, Wayne

    2010-01-01

    Depleted uranium (DU) is an emerging environmental pollutant that is introduced into the environment primarily by military activity. While depleted uranium is less radioactive than natural uranium, it still retains all the chemical toxicity associated with the original element. In large doses the kidney is the target organ for the acute chemical toxicity of this metal, producing potentially lethal tubular necrosis. In contrast, chronic low dose exposure to depleted uranium may not produce a clear and defined set of symptoms. Chronic low-dose, or subacute, exposure to depleted uranium alters the appearance of milestones in developing organisms. Adult animals that were exposed to depleted uranium during development display persistent alterations in behavior, even after cessation of depleted uranium exposure. Adult animals exposed to depleted uranium demonstrate altered behaviors and a variety of alterations to brain chemistry. Despite its reduced level of radioactivity evidence continues to accumulate that depleted uranium, if ingested, may pose a radiologic hazard. The current state of knowledge concerning DU is discussed. PMID:20195447

  5. Stratospheric ozone depletion

    PubMed Central

    Rowland, F. Sherwood

    2006-01-01

    Solar ultraviolet radiation creates an ozone layer in the atmosphere which in turn completely absorbs the most energetic fraction of this radiation. This process both warms the air, creating the stratosphere between 15 and 50 km altitude, and protects the biological activities at the Earth's surface from this damaging radiation. In the last half-century, the chemical mechanisms operating within the ozone layer have been shown to include very efficient catalytic chain reactions involving the chemical species HO, HO2, NO, NO2, Cl and ClO. The NOX and ClOX chains involve the emission at Earth's surface of stable molecules in very low concentration (N2O, CCl2F2, CCl3F, etc.) which wander in the atmosphere for as long as a century before absorbing ultraviolet radiation and decomposing to create NO and Cl in the middle of the stratospheric ozone layer. The growing emissions of synthetic chlorofluorocarbon molecules cause a significant diminution in the ozone content of the stratosphere, with the result that more solar ultraviolet-B radiation (290–320 nm wavelength) reaches the surface. This ozone loss occurs in the temperate zone latitudes in all seasons, and especially drastically since the early 1980s in the south polar springtime—the ‘Antarctic ozone hole’. The chemical reactions causing this ozone depletion are primarily based on atomic Cl and ClO, the product of its reaction with ozone. The further manufacture of chlorofluorocarbons has been banned by the 1992 revisions of the 1987 Montreal Protocol of the United Nations. Atmospheric measurements have confirmed that the Protocol has been very successful in reducing further emissions of these molecules. Recovery of the stratosphere to the ozone conditions of the 1950s will occur slowly over the rest of the twenty-first century because of the long lifetime of the precursor molecules. PMID:16627294

  6. Stratospheric ozone depletion.

    PubMed

    Rowland, F Sherwood

    2006-05-29

    Solar ultraviolet radiation creates an ozone layer in the atmosphere which in turn completely absorbs the most energetic fraction of this radiation. This process both warms the air, creating the stratosphere between 15 and 50 km altitude, and protects the biological activities at the Earth's surface from this damaging radiation. In the last half-century, the chemical mechanisms operating within the ozone layer have been shown to include very efficient catalytic chain reactions involving the chemical species HO, HO2, NO, NO2, Cl and ClO. The NOX and ClOX chains involve the emission at Earth's surface of stable molecules in very low concentration (N2O, CCl2F2, CCl3F, etc.) which wander in the atmosphere for as long as a century before absorbing ultraviolet radiation and decomposing to create NO and Cl in the middle of the stratospheric ozone layer. The growing emissions of synthetic chlorofluorocarbon molecules cause a significant diminution in the ozone content of the stratosphere, with the result that more solar ultraviolet-B radiation (290-320 nm wavelength) reaches the surface. This ozone loss occurs in the temperate zone latitudes in all seasons, and especially drastically since the early 1980s in the south polar springtime-the 'Antarctic ozone hole'. The chemical reactions causing this ozone depletion are primarily based on atomic Cl and ClO, the product of its reaction with ozone. The further manufacture of chlorofluorocarbons has been banned by the 1992 revisions of the 1987 Montreal Protocol of the United Nations. Atmospheric measurements have confirmed that the Protocol has been very successful in reducing further emissions of these molecules. Recovery of the stratosphere to the ozone conditions of the 1950s will occur slowly over the rest of the twenty-first century because of the long lifetime of the precursor molecules. PMID:16627294

  7. Depletion of the "gamma-type carbonic anhydrase-like" subunits of complex I affects central mitochondrial metabolism in Arabidopsis thaliana.

    PubMed

    Fromm, Steffanie; Göing, Jennifer; Lorenz, Christin; Peterhänsel, Christoph; Braun, Hans-Peter

    2016-01-01

    "Gamma-type carbonic anhydrase-like" (CAL) proteins form part of complex I in plants. Together with "gamma carbonic anhydrase" (CA) proteins they form an extra domain which is attached to the membrane arm of complex I on its matrix exposed side. In Arabidopsis two CAL and three CA proteins are present, termed CAL1, CAL2, CA1, CA2 and CA3. It has been proposed that the carbonic anhydrase domain of complex I is involved in a process mediating efficient recycling of mitochondrial CO2 for photosynthetic carbon fixation which is especially important during growth conditions causing increased photorespiration. Depletion of CAL proteins has been shown to significantly affect plant development and photomorphogenesis. To better understand CAL function in plants we here investigated effects of CAL depletion on the mitochondrial compartment. In mutant lines and cell cultures complex I amount was reduced by 90-95% but levels of complexes III and V were unchanged. At the same time, some of the CA transcripts were less abundant. Proteome analysis of CAL depleted cells revealed significant reduction of complex I subunits as well as proteins associated with photorespiration, but increased amounts of proteins participating in amino acid catabolism and stress response reactions. Developmental delay of the mutants was slightly alleviated if plants were cultivated at high CO2. Profiling of selected metabolites revealed defined changes in intermediates of the citric acid cycle and amino acid catabolism. It is concluded that CAL proteins are essential for complex I assembly and that CAL depletion specifically affects central mitochondrial metabolism. PMID:26482706

  8. Fibroblast Growth Factor (FGF) Signaling during Gastrulation Negatively Modulates the Abundance of MicroRNAs That Regulate Proteins Required for Cell Migration and Embryo Patterning*

    PubMed Central

    Bobbs, Alexander S.; Saarela, Aleksi V.; Yatskievych, Tatiana A.; Antin, Parker B.

    2012-01-01

    FGF signaling plays a pivotal role in regulating cell movements and lineage induction during gastrulation. Here we identify 44 microRNAs that are expressed in the primitive streak region of gastrula stage chicken embryos. We show that the primary effect of FGF signaling on microRNA abundance is to negatively regulate the levels of miR-let-7b, -9, -19b, -107, -130b, and -218. LIN28B inhibits microRNA processing and is positively regulated by FGF signaling. Gain- and loss-of-function experiments show that LIN28B negatively regulates the expression of miR-19b, -130b, and let-7b, whereas negative modulation of miR-9, -107, and -218 appears to be independent of LIN28B function. Predicted mRNA targets of the FGF-regulated microRNAs are over-represented in serine/threonine and tyrosine kinase receptors, including ACVR1, ACVR2B, PDGFRA, TGFBR1, and TGFBR3. Luciferase assays show that these and other candidates are targeted by FGF-regulated microRNAs. PDGFRA, a receptor whose activity is required for cell migration through the primitive streak, is a target of miR-130b and -218 in vivo. These results identify a novel mechanism by which FGF signaling regulates gene expression by negatively modulating microRNA abundance through both LIN28B-dependent and LIN28B-independent pathways. PMID:22995917

  9. Detection of depleted uranium in urine of veterans from the 1991 Gulf War.

    PubMed

    Gwiazda, R H; Squibb, K; McDiarmid, M; Smith, D

    2004-01-01

    American soldiers involved in "friendly fire" accidents during the 1991 Gulf War were injured with depleted-uranium-containing fragments or possibly exposed to depleted uranium via other routes such as inhalation, ingestion, and/or wound contamination. To evaluate the presence of depleted uranium in these soldiers eight years later, the uranium concentration and depleted uranium content of urine samples were determined by inductively coupled plasma mass spectrometry in (a) depleted uranium exposed soldiers with embedded shrapnel, (b) depleted uranium exposed soldiers with no shrapnel, and (c) a reference group of deployed soldiers not involved in the friendly fire incidents. Uranium isotopic ratios measured in many urine samples injected directly into the inductively coupled plasma mass spectrometer and analyzed at a mass resolution m/delta m of 300 appeared enriched in 235U with respect to natural abundance (0.72%) due to the presence of an interference of a polyatomic molecule of mass 234.81 amu that was resolved at a mass resolution m/delta m of 4,000. The 235U abundance measured on uranium separated from these urines by anion exchange chromatography was clearly natural or depleted. Urine uranium concentrations of soldiers with shrapnel were higher than those of the two other groups, and 16 out of 17 soldiers with shrapnel had detectable depleted uranium in their urine. In depleted uranium exposed soldiers with no shrapnel, depleted uranium was detected in urine samples of 10 out of 28 soldiers. The median uranium concentration of urines with depleted uranium from soldiers without shrapnel was significantly higher than in urines with no depleted uranium, though substantial overlap in urine uranium concentrations existed between the two groups. Accordingly, assessment of depleted uranium exposure using urine must rely on uranium isotopic analyses, since urine uranium concentration is not an unequivocal indicator of depleted uranium presence in soldiers with no

  10. Rapid Glucose Depletion Immobilizes Active Myosin-V on Stabilized Actin Cables

    PubMed Central

    Xu, Li; Bretscher, Anthony

    2014-01-01

    Summary Polarization of eukaryotic cells requires organelles and protein complexes to be transported to their proper destinations along the cytoskeleton [1]. When nutrients are abundant, budding yeast grows rapidly transporting secretory vesicles for localized growth and actively segregating organelles [2, 3]. This is mediated by myosin-Vs transporting cargos along F-actin bundles known as actin cables [4]. Actin cables are dynamic structures regulated by assembly, stabilization and disassembly [5]. Polarized growth and actin filament dynamics consume energy. For most organisms, glucose is the preferred energy source and generally represses alternative carbon source usage [6]. Thus upon abrupt glucose depletion, yeast shuts down pathways consuming large amounts of energy, including the vacuolar-ATPase [7, 8], translation [9] and phosphoinositide metabolism [10]. Here we show that glucose withdrawal rapidly (<1 min) depletes ATP levels and the yeast myosin V, Myo2, responds by relocalizing to actin cables, making it the fastest response documented. Myo2 immobilized on cables releases its secretory cargo, defining a new rigor-like state of a myosin-V in vivo. Only actively transporting Myo2 can be converted to the rigor-like state. Glucose depletion has differential effects on the actin cytoskeleton resulting in disassembly of actin patches with concomitant inhibition of endocytosis, and strong stabilization of actin cables, thereby revealing a selective and previously unappreciated ATP requirement for actin cable disassembly. A similar response is seen in HeLa cells to ATP depletion. These findings reveal a new fast-acting energy conservation strategy halting growth by immobilizing myosin-V in a newly described state on selectively stabilized actin cables. PMID:25308080

  11. The Abundance of Interstellar Fluorine

    NASA Technical Reports Server (NTRS)

    Lauroesch, James T.

    2005-01-01

    The primary objective of this program was to obtain FUSE observations of the interstellar absorption lines of F I at 951 and 954 Angstroms to derive the abundance of fluorine toward the star HD 164816. The nucleosynthetic source(s) of fluorine are still a matter of debate - the present day abundance of fluorine can potentially constrain models for pulsationally driven dredge-up in asymptotic giant branch stars. An accurate measure for the depletion behavior of fluorine will determine whether it may be detectable in QSO absorption line systems - an unambiguous detection of fluorine at suitably high redshifts would provide the best evidence to date for the neutrino process in massive stars. Furthermore, due to its extreme reactivity, measurement of the gas-phase interstellar fluorine abundance is important for models of grain chemistry. Despite the importance of measuring the interstellar fluorine abundance, at the time of our proposal only one previous detection has been made due to the low relative abundance of fluorine, the lack of lines outside the far-UV, and the blending of the available F I transitions with lines of Hz. The star HD 164816 is associated with the Lagoon nebula (M8), and at a distance of approximately 1.5 kpc probes both distant and local gas. Beginning April 8th, 2004 FUSE FP-Split observations of the star HD 164816 were obtained for this program. This data became available in the FUSE data archive May 21, 2004, and these observations were then downloaded and we began our analysis. Our analysis procedure has involved (1) fitting stellar models to the FUSE spectra, (2) using the multiple lines of Hz and N I at other wavelengths in the FUSE bandpass to derive column densities for the lines of H2 and N I which are blended with the F I features at 951 and 954 angstroms (3) the measurement of the column densities of F I and the species O I and C1 I which are important species for the dis-entangling of dust and nucleosynthetic effects. As discussed in

  12. The SLE variant Ala71Thr of BLK severely decreases protein abundance and binding to BANK1 through impairment of the SH3 domain function.

    PubMed

    Díaz-Barreiro, A; Bernal-Quirós, M; Georg, I; Marañón, C; Alarcón-Riquelme, M E; Castillejo-López, C

    2016-03-01

    The B-lymphocyte kinase (BLK) gene is associated genetically with several human autoimmune diseases including systemic lupus erythematosus. We recently described that the genetic risk is given by two haplotypes: one covering several strongly linked single-nucleotide polymorphisms within the promoter of the gene that correlated with low transcript levels, and a second haplotype that includes a rare nonsynonymous variant (Ala71Thr). Here we show that this variant, located within the BLK SH3 domain, is a major determinant of protein levels. In vitro analyses show that the 71Thr isoform is hyperphosphorylated and promotes kinase activation. As a consequence, BLK is ubiquitinated, its proteasomal degradation enhanced and the average life of the protein is reduced by half. Altogether, these findings suggest that an intrinsic autoregulatory mechanism previously unappreciated in BLK is disrupted by the 71Thr substitution. Because the SH3 domain is also involved in protein interactions, we sought for differences between the two isoforms in trafficking and binding to protein partners. We found that binding of the 71Thr variant to the adaptor protein BANK1 is severely reduced. Our study provides new insights on the intrinsic regulation of BLK activation and highlights the dominant role of its SH3 domain in BANK1 binding. PMID:26821283

  13. The absence of protein Y4yS affects negatively the abundance of T3SS Mesorhizobium loti secretin, RhcC2, in bacterial membranes

    PubMed Central

    Mercante, Virginia; Duarte, Cecilia M.; Sánchez, Cintia M.; Zalguizuri, Andrés; Caetano-Anollés, Gustavo; Lepek, Viviana C.

    2015-01-01

    Mesorhizobium loti MAFF303099 has a functional type III secretion system (T3SS) that is involved in the determination of nodulation competitiveness on Lotus. The M. loti T3SS cluster contains gene y4yS (mlr8765) that codes for a protein of unknown function (Y4yS). A mutation in the y4yS gene favors the M. loti symbiotic competitive ability on Lotus tenuis cv. Esmeralda and affects negatively the secretion of proteins through T3SS. Here we localize Y4yS in the bacterial membrane using a translational reporter peptide fusion. In silico analysis indicated that this protein presents a tetratricopeptide repeat (TPR) domain, a signal peptide and a canonical lipobox LGCC in the N-terminal sequence. These features that are shared with proteins required for the formation of the secretin complex in type IV secretion systems and in the Tad system, together with its localization, suggest that the y4yS-encoded protein is required for the formation of the M. loti T3SS secretin (RhcC2) complex. Remarkably, analysis of RhcC2 in the wild-type and M. loti y4yS mutant strains indicated that the absence of Y4yS affects negatively the accumulation of normal levels of RhcC2 in the membrane. PMID:25688250

  14. Interstellar magnesium abundances

    NASA Technical Reports Server (NTRS)

    Murray, M. J.; Dufton, P. L.; Hibbert, A.; York, D. G.

    1984-01-01

    An improved evaluation of the Mg II 1240 A doublet oscillator strength is used in conjunction with recently published Copernicus observations to derive accurate Mg II column densities toward 74 stars. These imply an average of 40 percent of interstellar magnesium is in the gaseous phase. Magnesium depletion is examined as a function of various interstellar extinction and density parameters, and the results are briefly discussed in terms of current depletion theories.

  15. Apical Na(+)-D-glucose cotransporter 1 (SGLT1) activity and protein abundance are expressed along the jejunal crypt-villus axis in the neonatal pig

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gut apical Na(+)-glucose cotransporter 1 (SGLT1) activity is high at the birth and during suckling, thus contributing substantially to neonatal glucose homeostasis. We hypothesize that neonates possess high SGLT1 maximal activity by expressing apical SGLT1 protein along the intestinal crypt-villus a...

  16. ARMS depletion facilitates UV irradiation induced apoptotic cell death in melanoma.

    PubMed

    Liao, Yi-Hua; Hsu, Su-Ming; Huang, Pei-Hsin

    2007-12-15

    Tumor cells often aberrantly reexpress molecules that mediate proper embryonic development for advantageous growth or survival. Here, we report that ankyrin repeat-rich membrane spanning (ARMS), a transmembrane protein abundant in the developing and adult neural tissues, is overexpressed in melanoma, a tumor ontogenetically originating from neural crest. Immunohistochemical study of 79 melanocytic lesions showed significantly increased expression of ARMS in primary malignant melanomas (92.9%) and metastatic melanoma (60.0%) in comparison with benign nevocellular nevi (26.7%). To investigate the role of ARMS in melanoma formation, murine B16F0 melanoma cells with stable knockdown of ARMS were established by RNA interference. Down-regulation of ARMS resulted in significant inhibition of anchorage-independent growth in soft agar and restrictive growth of melanoma in severe combined immunodeficient mice. Importantly, depletion of ARMS facilitated UVB-induced apoptosis in melanoma cells through inactivation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK. Addition of MEK inhibitor PD98059 further sensitized ARMS-depleted melanoma cells to UVB-induced apoptosis, whereas constitutively active MEK rescued ARMS-depleted cells from apoptosis. We further showed that BRAF, a downstream signaling molecule of ARMS in ERK pathway, is not mutated as a constitutively active form in acral lentiginous melanoma; in contrast, BRAF(T1799A) mutation, which leads to constitutive activation of ERK signaling, was detected in 57.1% of superficial spreading melanoma. Our study suggests that overexpression of ARMS per se serves as one mechanism to promote melanoma formation by preventing stress-induced apoptotic death mediated by the MEK/ERK signaling pathway, especially in acral lentiginous melanoma, most of which does not harbor BRAF mutation. PMID:18089783

  17. Ego depletion impairs implicit learning.

    PubMed

    Thompson, Kelsey R; Sanchez, Daniel J; Wesley, Abigail H; Reber, Paul J

    2014-01-01

    Implicit skill learning occurs incidentally and without conscious awareness of what is learned. However, the rate and effectiveness of learning may still be affected by decreased availability of central processing resources. Dual-task experiments have generally found impairments in implicit learning, however, these studies have also shown that certain characteristics of the secondary task (e.g., timing) can complicate the interpretation of these results. To avoid this problem, the current experiments used a novel method to impose resource constraints prior to engaging in skill learning. Ego depletion theory states that humans possess a limited store of cognitive resources that, when depleted, results in deficits in self-regulation and cognitive control. In a first experiment, we used a standard ego depletion manipulation prior to performance of the Serial Interception Sequence Learning (SISL) task. Depleted participants exhibited poorer test performance than did non-depleted controls, indicating that reducing available executive resources may adversely affect implicit sequence learning, expression of sequence knowledge, or both. In a second experiment, depletion was administered either prior to or after training. Participants who reported higher levels of depletion before or after training again showed less sequence-specific knowledge on the post-training assessment. However, the results did not allow for clear separation of ego depletion effects on learning versus subsequent sequence-specific performance. These results indicate that performance on an implicitly learned sequence can be impaired by a reduction in executive resources, in spite of learning taking place outside of awareness and without conscious intent. PMID:25275517

  18. Simulation of polar ozone depletion: An update

    NASA Astrophysics Data System (ADS)

    Solomon, Susan; Kinnison, Doug; Bandoro, Justin; Garcia, Rolando

    2015-08-01

    We evaluate polar ozone depletion chemistry using the specified dynamics version of the Whole Atmosphere Community Climate Model for the year 2011. We find that total ozone depletion in both hemispheres is dependent on cold temperatures (below 192 K) and associated heterogeneous chemistry on polar stratospheric cloud particles. Reactions limited to warmer temperatures above 192 K, or on binary liquid aerosols, yield little modeled polar ozone depletion in either hemisphere. An imposed factor of three enhancement in stratospheric sulfate increases ozone loss by up to 20 Dobson unit (DU) in the Antarctic and 15 DU in the Arctic in this model. Such enhanced sulfate loads are similar to those observed following recent relatively small volcanic eruptions since 2005 and imply impacts on the search for polar ozone recovery. Ozone losses are strongly sensitive to temperature, with a test case cooler by 2 K producing as much as 30 DU additional ozone loss in the Antarctic and 40 DU in the Arctic. A new finding of this paper is the use of the temporal behavior and variability of ClONO2 and HCl as indicators of the efficacy of heterogeneous chemistry. Transport of ClONO2 from the southern subpolar regions near 55-65°S to higher latitudes near 65-75°S provides a flux of NOx from more sunlit latitudes to the edge of the vortex and is important for ozone loss in this model. Comparisons between modeled and observed total column and profile ozone perturbations, ClONO2 abundances, and the rate of change of HCl bolster confidence in these conclusions.

  19. An abundant and ubiquitous homo-oligomeric ring-shaped ATPase particle related to the putative vesicle fusion proteins Sec18p and NSF.

    PubMed Central

    Peters, J M; Walsh, M J; Franke, W W

    1990-01-01

    We have discovered a ring-shaped particle of 12.5 nm diameter, 14.5S and apparent molecular weight of approximately 570,000 that displays 6-fold radial symmetry and is composed of a single kind of an acidic (pI approximately 5.5) polypeptide of Mr 97,000 (p97). Using antibodies to this protein we have detected its occurrence in a wide range of cells and tissues of diverse species from frog to man, including highly specialized cells such as mammalian erythrocytes and spermatozoa. In Xenopus laevis oocytes, the particle is found in both isolated nuclei and in manually enucleated ooplasms, which corresponds to immunofluorescence staining dispersed over both nucleoplasm and cytoplasm. The particle has a N-ethylmaleimide (NEM)-inhibitable Mg2(+)-ATPase activity, and its amino acid sequence, as deduced from cDNA clones, displays considerable homology to the mammalian NEM-sensitive fusion protein (NSF) and yeast Sec18p believed to be essential for vesicle fusion in secretory processes, indicating that these three proteins belong to the same multigene family. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2140770

  20. Solar abundance of osmium

    PubMed Central

    Jacoby, George; Aller, Lawrence H.

    1976-01-01

    The abundance parameter, log gfA, where g is the statistical weight of the lower level, f is the oscillator strength, and A is the abundance (by numbers of atoms with respect to hydrogen), has been derived for three lines of osmium by a method of spectrum synthesis. An apparent discordance of the derived abundance with that found from the carbonaceous chondrites is probably to be attributed primarily to errors in the f-values, and blending with unknown contributors. PMID:16592314

  1. Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes.

    PubMed

    Faca, Vitor; Pitteri, Sharon J; Newcomb, Lisa; Glukhova, Veronika; Phanstiel, Doug; Krasnoselsky, Alexei; Zhang, Qing; Struthers, Jason; Wang, Hong; Eng, Jimmy; Fitzgibbon, Matt; McIntosh, Martin; Hanash, Samir

    2007-09-01

    In-depth analysis of the serum and plasma proteomes by mass spectrometry is challenged by the vast dynamic range of protein abundance and substantial complexity. There is merit in reducing complexity through fractionation to facilitate mass spectrometry analysis of low-abundance proteins. However, fractionation reduces throughput and has the potential of diluting individual proteins or inducing their loss. Here, we have investigated the contribution of extensive fractionation of intact proteins to depth of analysis. Pooled serum depleted of abundant proteins was fractionated by an orthogonal two-dimensional system consisting of anion-exchange and reversed-phase chromatography. The resulting protein fractions were aliquotted; one aliquot was analyzed by shotgun LC-MS/MS, and another was further resolved into protein bands in a third dimension using SDS-PAGE. Individual gel bands were excised and subjected to in situ digestion and mass spectrometry. We demonstrate that increased fractionation results in increased depth of analysis based on total number of proteins identified in serum and based on representation in individual fractions of specific proteins identified in gel bands following a third-dimension SDS gel analysis. An intact protein analysis system (IPAS) based on a two-dimensional plasma fractionation schema was implemented that resulted in identification of 1662 proteins with high confidence with representation of protein isoforms that differed in their chromatographic mobility. Further increase in depth of analysis was accomplished by repeat analysis of aliquots from the same set of two-dimensional fractions resulting in overall identification of 2254 proteins. We conclude that substantial depth of analysis of proteins from milliliter quantities of serum or plasma and detection of isoforms are achieved with depletion of abundant proteins followed by two-dimensional protein fractionation and MS analysis of individual fractions. PMID:17696519

  2. Depleting depletion: Polymer swelling in poor solvent mixtures

    NASA Astrophysics Data System (ADS)

    Mukherji, Debashish; Marques, Carlos; Stuehn, Torsten; Kremer, Kurt

    A polymer collapses in a solvent when the solvent particles dislike monomers more than the repulsion between monomers. This leads to an effective attraction between monomers, also referred to as depletion induced attraction. This attraction is the key factor behind standard polymer collapse in poor solvents. Strikingly, even if a polymer exhibits poor solvent condition in two different solvents, it can also swell in mixtures of these two poor solvents. This collapse-swelling-collapse scenario is displayed by poly(methyl methacrylate) (PMMA) in aqueous alcohol. Using molecular dynamics simulations of a thermodynamically consistent generic model and theoretical arguments, we unveil the microscopic origin of this phenomenon. Our analysis suggests that a subtle interplay of the bulk solution properties and the local depletion forces reduces depletion effects, thus dictating polymer swelling in poor solvent mixtures.

  3. Perturbations of Amino Acid Metabolism Associated with Glyphosate-Dependent Inhibition of Shikimic Acid Metabolism Affect Cellular Redox Homeostasis and Alter the Abundance of Proteins Involved in Photosynthesis and Photorespiration1[W][OA

    PubMed Central

    Vivancos, Pedro Diaz; Driscoll, Simon P.; Bulman, Christopher A.; Ying, Liu; Emami, Kaveh; Treumann, Achim; Mauve, Caroline; Noctor, Graham; Foyer, Christine H.

    2011-01-01

    The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway. PMID:21757634

  4. Application of Natural Isotopic Abundance ¹H-¹³C- and ¹H-¹⁵N-Correlated Two-Dimensional NMR for Evaluation of the Structure of Protein Therapeutics.

    PubMed

    Arbogast, Luke W; Brinson, Robert G; Marino, John P

    2016-01-01

    Methods for characterizing the higher-order structure of protein therapeutics are in great demand for establishing consistency in drug manufacturing, for detecting drug product variations resulting from modifications in the manufacturing process, and for comparing a biosimilar to an innovator reference product. In principle, solution NMR can provide a robust approach for characterization of the conformation(s) of protein therapeutics in formulation at atomic resolution. However, molecular weight limitations and the perceived need for stable isotope labeling have to date limited its practical applications in the biopharmaceutical industry. Advances in NMR magnet and console technologies, cryogenically cooled probes, and new rapid acquisition methodologies, particularly selective optimized flip-angle short transient pulse schemes and nonuniform sampling, have greatly ameliorated these limitations. Here, we describe experimental methods for the collection and analysis of 2D (1)H(N)-(15)N-amide- and (1)H-(13)C-methyl-correlated spectra applied to protein drug products at natural isotopic abundance, including representatives from the rapidly growing class of monoclonal antibody (mAb) therapeutics. Practical aspects of experimental setup and data acquisition for both standard and rapid acquisition NMR techniques are described. Furthermore, strategies for the statistical comparison of 2D (1)H(N)-(15)N-amide- and (1)H-(13)C-methyl-correlated spectra are detailed. PMID:26791974

  5. Solar abundances as derived from solar energetic particles

    NASA Technical Reports Server (NTRS)

    Stone, E. C.

    1989-01-01

    Recent studies have shown that there are well defined average abundances of heavy (Z above 2) solar energetic particles (SEPs), with variations in the acceleration and propagation producing a systematic flare-to-flare fractionation that depends on the charge per unit mass of the ion. Correcting the average SEP abundances for this fractionation yields SEP-derived coronal abundances for 20 elements. High-resolution SEP studies have also provided isotopic abundances for five elements. SEP-derived abundances indicate that elements with high first ionization potentials (greater than 10 eV) are depleted in the corona relative to the photosphere and provide new information on the solar abundance of C and Ne-22.

  6. Beryllium in the Galactic halo - Surface abundances from standard, diffusive, and rotational stellar evolution, and implications

    NASA Technical Reports Server (NTRS)

    Deliyannis, Constantine P.; Pinsonneault, Marc H.

    1990-01-01

    The recently observed upper limits to the beryllium abundances in population II stars are much lower than population I detections. This difference reflects an intrinsic difference in the initial abundances and is not caused by different degrees of depletion driven by stellar evolution processes from similar initial abundances. Evolutionary sequences of models from the early premain sequence to beyond the turnoff that correspond to halo dwarfs with Fe/H abundances of -1.3, -2.3, and -3.3 are constructed, and standard, diffusive, and rotational mechanisms are used to estimate a maximal possible beryllium depletion. Halo star models in the T(eff) range 6000 to 5000 K might be rotationally depleted by a factor of 1.5-2, and the total depletion should be no more than (conservatively) a factor of 3. Implications for cosmology, cosmic-ray theory, and Galactic chemical evolution are discussed.

  7. Proteogenomics of a saxitoxin-producing and non-toxic strain of Anabaena circinalis (cyanobacteria) in response to extracellular NaCl and phosphate depletion.

    PubMed

    D'Agostino, Paul M; Song, Xiaomin; Neilan, Brett A; Moffitt, Michelle C

    2016-02-01

    In Australia, saxitoxin production is strain dependent within the bloom-forming freshwater cyanobacterium Anabaena circinalis. Freshwater cyanobacteria are exposed to rapid fluctuations in environmental nutrient concentrations, and their adaption is vital for competition, succession and dominance. Two elements of environmental significance, phosphorus and sodium chloride, are proposed to play a role in bloom development and saxitoxin biosynthesis respectively. The aim of our study was to comparatively analyse the model saxitoxin-producing A. circinalis AWQC131C and non-toxic A. circinalis AWQC310F at the genomic level and proteomic level, in response to phosphate depletion and increased extracellular NaCl. When challenged, photosynthesis, carbon/nitrogen metabolisms, transcription/translation, oxidative stress and nutrient transport functional categories demonstrated the largest changes in protein abundance. In response to increased NaCl, SxtC, a protein conserved in all known saxitoxin biosynthetic pathways, was downregulated. Additionally, toxin quantification revealed a decrease in total saxitoxin and decarbomoyl-gonyautoxin2/3 content in response to the NaCl treatment. In response to phosphate depletion, the toxic and non-toxic strain displayed similar proteomic profiles, although the toxic strain did not alter the abundance of as many proteins as the non-toxic strain. These findings have important implications for the future, since response and adaption mechanisms are directly related to in situ dominance of cyanobacteria. PMID:26568470

  8. Be ABUNDANCES IN COOL MAIN-SEQUENCE STARS WITH EXOPLANETS

    SciTech Connect

    Delgado Mena, E.; Israelian, G.; Gonzalez Hernandez, J. I.; Rebolo, R.; Santos, N. C.

    2012-02-10

    We present new Ultraviolet and Visual Echelle Spectrograph (UVES) spectra of a sample of 15 cool unevolved stars with and without detected planetary companions. Together with previous determinations, we study Be depletion and possible differences in Be abundances between the two groups of stars. We obtain a final sample of 89 and 40 stars with and without planets, respectively, which covers a wide range of effective temperatures, from 4700 K to 6400 K, and includes several cool dwarf stars for the first time. We determine Be abundances for these stars and find that for most of them (the coolest ones) the Be II resonance lines are often undetectable, implying significant Be depletion. While for hot stars Be abundances are approximately constant, with a slight fall as T{sub eff} decreases and the Li-Be gap around 6300 K, we find a steep drop of Be content as T{sub eff} decreases for T{sub eff} < 5500 K, confirming the results of previous papers. Therefore, for these stars there is an unknown mechanism destroying Be that is not reflected in current models of Be depletion. Moreover, this strong Be depletion in cool objects takes place for all the stars regardless of the presence of planets; thus, the effect of extra Li depletion in solar-type stars with planets when compared with stars without detected planets does not seem to be present for Be, although the number of stars at those temperatures is still small to reach a final conclusion.

  9. Depletable externalities and Pigouvian taxation

    SciTech Connect

    Freeman, A.M. III

    1984-06-01

    In their book Baumol and Oates (The Theory of Environmental Policy: Externalities, Public Outlays, and the Quality of Life; Prentice-Hall, Englewood Cliffs, NJ (1975).) argue that whether an externality is depletable (private) or undepletable (public) is the key characteristic in determining the optimal pricing pattern. They argue that unlike the undepletable case a negative depletable externality requires not only a charge or tax on the generator of the externality but a payment or compensation to the victim in order to achieve Pareto optimality. It is shown that the key characteristic determining whether compensation of victims is required for efficiency is not the depletability of the externality but whether the victim can costlessly control or limit the amount of the damaging substance received. 6 references.

  10. Fully depleted back illuminated CCD

    DOEpatents

    Holland, Stephen Edward

    2001-01-01

    A backside illuminated charge coupled device (CCD) is formed of a relatively thick high resistivity photon sensitive silicon substrate, with frontside electronic circuitry, and an optically transparent backside ohmic contact for applying a backside voltage which is at least sufficient to substantially fully deplete the substrate. A greater bias voltage which overdepletes the substrate may also be applied. One way of applying the bias voltage to the substrate is by physically connecting the voltage source to the ohmic contact. An alternate way of applying the bias voltage to the substrate is to physically connect the voltage source to the frontside of the substrate, at a point outside the depletion region. Thus both frontside and backside contacts can be used for backside biasing to fully deplete the substrate. Also, high resistivity gaps around the CCD channels and electrically floating channel stop regions can be provided in the CCD array around the CCD channels. The CCD array forms an imaging sensor useful in astronomy.

  11. Reconstructing ecosystem functions of the active microbial community of the Baltic Sea oxygen depleted sediments.

    PubMed

    Thureborn, Petter; Franzetti, Andrea; Lundin, Daniel; Sjöling, Sara

    2016-01-01

    Baltic Sea deep water and sediments hold one of the largest anthropogenically induced hypoxic areas in the world. High nutrient input and low water exchange result in eutrophication and oxygen depletion below the halocline. As a consequence at Landsort Deep, the deepest point of the Baltic Sea, anoxia in the sediments has been a persistent condition over the past decades. Given that microbial communities are drivers of essential ecosystem functions we investigated the microbial community metabolisms and functions of oxygen depleted Landsort Deep sediments by metatranscriptomics. Results show substantial expression of genes involved in protein metabolism demonstrating that the Landsort Deep sediment microbial community is active. Identified expressed gene suites of metabolic pathways with importance for carbon transformation including fermentation, dissimilatory sulphate reduction and methanogenesis were identified. The presence of transcripts for these metabolic processes suggests a potential for heterotrophic-autotrophic community synergism and indicates active mineralisation of the organic matter deposited at the sediment as a consequence of the eutrophication process. Furthermore, cyanobacteria, probably deposited from the water column, are transcriptionally active in the anoxic sediment at this depth. Results also reveal high abundance of transcripts encoding integron integrases. These results provide insight into the activity of the microbial community of the anoxic sediment at the deepest point of the Baltic Sea and its possible role in ecosystem functioning. PMID:26823996

  12. Reconstructing ecosystem functions of the active microbial community of the Baltic Sea oxygen depleted sediments

    PubMed Central

    Franzetti, Andrea; Lundin, Daniel; Sjöling, Sara

    2016-01-01

    Baltic Sea deep water and sediments hold one of the largest anthropogenically induced hypoxic areas in the world. High nutrient input and low water exchange result in eutrophication and oxygen depletion below the halocline. As a consequence at Landsort Deep, the deepest point of the Baltic Sea, anoxia in the sediments has been a persistent condition over the past decades. Given that microbial communities are drivers of essential ecosystem functions we investigated the microbial community metabolisms and functions of oxygen depleted Landsort Deep sediments by metatranscriptomics. Results show substantial expression of genes involved in protein metabolism demonstrating that the Landsort Deep sediment microbial community is active. Identified expressed gene suites of metabolic pathways with importance for carbon transformation including fermentation, dissimilatory sulphate reduction and methanogenesis were identified. The presence of transcripts for these metabolic processes suggests a potential for heterotrophic-autotrophic community synergism and indicates active mineralisation of the organic matter deposited at the sediment as a consequence of the eutrophication process. Furthermore, cyanobacteria, probably deposited from the water column, are transcriptionally active in the anoxic sediment at this depth. Results also reveal high abundance of transcripts encoding integron integrases. These results provide insight into the activity of the microbial community of the anoxic sediment at the deepest point of the Baltic Sea and its possible role in ecosystem functioning. PMID:26823996

  13. Exploring the limit of metazoan thermal tolerance via comparative proteomics: thermally induced changes in protein abundance by two hydrothermal vent polychaetes

    PubMed Central

    Dilly, Geoffrey F.; Young, C. Robert; Lane, William S.; Pangilinan, Jasmyn; Girguis, Peter R.

    2012-01-01

    Temperatures around hydrothermal vents are highly variable, ranging from near freezing up to 300°C. Nevertheless, animals thrive around vents, some of which live near the known limits of animal thermotolerance. Paralvinella sulfincola, an extremely thermotolerant vent polychaete, and Paralvinella palmiformis, a cooler-adapted congener, are found along the Juan de Fuca Ridge in the northwestern Pacific. We conducted shipboard high-pressure thermotolerance experiments on both species to characterize the physiological adaptations underlying P. sulfincola's pronounced thermotolerance. Quantitative proteomics, expressed sequence tag (EST) libraries and glutathione assays revealed that P. sulfincola (i) exhibited an upregulation in the synthesis and recycling of glutathione with increasing temperature, (ii) downregulated nicotinamide adenine dinucleotide (NADH) and succinate dehydrogenases (key enzymes in oxidative phosphorylation) with increasing temperature, and (iii) maintained elevated levels of heat shock proteins (HSPs) across all treatments. In contrast, P. palmiformis exhibited more typical responses to increasing temperatures (e.g. increasing HSPs at higher temperatures). These data reveal differences in how a mesotolerant and extremely thermotolerant eukaryote respond to thermal stress, and suggest that P. sulfincola's capacity to mitigate oxidative stress via increased synthesis of antioxidants and decreased flux through the mitochondrial electron transport chain enable pronounced thermotolerance. Ultimately, oxidative stress may be the key factor in limiting all metazoan thermotolerance. PMID:22553092

  14. Robust Abundance Estimation in Animal Surveys with Imperfect Detection

    EPA Science Inventory

    Surveys of animal abundance are central to the conservation and management of living natural resources. However, detection uncertainty complicates the sampling process of many species. One sampling method employed to deal with this problem is depletion (or removal) surveys in whi...

  15. Enhanced Detection of Low-Abundance Host Cell Protein Impurities in High-Purity Monoclonal Antibodies Down to 1 ppm Using Ion Mobility Mass Spectrometry Coupled with Multidimensional Liquid Chromatography.

    PubMed

    Doneanu, Catalin E; Anderson, Malcolm; Williams, Brad J; Lauber, Matthew A; Chakraborty, Asish; Chen, Weibin

    2015-10-20

    The enormous dynamic range of proteinaceous species present in protein biotherapeutics poses a significant challenge for current mass spectrometry (MS)-based methods to detect low-abundance HCP impurities. Previously, an HCP assay based on two-dimensional chromatographic separation (high pH/low pH) coupled to high-resolution quadrupole time-of-flight (QTOF) mass spectrometry and developed in the author's laboratory has been shown to achieve a detection limit of about 50 ppm (parts per milion) for the identification and quantification of HCPs present in monoclonal antibodies following Protein A purification.1 To improve the HCP detection limit we have explored the utility of several new analytical techniques for HCP analysis and thereby developed an improved liquid chromatography-mass spectrometry (LC-MS) methodology for enhanced detection of HCPs. The new method includes (1) the use of a new charge-surface-modified (CSH) C18 stationary phase to mitigate the challenges of column saturation, peak tailing, and distortion that are commonly observed in the HCP analysis; (2) the incorporation of traveling-wave ion mobility (TWIM) separation of coeluting peptide precursors, and (3) the improvement of fragmentation efficiency of low-abundance HCP peptides by correlating the collision energy used for precursor fragmentation with their mobility drift time. As a result of these improvements, the detection limit of the new methodology was greatly improved, and HCPs present at a concentration as low as 1 ppm (1 ng HCP/mg mAb) were successfully identified and quantified. The newly developed method was applied to analyze two high-purity mAbs (NIST mAb and Infliximab) expressed in a murine cell line. For both samples, low-abundance HCPs (down to 1 ppm) were confidently identified, and the identities of the HCPs were further confirmed by targeted MS/MS experiments. In addition, the performance of the assay was evaluated by an interlaboratory study in which three independent

  16. Evaluation of Multi-Protein Immunoaffinity Subtraction for Plasma Proteomics and Candidate Biomarker Discovery Using Mass Spectrometry

    SciTech Connect

    Liu, Tao; Qian, Weijun; Mottaz, Heather M.; Gritsenko, Marina A.; Norbeck, Angela D.; Moore, Ronald J.; Purvine, Samuel O.; Camp, David G.; Smith, Richard D.

    2006-11-01

    The detection of low-abundance protein disease biomarkers from human blood poses significant challenges due to the high dynamic range of protein concentrations that span more than 10 orders of magnitude, as well as the extreme complexity of the serum/plasma proteome. Therefore, experimental strategies that include the removal of high-abundance proteins have been increasingly utilized in proteomic studies of serum, plasma, and other body fluids to enhance detection of low-abundance proteins and achieve broader proteome coverage. However, both the specificity and reproducibility of the high-abundance protein depletion process represent common concerns. Here, we report a detailed evaluation of the performance of two commercially available immunoaffinity subtraction systems commonly used in human serum/plasma proteome characterization by high resolution LC-MS/MS. One system uses mammalian IgG antibodies to remove six of the most abundant plasma proteins, and the other uses chicken immunoglobulin yolk (IgY) antibodies to remove twelve of the most abundant plasma proteins. Plasma samples were repeatedly processed using these two systems, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. Removal of target proteins by both immunoaffinity subtraction systems proved reproducible and efficient. Nontarget proteins, including spiked protein standards, were also observed to bind to the columns, but in a fairly reproducible manner. The results suggest that these multi-protein immunoaffinity subtraction systems are both highly effective and reproducible for removing high-abundance proteins and therefore, can be readily integrated into quantitative strategies to enhance detection of low-abundance proteins in biomarker discovery studies.

  17. Global transcriptome analysis of Human Bone Marrow Stromal Cells (BMSCs) reveals proliferative, mobile, and Interactive cells that produce abundant extracellular matrix proteins, some of which may affect BMSC Potency

    PubMed Central

    Ren, Jiaqiang; Jin, Ping; Sabatino, Marianna; Balakumaran, Arun; Feng, Ji; Kuznetsov, Sergei A.; Klein, Harvey G.; Robey, Pamela G.; Stroncek, David F.

    2012-01-01

    Background Bone marrow stromal cells (BMSCs) are being used for immune modulatory, anti-inflammatory and tissue engineering applications, but the properties responsible for these effects are not completely understood. Human BMSCs were characterized to identify factors that might be responsible for their clinical effects and biomarkers for assessing their quality. Methods Early passage BMSCs prepared from marrow aspirates of 4 healthy subjects were compared to 3 human embryonic stem cell (hESC) samples, CD34+ cells from 3 healthy subjects and 3 fibroblast cell lines. The cells were analyzed with oligonucleotide expression microarrays with more than 35,000 probes. Results BMSC gene expression signatures of BMSCs differed from those of hematopoietic stem cells (HSCs), hESCs and fibroblasts. Genes up-regulated in BMSCs were involved with cell movement, cell-to-cell signaling and interaction and proliferation. The up-regulated genes most likely belonged to pathways for integrin signaling, integrin linked kinase (ILK) signaling, NFR2-mediated oxidative stress response, regulation of actin-based motility by Rho, actin cytoskeletal signaling, caveolar-mediated endocytosis, clathrin-mediated endocytosis and Wnt/β catenin signaling. Among the most highly up-regulated genes were structural extracellular (ECM) proteins:α5 and β 5 integrin chains, fibronectin, collagen type IIIα1 and Vα1; and functional EMC proteins: connective tissue growth factor (CTGF), transforming growth factor beta induced protein (TGFBI) and ADAM12. Conclusions Global analysis of human BMSCs suggests that they are mobile, metabolically active, proliferative and interactive cells that make use of integrins and integrin signaling. They produce abundant ECM proteins that may contribute to their clinical immune modulatory and anti-inflammatory effects. PMID:21250865

  18. Altered expression and subcellular distribution of GRK subtypes in the dopamine-depleted rat basal ganglia is not normalized by L-DOPA treatment

    PubMed Central

    Ahmed, M. Rafiuddin; Bychkov, Evgeny; Gurevich, Vsevolod V.; Benovic, Jeffrey L.; Gurevich, Eugenia V.

    2009-01-01

    Dysregulation of dopamine (DA) receptors is believed to underlie Parkinson’s disease pathology and L-DOPA-induced motor complications. DA receptors are subject to regulation by G protein-coupled receptor kinases (GRKs) and arrestins. DA lesion with 6-hydroxydopamine caused multiple protein- and brain region-specific changes in the expression of GRKs. In the globus pallidus, all four GRK isoforms (GRK2, 3, 5, 6) were reduced in the lesioned hemisphere. In the caudal caudate-putamen (cCPu) three GRK isoforms (GRK2, 3, 6) were decreased by DA depletion. The decrease in GRK proteins in globus pallidus, but not cCPu, was mirrored by reduction in mRNA. GRK3 protein was reduced in the rostral caudate-putamen (rCPu), whereas other isoforms were either unchanged or up-regulated. GRK6 protein and mRNA were up-regulated in rCPu and nucleus accumbens. L-DOPA (25 mg/kg, twice daily for 10 days) failed to reverse changes caused by DA depletion, whereas D2/D3 agonist pergolide (0.25 mg/kg daily for 10 days) restored normal levels of expression of GRK5 and 6. In rCPu, GRK2 protein was increased in most subcellular fractions by L-DOPA but not by DA depletion alone. Similarly, L-DOPA up-regulated arrestin3 in membrane fractions in both regions. GRK5 was down-regulated by L-DOPA in cCPu in the light membrane fraction, where this isoform is the most abundant. The data suggest that alterations in the expression and subcellular distribution of arrestins and GRKs contribute to pathophysiology of Parkinson’s disease. Thus, these proteins may be targets for antiparkinsonian therapy. PMID:17996024

  19. The abundant elements in interstellar dust

    NASA Technical Reports Server (NTRS)

    Sofia, Ulysses J.; Cardelli, Jason A.; Savage, Blair D.

    1994-01-01

    We explore the incorporation of the cosmically abundant species O, C, N, Mg, Si, Fe, and S into interstellar dust. Column densities based on Goddard High Resolution Spectrograph 3.5 km/s resolution measurements from the literature for eight individual absorbing regions toward five lines of sight are used. Corrections are applied as needed in order to account for recent improvements in oscillator strengths. In order to acquire the most accurate column densities, and check on the accuracy of the oscillator strengths, we compare column densities based on the very strong Lorentzian damped lines of C II, O I, N I, and Mg II with results for the weak lines of these species, and confirm the previously determined f-values for O I lambda 1335, C II lambda 2325, and N I lambda lambda 1159, 1160. New empirical f-values of 1.25 x 10(exp -3) and 6.25 x 10(exp -4), respectively, are derived for the Mg II weak doublet at 1239 and 1240 A. Assuming a cosmic reference abundance based on solar and B star values, we derive depletions and dust-phase abundances which suggest that more than 70% of the available Mg and Fe is incorporated into dust-grain cores, whereas only 35% of the silicon is. This implies that oxides are important constituents of the grain core population. Mg and Fe atoms are mantled onto grain cores in a ratio of 1.8 to 1, whereas approximately 4.0 Si atoms are in the mantle per Fe atom. Since Si is not expected to accrete onto silicate or graphite grains, other grain cores, perhaps oxides and/or metallic Fe, may provide mantling sites for this species. The abundances of Fe and Mg in mantles would imply that graphite grains must have a substantial coating unless oxides provide significant mantling sites for these species. The abundance of O and N in the dust phase as implied by the solar reference abundance values are difficult to reconcile with the fact that these elements are not expected to participate in mantle formation, and the 3.1 micrometer H2O ice feature is

  20. Purification and Characterization of an Endophytic Fungal Proteinase That Is Abundantly Expressed in the Infected Host Grass.

    PubMed Central

    Lindstrom, J. T.; Belanger, F. C.

    1994-01-01

    A novel Acremonium typhinum proteinase that is expressed during endophytic infection of the grass Poa ampla Merr. was purified from endophyte-infected leaf sheath tissue. It is a thiol-containing serine alkaline endoproteinase with bound carbohydrate. In the infected host tissue, this proteinase is an abundant protein localized within fungal membrane vesicles and in the plant and/or fungal cell walls. This proteinase was not expressed constitutively during fungus culture. Rather, its expression appeared to be induced by nutrient depletion. Expression of an antigenically similar proteinase was detected in five other endophyte-infected Poa species. The regulated expression of the proteinase in culture and its abundance in infected plant tissue suggest that its expression may be involved in the symbiotic interaction of the plant and the fungus. PMID:12232300

  1. DOPAMINE DEPLETION SLOWS RETINAL TRANSMISSION

    EPA Science Inventory

    In male hooded rats, depletion of norepinephrine and dopamine by a-methyl-paratyrosine (AMT) significantly increased the latencies of early peaks in flash-evoked potentials recorded from the visual cortex, lateral geniculate nucleus, and optic tract. These effects were not produc...

  2. OXYGEN ABUNDANCES IN CEPHEIDS

    SciTech Connect

    Luck, R. E.; Andrievsky, S. M.; Korotin, S. N.; Kovtyukh, V. V. E-mail: serkor@skyline.od.ua E-mail: scan@deneb1.odessa.ua

    2013-07-01

    Oxygen abundances in later-type stars, and intermediate-mass stars in particular, are usually determined from the [O I] line at 630.0 nm, and to a lesser extent, from the O I triplet at 615.7 nm. The near-IR triplets at 777.4 nm and 844.6 nm are strong in these stars and generally do not suffer from severe blending with other species. However, these latter two triplets suffer from strong non-local thermodynamic equilibrium (NLTE) effects and thus see limited use in abundance analyses. In this paper, we derive oxygen abundances in a large sample of Cepheids using the near-IR triplets from an NLTE analysis, and compare those abundances to values derived from a local thermodynamic equilibrium (LTE) analysis of the [O I] 630.0 nm line and the O I 615.7 nm triplet as well as LTE abundances for the 777.4 nm triplet. All of these lines suffer from line strength problems making them sensitive to either measurement complications (weak lines) or to line saturation difficulties (strong lines). Upon this realization, the LTE results for the [O I] lines and the O I 615.7 nm triplet are in adequate agreement with the abundance from the NLTE analysis of the near-IR triplets.

  3. THE OXYGEN ABUNDANCE IN THE SOLAR NEIGHBORHOOD

    SciTech Connect

    RodrIguez, Monica; Delgado-Inglada, Gloria E-mail: gloria@inaoep.mx

    2011-06-01

    We present a homogeneous analysis of the oxygen abundance in five H II regions and eight planetary nebulae (PNe) located at distances lower than 2 kpc and with available spectra of high quality. We find that both the collisionally excited lines (CELs) and recombination lines imply that the PNe are overabundant in oxygen by about 0.2 dex. An explanation that reconciles the oxygen abundances derived with CELs for H II regions and PNe with the values found for B stars, the Sun, and the diffuse interstellar medium (ISM) requires the presence in H II regions of an organic refractory dust component that is not present in PNe. This dust component has already been invoked to explain the depletion of oxygen in molecular clouds and in the diffuse ISM.

  4. Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling