46 CFR 252.33 - Hull and machinery insurance.

Science.gov (United States)

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Hull and machinery insurance. 252.33 Section 252.33... Subsidy Rates § 252.33 Hull and machinery insurance. (a) Subsidy items. The fair and reasonable net premium costs (including stamp taxes) of hull and machinery, increased value, excess general average...

Structure, Function and Regulation of the Hsp90 Machinery

Directory of Open Access Journals (Sweden)

Jing Li

2013-06-01

Full Text Available Heat shock protein 90 (Hsp90 is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction, intracellular transport, and protein degradation, it became an interesting target for cancer therapy. Structurally, Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function, Hsp90 works together with a large group of cofactors, termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90, which facilitate the maturation of client proteins. In addition, posttranslational modifications of Hsp90, such as phosphorylation and acetylation, provide another level of regulation. They influence the conformational cycle, co-chaperone interaction, and inter-domain communications. In this review, we discuss the recent progress made in understanding the Hsp90 machinery.

46 CFR 282.23 - Hull and machinery insurance.

Science.gov (United States)

2010-10-01

... 46 Shipping 8 2010-10-01 2010-10-01 false Hull and machinery insurance. 282.23 Section 282.23... COMMERCE OF THE UNITED STATES Calculation of Subsidy Rates § 282.23 Hull and machinery insurance. (a) Subsidy items. The fair and reasonable net premium costs (including stamp taxes) of hull and machinery...

Bacterial mitotic machineries

DEFF Research Database (Denmark)

Gerdes, Kenn; Møller-Jensen, Jakob; Ebersbach, Gitte

2004-01-01

Here, we review recent progress that yields fundamental new insight into the molecular mechanisms behind plasmid and chromosome segregation in prokaryotic cells. In particular, we describe how prokaryotic actin homologs form mitotic machineries that segregate DNA before cell division. Thus, the P......M protein of plasmid R1 forms F actin-like filaments that separate and move plasmid DNA from mid-cell to the cell poles. Evidence from three different laboratories indicate that the morphogenetic MreB protein may be involved in segregation of the bacterial chromosome.......Here, we review recent progress that yields fundamental new insight into the molecular mechanisms behind plasmid and chromosome segregation in prokaryotic cells. In particular, we describe how prokaryotic actin homologs form mitotic machineries that segregate DNA before cell division. Thus, the Par...

46 CFR 97.30-5 - Accidents to machinery.

Science.gov (United States)

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Accidents to machinery. 97.30-5 Section 97.30-5 Shipping... Reports of Accidents, Repairs, and Unsafe Equipment § 97.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use of...

46 CFR 196.30-5 - Accidents to machinery.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Accidents to machinery. 196.30-5 Section 196.30-5... Reports of Accidents, Repairs, and Unsafe Equipment § 196.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use of...

Phosphorylation of Lbx1 controls lateral myoblast migration into the limb.

Science.gov (United States)

Masselink, Wouter; Masaki, Megumi; Sieiro, Daniel; Marcelle, Christophe; Currie, Peter D

2017-10-15

The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage. It acts as a core regulator of the limb myoblast migration machinery, controlled by Pax3. A secondary role for Lbx1 in the differentiation and commitment of limb musculature has also been proposed (Brohmann et al., 2000; Uchiyama et al., 2000). Here we show that lateral migration, but not differentiation or commitment of limb myoblasts, is controlled by the phosphorylation of three adjacent serine residues of LBX1. Electroporation of limb level somites in the chick embryo with a dephosphomimetic form of Lbx1 results in a specific defect in the lateral migration of limb myoblasts. Although the initial delamination and migration of myoblasts is unaffected, migration into the distal limb bud is severely disrupted. Interestingly, myoblasts undergo normal differentiation independent of their migratory status, suggesting that the differentiation potential of hypaxial muscle is not regulated by the phosphorylation state of LBX1. Furthermore, we show that FGF8 and ERK mediated signal transduction, both critical regulators of the developing limb bud, have the capacity to induce the phosphorylation of LBX1 at these residues. Overall, this suggests a mechanism whereby the phosphorylation of LBX1, potentially through FGF8 and ERK signalling, controls the lateral migration of myoblasts into the distal limb bud. Copyright © 2017. Published by Elsevier Inc.

Rab11 is phosphorylated by classical and novel protein kinase C isoenzymes upon sustained phorbol ester activation.

Science.gov (United States)

Pavarotti, Martín; Capmany, Anahí; Vitale, Nicolas; Colombo, María Isabel; Damiani, María Teresa

2012-02-01

Rab11 is a small GTPase that controls diverse intracellular trafficking pathways. However, the molecular machinery that regulates the participation of Rab11 in those different transport events is poorly understood. In resting cells, Rab11 localizes at the endocytic recycling compartment (ERC), whereas the different protein kinase C (PKC) isoforms display a cytosolic distribution. Sustained phorbol ester stimulation induces the translocation of the classical PKCα and PKCβII isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKCε and atypical PKCζ isoenzymes neither redistribute to the perinucleus nor modify transferrin recycling transport after phorbol ester stimulation. Although several Rabs have been shown to be phosphorylated, there is to date no evidence indicating Rab11 as a kinase substrate. In this report, we show that Rab11 appears phosphorylated in vivo in phorbol ester-stimulated cells. A bioinformatic analysis of Rab11 allowed us to identify several high-probability Ser/Thr kinase phosphorylation sites. Our results demonstrate that classical PKC (PKCα and PKCβII but not PKCβI) directly phosphorylate Rab11 in vitro. In addition, novel PKCε and PKCη but not PKCδ isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKCβII or PKCε. In agreement, the phosphomimetic mutant, Rab11 S177D, retains transferrin at the ERC in the absence of phorbol-12-myristate-13-acetate stimulus. This report shows for the first time that Rab11 is differentially phosphorylated by distinct PKC isoenzymes and that this post-translational modification might be a regulatory mechanism of intracellular trafficking. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

46 CFR 109.205 - Inspection of boilers and machinery.

Science.gov (United States)

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Inspection of boilers and machinery. 109.205 Section 109... OPERATIONS Tests, Drills, and Inspections § 109.205 Inspection of boilers and machinery. The chief engineer or engineer in charge, before he assumes charge of the boilers and machinery of a unit shall inspect...

46 CFR 185.208 - Accidents to machinery.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Accidents to machinery. 185.208 Section 185.208 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) OPERATIONS Marine Casualties and Voyage Records § 185.208 Accidents to machinery. The owner, managing...

46 CFR 122.208 - Accidents to machinery.

Science.gov (United States)

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Accidents to machinery. 122.208 Section 122.208 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS CARRYING MORE THAN 150... Voyage Records § 122.208 Accidents to machinery. The owner, managing operator, or master shall report...

29 CFR 1915.164 - Ship's propulsion machinery.

Science.gov (United States)

2010-07-01

... 29 Labor 7 2010-07-01 2010-07-01 false Ship's propulsion machinery. 1915.164 Section 1915.164 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) OCCUPATIONAL SAFETY AND HEALTH STANDARDS FOR SHIPYARD EMPLOYMENT Ship's Machinery and Piping Systems § 1915.164 Ship's...

Vibration vector monitoring of rotating machinery: A predictive/preventative maintenance technique

International Nuclear Information System (INIS)

Humes, B.R.

1990-01-01

Monitoring of overall vibration amplitudes to indicate machinery faults is a standard practice in most industries. The appearance of shaft cracks in machines retrofitted for extended life have prompted development of higher levels of machinery monitoring. Part 1 of this paper discusses vibration vector monitoring for machinery malfunction prediction and failure prevention. Machinery faults which can be diagnosed by this type of monitoring, such as rotor rubs, loose parts, shaft cracks, ..., are presented along with their most common characteristics. The newest, most effective methods of permanent machinery monitoring are presented and critiqued. An extensive case history is presented in Part 2 in which a potentially disastrous machinery fault was predicted using vibration vector monitoring and analysis. The addition of vector monitoring to the normal, overall vibration monitoring proved more effective in diagnosing the machinery fault and predicting impending failure

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.

Science.gov (United States)

Belluzzi, Elisa; Gonnelli, Adriano; Cirnaru, Maria-Daniela; Marte, Antonella; Plotegher, Nicoletta; Russo, Isabella; Civiero, Laura; Cogo, Susanna; Carrion, Maria Perèz; Franchin, Cinzia; Arrigoni, Giorgio; Beltramini, Mariano; Bubacco, Luigi; Onofri, Franco; Piccoli, Giovanni; Greggio, Elisa

2016-01-13

Lrrk2, a gene linked to Parkinson's disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson's disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

Risk assessments and safe machinery ensuring compliance with the EU directives

CERN Document Server

Jespen, Torben

2016-01-01

This book describes the prerequisites for the placing on the market and the safe use of machinery in compliance with the relevant EU Directives, especially the Machinery Directive 2006/42. It provides readers with high-level knowledge concerning the Essential Health and Safety Requirements (EHSR) that machinery must fulfill. The approach and principles of the Machinery Directive were most recently made worldwide acknowledged in the ILO code of practice on safe machinery, released in 2013. The book addresses that code, as well as providing valuable insight into other EU Product and Workplace legislation. Focusing on the key aspect of safe machinery, the “machinery safety risk assessment”, which allows readers to better understand the more difficult aspects of risk assessments, the book equips readers to tackle problems at the manufacturing stage and in different use scenarios, introducing them to risk reduction techniques and functional safety aspects.

Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation.

Science.gov (United States)

Ferreira, Filipe; Foley, Matthew; Cooke, Alex; Cunningham, Margaret; Smith, Gemma; Woolley, Robert; Henderson, Graeme; Kelly, Eamonn; Mundell, Stuart; Smythe, Elizabeth

2012-08-07

Signaling by transmembrane receptors such as G protein-coupled receptors (GPCRs) occurs at the cell surface and throughout the endocytic pathway, and signaling from the cell surface may differ in magnitude and downstream output from intracellular signaling. As a result, the rate at which signaling molecules traverse the endocytic pathway makes a significant contribution to downstream output. Modulation of the core endocytic machinery facilitates differential uptake of individual cargoes. Clathrin-coated pits are a major entry portal where assembled clathrin forms a lattice around invaginating buds that have captured endocytic cargo. Clathrin assembles into triskelia composed of three clathrin heavy chains and associated clathrin light chains (CLCs). Despite the identification of clathrin-coated pits at the cell surface over 30 years ago, the functions of CLCs in endocytosis have been elusive. In this work, we identify a novel role for CLCs in the regulated endocytosis of specific cargoes. Small interfering RNA-mediated knockdown of either CLCa or CLCb inhibits the uptake of GPCRs. Moreover, we demonstrate that phosphorylation of Ser204 in CLCb is required for efficient endocytosis of a subset of GPCRs and identify G protein-coupled receptor kinase 2 (GRK2) as a kinase that can phosphorylate CLCb on Ser204. Overexpression of CLCb(S204A) specifically inhibits the endocytosis of those GPCRs whose endocytosis is GRK2-dependent. Together, these results indicate that CLCb phosphorylation acts as a discriminator for the endocytosis of specific GPCRs. Copyright © 2012 Elsevier Ltd. All rights reserved.

46 CFR 182.465 - Ventilation of spaces containing diesel machinery.

Science.gov (United States)

2010-10-01

... furnish natural or powered supply and exhaust ventilation. The total inlet area and the total outlet area... 46 Shipping 7 2010-10-01 2010-10-01 false Ventilation of spaces containing diesel machinery. 182... Ventilation of spaces containing diesel machinery. (a) A space containing diesel machinery must be fitted with...

The Research of Computer Aided Farm Machinery Designing Method Based on Ergonomics

Science.gov (United States)

Gao, Xiyin; Li, Xinling; Song, Qiang; Zheng, Ying

Along with agricultural economy development, the farm machinery product type Increases gradually, the ergonomics question is also getting more and more prominent. The widespread application of computer aided machinery design makes it possible that farm machinery design is intuitive, flexible and convenient. At present, because the developed computer aided ergonomics software has not suitable human body database, which is needed in view of farm machinery design in China, the farm machinery design have deviation in ergonomics analysis. This article puts forward that using the open database interface procedure in CATIA to establish human body database which aims at the farm machinery design, and reading the human body data to ergonomics module of CATIA can product practical application virtual body, using human posture analysis and human activity analysis module to analysis the ergonomics in farm machinery, thus computer aided farm machinery designing method based on engineering can be realized.

46 CFR 97.15-15 - Examination of boilers and machinery.

Science.gov (United States)

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Examination of boilers and machinery. 97.15-15 Section... VESSELS OPERATIONS Tests, Drills, and Inspections § 97.15-15 Examination of boilers and machinery. It shall be the duty of the chief engineer when assuming charge of the boilers and machinery of a vessel to...

46 CFR 32.35-1 - Boilers and machinery-TB/ALL.

Science.gov (United States)

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Boilers and machinery-TB/ALL. 32.35-1 Section 32.35-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS SPECIAL EQUIPMENT, MACHINERY, AND HULL REQUIREMENTS Main and Auxiliary Machinery § 32.35-1 Boilers and machinery—TB/ALL. Boilers, main and auxiliary...

46 CFR 78.17-30 - Examination of boilers and machinery.

Science.gov (United States)

2010-10-01

... 46 Shipping 3 2010-10-01 2010-10-01 false Examination of boilers and machinery. 78.17-30 Section... OPERATIONS Tests, Drills, and Inspections § 78.17-30 Examination of boilers and machinery. It shall be the duty of the chief engineer when assuming charge of the boilers and machinery of a vessel to examine...

Machinery condition monitoring principles and practices

CERN Document Server

Mohanty, Amiya Ranjan

2015-01-01

Find the Fault in the MachinesDrawing on the author's more than two decades of experience with machinery condition monitoring and consulting for industries in India and abroad, Machinery Condition Monitoring: Principles and Practices introduces the practicing engineer to the techniques used to effectively detect and diagnose faults in machines. Providing the working principle behind the instruments, the important elements of machines as well as the technique to understand their conditions, this text presents every available method of machine fault detection occurring in machines in general, an

46 CFR 196.15-15 - Examination of boilers and machinery.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Examination of boilers and machinery. 196.15-15 Section... VESSELS OPERATIONS Test, Drills, and Inspections § 196.15-15 Examination of boilers and machinery. (a) It shall be the duty of the chief engineer when he assumes charge of the boilers and machinery of a vessel...

Vibration of hydraulic machinery

CERN Document Server

Wu, Yulin; Liu, Shuhong; Dou, Hua-Shu; Qian, Zhongdong

2013-01-01

Vibration of Hydraulic Machinery deals with the vibration problem which has significant influence on the safety and reliable operation of hydraulic machinery. It provides new achievements and the latest developments in these areas, even in the basic areas of this subject. The present book covers the fundamentals of mechanical vibration and rotordynamics as well as their main numerical models and analysis methods for the vibration prediction. The mechanical and hydraulic excitations to the vibration are analyzed, and the pressure fluctuations induced by the unsteady turbulent flow is predicted in order to obtain the unsteady loads. This book also discusses the loads, constraint conditions and the elastic and damping characters of the mechanical system, the structure dynamic analysis, the rotor dynamic analysis and the system instability of hydraulic machines, including the illustration of monitoring system for the instability and the vibration in hydraulic units. All the problems are necessary for vibration pr...

46 CFR 78.33-5 - Accidents to machinery.

Science.gov (United States)

2010-10-01

... 46 Shipping 3 2010-10-01 2010-10-01 false Accidents to machinery. 78.33-5 Section 78.33-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) PASSENGER VESSELS OPERATIONS Reports of Accidents, Repairs, and Unsafe Equipment § 78.33-5 Accidents to machinery. (a) In the event of an accident...

Open innovation and supply chain management in food machinery ...

African Journals Online (AJOL)

Open innovation and supply chain management in food machinery supply chain: a ... This paradigm describes a new approach to internal R&D management, which ... a picture of the adoption of open innovation in the food machinery industry.

46 CFR 169.629 - Compartments containing gasoline machinery or fuel tanks.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Compartments containing gasoline machinery or fuel tanks. 169.629 Section 169.629 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL... gasoline machinery or fuel tanks. Spaces containing gasoline machinery or fuel tanks must have natural...

46 CFR 116.620 - Ventilation of machinery and fuel tank spaces.

Science.gov (United States)

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Ventilation of machinery and fuel tank spaces. 116.620... AND ARRANGEMENT Ventilation § 116.620 Ventilation of machinery and fuel tank spaces. In addition to the requirements of this subpart, ventilation systems for spaces containing machinery or fuel tanks...

46 CFR 91.15-1 - Standards in inspection of hulls, boilers, and machinery.

Science.gov (United States)

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Standards in inspection of hulls, boilers, and machinery... hulls, boilers, and machinery. In the inspection of hulls, boilers, and machinery of vessels, the..., respecting material and inspection of hulls, boilers, and machinery, and the certificate of classification...

46 CFR 189.15-1 - Standards in inspection of hulls, boilers, and machinery.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Standards in inspection of hulls, boilers, and machinery... inspection of hulls, boilers, and machinery. In the inspection of hulls, boilers, and machinery of vessels... chapter, respecting material and construction of hulls, boilers, and machinery, and certificate of...

46 CFR 71.15-1 - Standards in inspection of hulls, boilers, and machinery.

Science.gov (United States)

2010-10-01

... 46 Shipping 3 2010-10-01 2010-10-01 false Standards in inspection of hulls, boilers, and machinery..., boilers, and machinery. In the inspection of hulls, boilers, and machinery of vessels, the standards... and inspection of hulls, boilers, and machinery, and the certificate of classification referring...

49 CFR 1242.47 - Machinery (account XX-27-40).

Science.gov (United States)

2010-10-01

... 49 Transportation 9 2010-10-01 2010-10-01 false Machinery (account XX-27-40). 1242.47 Section 1242...-Equipment § 1242.47 Machinery (account XX-27-40). Separate common expenses on the basis of the freight/passenger separation of administration (account XX-27-01). ...

Phosphorylated nano-diamond/ Polyimide Nanocomposites

International Nuclear Information System (INIS)

Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Kahraman, Memet Vezir

2014-01-01

In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased

46 CFR 169.315 - Ventilation (other than machinery spaces).

Science.gov (United States)

2010-10-01

... section is satisfied, a vessel having only a natural ventilation system must satisfy the following: V/A≥1... 46 Shipping 7 2010-10-01 2010-10-01 false Ventilation (other than machinery spaces). 169.315... SCHOOL VESSELS Construction and Arrangement Hull Structure § 169.315 Ventilation (other than machinery...

Budding yeast ATM/ATR control meiotic double-strand break (DSB levels by down-regulating Rec114, an essential component of the DSB-machinery.

Directory of Open Access Journals (Sweden)

Jesús A Carballo

2013-06-01

Full Text Available An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs. Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks.

Budding Yeast ATM/ATR Control Meiotic Double-Strand Break (DSB) Levels by Down-Regulating Rec114, an Essential Component of the DSB-machinery

Science.gov (United States)

Carballo, Jesús A.; Panizza, Silvia; Serrentino, Maria Elisabetta; Johnson, Anthony L.; Geymonat, Marco; Borde, Valérie; Klein, Franz; Cha, Rita S.

2013-01-01

An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or “DSB homeostasis”, might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks. PMID:23825959

40 CFR 180.521 - Fumigants for grain-mill machinery; tolerances for residues.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Fumigants for grain-mill machinery... Tolerances § 180.521 Fumigants for grain-mill machinery; tolerances for residues. (a) General. Fumigants may be safely used in or on grain-mill machinery in accordance with the following prescribed conditions...

Genome-scale modeling of the protein secretory machinery in yeast

DEFF Research Database (Denmark)

Feizi, Amir; Österlund, Tobias; Petranovic, Dina

2013-01-01

The protein secretory machinery in Eukarya is involved in post-translational modification (PTMs) and sorting of the secretory and many transmembrane proteins. While the secretory machinery has been well-studied using classic reductionist approaches, a holistic view of its complex nature is lacking....... Here, we present the first genome-scale model for the yeast secretory machinery which captures the knowledge generated through more than 50 years of research. The model is based on the concept of a Protein Specific Information Matrix (PSIM: characterized by seven PTMs features). An algorithm...

Oxidative phosphorylation revisited

DEFF Research Database (Denmark)

Nath, Sunil; Villadsen, John

2015-01-01

The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic...

Machinery failure analysis and troubleshooting practical machinery management for process plants

CERN Document Server

Bloch, Heinz P

2012-01-01

Solve the machinery failure problems costing you time and money with this classic, comprehensive guide to analysis and troubleshooting  Provides detailed, complete and accurate information on anticipating risk of component failure and avoiding equipment downtime Includes numerous photographs of failed parts to ensure you are familiar with the visual evidence you need to recognize Covers proven approaches to failure definition and offers failure identification and analysis methods that can be applied to virtually all problem situations Demonstr

Radiation technologies in metallurgy and machinery

International Nuclear Information System (INIS)

Meshkov, I.N.

1990-01-01

Applications of electron beam accelerators for technologies in metallurgy and machinery are discussed. Processes described are provided with special industrial accelerators, developed in the Institute of Nuclear Physics, Novosibirsk. (author)

Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation.

Science.gov (United States)

Swetha, Medchalmi; Ramaiah, Kolluru V A

2015-11-01

Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells. Copyright © 2015 Elsevier Inc. All rights reserved.

FOREWORD: 26th IAHR Symposium on Hydraulic Machinery and Systems

Science.gov (United States)

Wu, Yulin; Wang, Zhengwei; Liu, Shuhong; Yuan, Shouqi; Luo, Xingqi; Wang, Fujun

2012-11-01

The 26th IAHR Symposium on Hydraulic Machinery and Systems, will be held in Beijing, China, 19-23 August 2012. It is jointly organized by Tsinghua University, State Key Laboratory of Hydro Science and Hydraulic Engineering, China, Jiangsu University, Xi'an University of Technology, China Agricultural University, National Engineering Research Center of Hydropower Equipment and Dongfang Electric Machinery Co., Ltd. It is the second time that China hosts such a symposium. By the end of 2011, the China electrical power system had a total of 1 050 GW installed power, out of which 220 GW was in hydropower plants. The energy produced in hydropower facilities was 662.6 TWh from a total of 4,720 TWh electrical energy production in 2011. Moreover, in 2020, new hydropower capacities are going to be developed, with a total of 180 GW installed power and an estimated 708 TWh/year energy production. And in 2011, the installed power of pumped storage stations was about 25GW. In 2020, the data will be 70GW. At the same time, the number of pumps used in China is increasing rapidly. China produces about 29,000,000 pumps with more than 220 series per year. By the end of 2011, the Chinese pumping system has a total of 950 GW installed power. The energy consumed in pumping facilities was 530 TWh in 2011. The pump energy consumption accounted for about 12% of the national electrical energy production. Therefore, there is a large market in the field of hydraulic machinery including water turbines, pump turbines and a variety of pumps in China. There are also many research projects in this field. For example, we have conducted National Key Research Projects on 1000 MW hydraulic turbine, and on the pump turbines with high head, as well as on the large capacity pumps for water supply. Tsinghua University of Beijing is proud to host the 26th IAHR Symposium on Hydraulic Machinery and Systems. Tsinghua University was established in 1911, after the founding of the People's Republic of China. It

Fault size classification of rotating machinery using support vector machine

Energy Technology Data Exchange (ETDEWEB)

Kim, Y. S.; Lee, D. H.; Park, S. K. [Korea Hydro and Nuclear Power Co. Ltd., Daejeon (Korea, Republic of)

2012-03-15

Studies on fault diagnosis of rotating machinery have been carried out to obtain a machinery condition in two ways. First is a classical approach based on signal processing and analysis using vibration and acoustic signals. Second is to use artificial intelligence techniques to classify machinery conditions into normal or one of the pre-determined fault conditions. Support Vector Machine (SVM) is well known as intelligent classifier with robust generalization ability. In this study, a two-step approach is proposed to predict fault types and fault sizes of rotating machinery in nuclear power plants using multi-class SVM technique. The model firstly classifies normal and 12 fault types and then identifies their sizes in case of predicting any faults. The time and frequency domain features are extracted from the measured vibration signals and used as input to SVM. A test rig is used to simulate normal and the well-know 12 artificial fault conditions with three to six fault sizes of rotating machinery. The application results to the test data show that the present method can estimate fault types as well as fault sizes with high accuracy for bearing an shaft-related faults and misalignment. Further research, however, is required to identify fault size in case of unbalance, rubbing, looseness, and coupling-related faults.

Fault size classification of rotating machinery using support vector machine

International Nuclear Information System (INIS)

Kim, Y. S.; Lee, D. H.; Park, S. K.

2012-01-01

Studies on fault diagnosis of rotating machinery have been carried out to obtain a machinery condition in two ways. First is a classical approach based on signal processing and analysis using vibration and acoustic signals. Second is to use artificial intelligence techniques to classify machinery conditions into normal or one of the pre-determined fault conditions. Support Vector Machine (SVM) is well known as intelligent classifier with robust generalization ability. In this study, a two-step approach is proposed to predict fault types and fault sizes of rotating machinery in nuclear power plants using multi-class SVM technique. The model firstly classifies normal and 12 fault types and then identifies their sizes in case of predicting any faults. The time and frequency domain features are extracted from the measured vibration signals and used as input to SVM. A test rig is used to simulate normal and the well-know 12 artificial fault conditions with three to six fault sizes of rotating machinery. The application results to the test data show that the present method can estimate fault types as well as fault sizes with high accuracy for bearing an shaft-related faults and misalignment. Further research, however, is required to identify fault size in case of unbalance, rubbing, looseness, and coupling-related faults

The Autophagic Machinery in Enterovirus Infection.

Science.gov (United States)

Lai, Jeffrey K F; Sam, I-Ching; Chan, Yoke Fun

2016-01-27

The Enterovirus genus of the Picornaviridae family comprises many important human pathogens, including polioviruses, rhinovirus, enterovirus A71, and enterovirus D68. They cause a wide variety of diseases, ranging from mild to severe life-threatening diseases. Currently, no effective vaccine is available against enteroviruses except for poliovirus. Enteroviruses subvert the autophagic machinery to benefit their assembly, maturation, and exit from host. Some enteroviruses spread between cells via a process described as autophagosome-mediated exit without lysis (AWOL). The early and late phases of autophagy are regulated through various lipids and their metabolizing enzymes. Some of these lipids and enzymes are specifically regulated by enteroviruses. In the present review, we summarize the current understanding of the regulation of autophagic machinery by enteroviruses, and provide updates on recent developments in this field.

A Survey of Fish Production and Processing Machinery in Rivers ...

African Journals Online (AJOL)

Survey of fish production and processing machinery in Port Harcourt City Local Government Area of Rivers State, Nigeria was carried out to evaluate the followings: different machines used for fish production and processing, the most acceptable machine, effect of cost of machinery on the fish farmer, whether gender has ...

Coordinate to Guard: Crosstalk of Phosphorylation, Sumoylation, and Ubiquitylation in DNA Damage Response

International Nuclear Information System (INIS)

Kuo, Ching-Ying; Shieh, Christine; Cai, Fei; Ann, David Kong

2012-01-01

Small ubiquitin-like modifier-1/2/3 (SUMO-1/2/3) and ubiquitin share similar structure and utilize analogous machinery for protein lysine conjugation. Although sumoylation and ubiquitylation have distinct functions, they are often tightly associated with each other to fine-tune protein fate in transducing signals to regulate a wide variety of cellular functions, including DNA damage response, cell proliferation, DNA replication, embryonic development, and cell differentiation. In this Perspective, we specifically highlight the role of sumoylation and ubiquitylation in ataxia-telangiectasia mutated (ATM) signaling in response to DNA double-strand breaks and hypothesize that ATM-induced phosphorylation is a unique node in regulating SUMO-targeted ubiquitylation in mammalian cells to combat DNA damage and to maintain genome integrity. A potential role for the coordination of three types of post-translational modification in dictating the tempo and extent of cellular response to genotoxic stress is speculated.

Coordinate to Guard: Crosstalk of Phosphorylation, Sumoylation, and Ubiquitylation in DNA Damage Response

Energy Technology Data Exchange (ETDEWEB)

Kuo, Ching-Ying [Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA (United States); Department of Molecular Pharmacology, Beckman Research Institute of City of Hope, Duarte, CA (United States); Shieh, Christine; Cai, Fei [Eugene and Ruth Roberts Summer Student Academy, Beckman Research Institute of City of Hope, Duarte, CA (United States); Ann, David Kong, E-mail: dann@coh.org [Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA (United States); Department of Molecular Pharmacology, Beckman Research Institute of City of Hope, Duarte, CA (United States); Eugene and Ruth Roberts Summer Student Academy, Beckman Research Institute of City of Hope, Duarte, CA (United States)

2012-01-19

Small ubiquitin-like modifier-1/2/3 (SUMO-1/2/3) and ubiquitin share similar structure and utilize analogous machinery for protein lysine conjugation. Although sumoylation and ubiquitylation have distinct functions, they are often tightly associated with each other to fine-tune protein fate in transducing signals to regulate a wide variety of cellular functions, including DNA damage response, cell proliferation, DNA replication, embryonic development, and cell differentiation. In this Perspective, we specifically highlight the role of sumoylation and ubiquitylation in ataxia-telangiectasia mutated (ATM) signaling in response to DNA double-strand breaks and hypothesize that ATM-induced phosphorylation is a unique node in regulating SUMO-targeted ubiquitylation in mammalian cells to combat DNA damage and to maintain genome integrity. A potential role for the coordination of three types of post-translational modification in dictating the tempo and extent of cellular response to genotoxic stress is speculated.

Detection of Early Faults in Rotating Machinery Based on Wavelet Analysis

Directory of Open Access Journals (Sweden)

Meng Hee Lim

2013-01-01

Full Text Available This paper explores the application of wavelet analysis for the detection of early changes in rotor dynamics caused by common machinery faults, namely, rotor unbalance and minor blade rubbing conditions. In this paper, the time synchronised wavelet analysis method was formulated and its effectiveness to detect machinery faults at the early stage was evaluated based on signal simulation and experimental study. The proposed method provides a more standardised approach to visualise the current state of rotor dynamics of a rotating machinery by taking into account the effects of time shift, wavelet edge distortion, and system noise suppression. The experimental results showed that this method is able to reveal subtle changes of the vibration signal characteristics in both the frequency content distribution and the amplitude distortion caused by minor rotor unbalance and blade rubbing conditions. Besides, this method also appeared to be an effective tool to diagnose and to discriminate the different types of machinery faults based on the unique pattern of the wavelet contours. This study shows that the proposed wavelet analysis method is promising to reveal machinery faults at early stage as compared to vibration spectrum analysis.

Signifiance éthique et corporéité dans Autrement qu’être ou au-delà de l’essence

Czech Academy of Sciences Publication Activity Database

Bierhanzl, Jan

2012-01-01

Roč. 12, č. 1 (2012), s. 85-107 ISSN 1582-5647 R&D Projects: GA ČR(CZ) GAP401/10/1164 Institutional support: RVO:67985955 Keywords : ethics * signifying corporeity * Levinas * Selfhood Subject RIV: AA - Philosophy ; Religion

The capacity of sugar beet farms’ machinery and equipment

Directory of Open Access Journals (Sweden)

Małgorzata BZOWSKA – BAKALARZ

2012-12-01

Full Text Available The survey investigates into equipment of sugar beet farms of the Lublin region, Poland, with machinery – with reference to plantation size and yields. To assess the production potential of the farms, the authors determined the age structure of the machinery owned by the farmers and established the scale of investment in new equipment. The machinery most important for sugar beet production are pre-sowing and post-harvest tillage units, sprayers, seed drills, combine harvesters and self-unloading trailers. In most cases, the surveyed farmers own most of these machines, but they are often obsolete: 37% of them is in operation for more than 15 years. As for the machines dedicated solely to sugar beet growing (harvesters and seeders, their age structure is most unfavourable – 70% of them have been used for over 15 years. A trend towards increasing plantation sizes provides incentives for introducing innovation to cultivation methods. However, the scale of investment in new machinery is small, especially in the case of small and medium-sized farms that dominate in the region. The authors surveyed also the scale of using professional services in the field of tillage processes to determine changes in farming practices.

PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

International Nuclear Information System (INIS)

Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

2015-01-01

Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression

Multiscale singular value manifold for rotating machinery fault diagnosis

Energy Technology Data Exchange (ETDEWEB)

Feng, Yi; Lu, BaoChun; Zhang, Deng Feng [School of Mechanical Engineering, Nanjing University of Science and Technology,Nanjing (United States)

2017-01-15

Time-frequency distribution of vibration signal can be considered as an image that contains more information than signal in time domain. Manifold learning is a novel theory for image recognition that can be also applied to rotating machinery fault pattern recognition based on time-frequency distributions. However, the vibration signal of rotating machinery in fault condition contains cyclical transient impulses with different phrases which are detrimental to image recognition for time-frequency distribution. To eliminate the effects of phase differences and extract the inherent features of time-frequency distributions, a multiscale singular value manifold method is proposed. The obtained low-dimensional multiscale singular value manifold features can reveal the differences of different fault patterns and they are applicable to classification and diagnosis. Experimental verification proves that the performance of the proposed method is superior in rotating machinery fault diagnosis.

SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides

DEFF Research Database (Denmark)

Thingholm, Tine E; Jensen, Ole N; Robinson, Phillip J

2008-01-01

spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from...... and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides....

STUDY OF THE PARAMETERS OF EFFICIENCY IN CENTRES FOR REPAIR OF AGRICULTURAL MACHINERY

Directory of Open Access Journals (Sweden)

Natalia Stoyanova

2015-06-01

Full Text Available The paper makes a thorough study of the parameters of efficiency in the centers for the repair of agricultural machinery, considering production and technological structure, the basic principles for design of the process of service, the quantitative indicators for servicing. It presents a theoretical model for the management of services in the service business, taking into account the basic system requirements for maintenance of agricultural machinery, the main elements of the standards of customer service, choice of forms for maintenance of agricultural machinery. Opportunities are proposed for the optimization of repair activities in the centers for repair of agricultural machinery.

Recruitment of TREX to the transcription machinery by its direct binding to the phospho-CTD of RNA polymerase II.

Directory of Open Access Journals (Sweden)

Dominik M Meinel

2013-11-01

Full Text Available Messenger RNA (mRNA synthesis and export are tightly linked, but the molecular mechanisms of this coupling are largely unknown. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to mRNA export and mediates mRNP formation. Here, we show that TREX is recruited to the transcription machinery by direct interaction of its subcomplex THO with the serine 2-serine 5 (S2/S5 diphosphorylated CTD of RNA polymerase II. S2 and/or tyrosine 1 (Y1 phosphorylation of the CTD is required for TREX occupancy in vivo, establishing a second interaction platform necessary for TREX recruitment in addition to RNA. Genome-wide analyses show that the occupancy of THO and the TREX components Sub2 and Yra1 increases from the 5' to the 3' end of the gene in accordance with the CTD S2 phosphorylation pattern. Importantly, in a mutant strain, in which TREX is recruited to genes but does not increase towards the 3' end, the expression of long transcripts is specifically impaired. Thus, we show for the first time that a 5'-3' increase of a protein complex is essential for correct expression of the genome. In summary, we provide insight into how the phospho-code of the CTD directs mRNP formation and export through TREX recruitment.

Recruitment of TREX to the transcription machinery by its direct binding to the phospho-CTD of RNA polymerase II.

Science.gov (United States)

Meinel, Dominik M; Burkert-Kautzsch, Cornelia; Kieser, Anja; O'Duibhir, Eoghan; Siebert, Matthias; Mayer, Andreas; Cramer, Patrick; Söding, Johannes; Holstege, Frank C P; Sträßer, Katja

2013-11-01

Messenger RNA (mRNA) synthesis and export are tightly linked, but the molecular mechanisms of this coupling are largely unknown. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to mRNA export and mediates mRNP formation. Here, we show that TREX is recruited to the transcription machinery by direct interaction of its subcomplex THO with the serine 2-serine 5 (S2/S5) diphosphorylated CTD of RNA polymerase II. S2 and/or tyrosine 1 (Y1) phosphorylation of the CTD is required for TREX occupancy in vivo, establishing a second interaction platform necessary for TREX recruitment in addition to RNA. Genome-wide analyses show that the occupancy of THO and the TREX components Sub2 and Yra1 increases from the 5' to the 3' end of the gene in accordance with the CTD S2 phosphorylation pattern. Importantly, in a mutant strain, in which TREX is recruited to genes but does not increase towards the 3' end, the expression of long transcripts is specifically impaired. Thus, we show for the first time that a 5'-3' increase of a protein complex is essential for correct expression of the genome. In summary, we provide insight into how the phospho-code of the CTD directs mRNP formation and export through TREX recruitment.

Condition Monitoring of Machinery in Non-Stationary Operations : Proceedings of the Second International Conference "Condition Monitoring of Machinery in Non-Stationnary Operations"

CERN Document Server

Bartelmus, Walter; Chaari, Fakher; Zimroz, Radoslaw; Haddar, Mohamed

2012-01-01

Condition monitoring of machines in non-stationary operations (CMMNO) can be seen as the major challenge for research in the field of machinery diagnostics. Condition monitoring of machines in non-stationary operations is the title of the presented book and the title of the Conference held in Hammamet - Tunisia March 26 – 28, 2012. It is the second conference under this title, first took place in Wroclaw - Poland , March 2011. The subject CMMNO comes directly from industry needs and observation of real objects. Most monitored and diagnosed objects used in industry works in non-stationary operations condition. The non-stationary operations come from fulfillment of machinery tasks, for which they are designed for. All machinery used in different kind of mines, transport systems, vehicles like: cars, buses etc, helicopters, ships and battleships and so on work in non-stationary operations. The papers included in the book are shaped by the organizing board of the conference and authors of the papers. The papers...

Design and Realization of Rotating Machinery Conditions Monitoring System Based on Labview

Science.gov (United States)

Fan, Qiyuan

Nonlinear dynamic analysis of rotating machinery system has always been the hot spot of the rotational dynamics research. This article sets up a rotating machinery condition monitoring system to realize the measurement of system dynamic characteristic parameters based on NI(National Instruments) virtual instruments technology. The measurement of vibration signal of rotating machinery system is achieved by using NI company general data acquisition module of NI company. Meanwhile, by analyzing and processing the acquired data using Labview 2012, the dynamic characteristics, such as .the speed of the rotating machinery system, the axis trajectory, spectrum parameters, are attained. The measurement results show that the rotating machinery condition monitoring system based on Labview is easy to operate, easy to realize the function extension and maintenance, and that it can be used in the industrial engineering projects with rotation characteristics. Labview as the development tools used by virtual instrument function, is very powerful data acquisition software products support is one of the features of it, so using Labview programming and data acquisition is simple and convenient [1].

Emergence and modular evolution of a novel motility machinery in bacteria.

Directory of Open Access Journals (Sweden)

Jennifer Luciano

2011-09-01

Full Text Available Bacteria glide across solid surfaces by mechanisms that have remained largely mysterious despite decades of research. In the deltaproteobacterium Myxococcus xanthus, this locomotion allows the formation stress-resistant fruiting bodies where sporulation takes place. However, despite the large number of genes identified as important for gliding, no specific machinery has been identified so far, hampering in-depth investigations. Based on the premise that components of the gliding machinery must have co-evolved and encode both envelope-spanning proteins and a molecular motor, we re-annotated known gliding motility genes and examined their taxonomic distribution, genomic localization, and phylogeny. We successfully delineated three functionally related genetic clusters, which we proved experimentally carry genes encoding the basal gliding machinery in M. xanthus, using genetic and localization techniques. For the first time, this study identifies structural gliding motility genes in the Myxobacteria and opens new perspectives to study the motility mechanism. Furthermore, phylogenomics provide insight into how this machinery emerged from an ancestral conserved core of genes of unknown function that evolved to gliding by the recruitment of functional modules in Myxococcales. Surprisingly, this motility machinery appears to be highly related to a sporulation system, underscoring unsuspected common mechanisms in these apparently distinct morphogenic phenomena.

A novel ATP-generating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation.

Science.gov (United States)

Auger, Christopher; Appanna, Vasu D

2015-01-01

It is well-known that elevated amounts of nitric oxide and other reactive nitrogen species (RNS) impact negatively on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. These perturbations severely compromise O2-dependent energy production. While bacteria are known to adapt to RNS, a key tool employed by macrophages to combat infections, the exact mechanisms are unknown. The bacterium was cultured in a defined mineral medium and cell-free extracts obtained at the same growth phase were utilized for various biochemical studies Blue native polyacrylamide gel electrophoresis followed by in-gel activity assays, high performance liquid chromatography and co-immunoprecipitaton are applied to investigate the effects of RNS on the model microbe Pseudomonas fluorescens. Citrate is channeled away from the tricarboxylic acid cycle using a novel metabolon consisting of citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK). This metabolic engine comprising three disparate enzymes appears to transiently assemble as a supercomplex aimed at ATP synthesis. The up-regulation in the activities of adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK) ensured the efficacy of this ATP-making machine. Microbes may escape the effects of nitrosative stress by re-engineering metabolic networks in order to generate and store ATP anaerobically when the electron transport chain is defective. The molecular configuration described herein provides further understanding of how metabolism plays a key role in the adaptation to nitrosative stress and reveals novel targets that will inform the development of antimicrobial agents to counter RNS-resistant pathogens. Copyright © 2014 Elsevier B.V. All rights reserved.

46 CFR 169.631 - Separation of machinery and fuel tank spaces from accommodation spaces.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Separation of machinery and fuel tank spaces from accommodation spaces. 169.631 Section 169.631 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED... machinery and fuel tank spaces from accommodation spaces. (a) Machinery and fuel tank spaces must be...

Aquí y en el otro lado. Los signifi cados socioculturales de la sexualidad y sus implicaciones en la salud sexual de los migrantes mexicanos

OpenAIRE

Imelda Hidalgo; Fabiola García; Ángel Flores; Xóchitl Castañeda; George F. Lemp; Juan Ruiz

2008-01-01

Se analizan los signifi cados culturales sobre la sexualidad y el género que permean las actividades sexuales de los migrantes temporales en Estados Unidos, así como las condiciones sociales que favorecen su involucramiento en prácticas de riesgo. Los resultados muestran cómo la construcción social de los signifi cados en las comunidades de origen y las condiciones sociales subyacentes al movimiento migratorio, incluyendo los procesos de hibridación cultural, favorecen el ejerc...

Application of DNA Machineries for the Barcode Patterned Detection of Genes or Proteins.

Science.gov (United States)

Zhou, Zhixin; Luo, Guofeng; Wulf, Verena; Willner, Itamar

2018-06-05

The study introduces an analytical platform for the detection of genes or aptamer-ligand complexes by nucleic acid barcode patterns generated by DNA machineries. The DNA machineries consist of nucleic acid scaffolds that include specific recognition sites for the different genes or aptamer-ligand analytes. The binding of the analytes to the scaffolds initiate, in the presence of the nucleotide mixture, a cyclic polymerization/nicking machinery that yields displaced strands of variable lengths. The electrophoretic separation of the resulting strands provides barcode patterns for the specific detection of the different analytes. Mixtures of DNA machineries that yield, upon sensing of different genes (or aptamer ligands), one-, two-, or three-band barcode patterns are described. The combination of nucleic acid scaffolds acting, in the presence of polymerase/nicking enzyme and nucleotide mixture, as DNA machineries, that generate multiband barcode patterns provide an analytical platform for the detection of an individual gene out of many possible genes. The diversity of genes (or other analytes) that can be analyzed by the DNA machineries and the barcode patterned imaging is given by the Pascal's triangle. As a proof-of-concept, the detection of one of six genes, that is, TP53, Werner syndrome, Tay-Sachs normal gene, BRCA1, Tay-Sachs mutant gene, and cystic fibrosis disorder gene by six two-band barcode patterns is demonstrated. The advantages and limitations of the detection of analytes by polymerase/nicking DNA machineries that yield barcode patterns as imaging readout signals are discussed.

46 CFR 177.620 - Ventilation of machinery and fuel tank spaces.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Ventilation of machinery and fuel tank spaces. 177.620... fuel tank spaces. In addition to the requirements of this subpart, ventilation systems for spaces containing machinery or fuel tanks must comply with the requirements of part 182 of this chapter. ...

Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

International Nuclear Information System (INIS)

Smiley, R.M.; Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J.

1990-01-01

The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with 32 PO 4 in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the β-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M r 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the α-2 agonist clonidine. Epinephrine, a combined α and β agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the α-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes

Structural design of nuclear reactor machinery and equipment

International Nuclear Information System (INIS)

Hara, Hideki

1992-01-01

Since the machinery, equipment and piping which compose nuclear power station facilities are diverse, when those are designed, consideration is given sufficiently to the objective of use and the importance of the object machinery and equipment so that those can maintain the soundness over the design life. In this report, on the contents and the design standard in the design techniques for nuclear reactor machinery and equipment, the way of thinking is shown, taking an example of reactor pressure vessel which is stipulated as the vessel kind 1 in the 'Technical standard of structures and others regarding nuclear facilities for electric power generation', Notice No. 501 of the Ministry of International Trade and Industry. The reactor pressure vessel of 1350 MWe improved type BWR (ABWR) is used under the condition of 87.9 kg/cm 2 and 302 degC, and the inside diameter is about 7.2 m, the inside height is about 21 m, and the wall thickness is about 170 mm. The design standard for reactor pressure vessels and its way of thinking, breakdown prevention design and the design techniques for reactor pressure vessels are described. (K.I.)

Targeting Cell Polarity Machinery to Exhaust Breast Cancer Stem Cells

Science.gov (United States)

2017-10-01

AWARD NUMBER: W81XWH-15-1-0644 TITLE: Targeting Cell Polarity Machinery to Exhaust Breast Cancer Stem Cells PRINCIPAL INVESTIGATOR: Chun-Ju...Targeting Cell Polarity Machinery to Exhaust Breast Cancer Stem Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-15-1-0644 5c. PROGRAM ELEMENT...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Cancer stem cells (CSCs), a cell population with acquired perpetuating self-renewal properties which

Phosphorylation of chicken growth hormone

International Nuclear Information System (INIS)

Aramburo, C.; Montiel, J.L.; Donoghue, D.; Scanes, C.G.; Berghman, L.R.

1990-01-01

The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and γ- 32 P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of 32 P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of 32 P-phosphate labeled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer

Fault Diagnosis for Rotating Machinery: A Method based on Image Processing.

Directory of Open Access Journals (Sweden)

Chen Lu

Full Text Available Rotating machinery is one of the most typical types of mechanical equipment and plays a significant role in industrial applications. Condition monitoring and fault diagnosis of rotating machinery has gained wide attention for its significance in preventing catastrophic accident and guaranteeing sufficient maintenance. With the development of science and technology, fault diagnosis methods based on multi-disciplines are becoming the focus in the field of fault diagnosis of rotating machinery. This paper presents a multi-discipline method based on image-processing for fault diagnosis of rotating machinery. Different from traditional analysis method in one-dimensional space, this study employs computing method in the field of image processing to realize automatic feature extraction and fault diagnosis in a two-dimensional space. The proposed method mainly includes the following steps. First, the vibration signal is transformed into a bi-spectrum contour map utilizing bi-spectrum technology, which provides a basis for the following image-based feature extraction. Then, an emerging approach in the field of image processing for feature extraction, speeded-up robust features, is employed to automatically exact fault features from the transformed bi-spectrum contour map and finally form a high-dimensional feature vector. To reduce the dimensionality of the feature vector, thus highlighting main fault features and reducing subsequent computing resources, t-Distributed Stochastic Neighbor Embedding is adopt to reduce the dimensionality of the feature vector. At last, probabilistic neural network is introduced for fault identification. Two typical rotating machinery, axial piston hydraulic pump and self-priming centrifugal pumps, are selected to demonstrate the effectiveness of the proposed method. Results show that the proposed method based on image-processing achieves a high accuracy, thus providing a highly effective means to fault diagnosis for

Fault Diagnosis for Rotating Machinery: A Method based on Image Processing.

Science.gov (United States)

Lu, Chen; Wang, Yang; Ragulskis, Minvydas; Cheng, Yujie

2016-01-01

Rotating machinery is one of the most typical types of mechanical equipment and plays a significant role in industrial applications. Condition monitoring and fault diagnosis of rotating machinery has gained wide attention for its significance in preventing catastrophic accident and guaranteeing sufficient maintenance. With the development of science and technology, fault diagnosis methods based on multi-disciplines are becoming the focus in the field of fault diagnosis of rotating machinery. This paper presents a multi-discipline method based on image-processing for fault diagnosis of rotating machinery. Different from traditional analysis method in one-dimensional space, this study employs computing method in the field of image processing to realize automatic feature extraction and fault diagnosis in a two-dimensional space. The proposed method mainly includes the following steps. First, the vibration signal is transformed into a bi-spectrum contour map utilizing bi-spectrum technology, which provides a basis for the following image-based feature extraction. Then, an emerging approach in the field of image processing for feature extraction, speeded-up robust features, is employed to automatically exact fault features from the transformed bi-spectrum contour map and finally form a high-dimensional feature vector. To reduce the dimensionality of the feature vector, thus highlighting main fault features and reducing subsequent computing resources, t-Distributed Stochastic Neighbor Embedding is adopt to reduce the dimensionality of the feature vector. At last, probabilistic neural network is introduced for fault identification. Two typical rotating machinery, axial piston hydraulic pump and self-priming centrifugal pumps, are selected to demonstrate the effectiveness of the proposed method. Results show that the proposed method based on image-processing achieves a high accuracy, thus providing a highly effective means to fault diagnosis for rotating machinery.

Modern principles used in conformity assessment of machinery from forestry sector

Directory of Open Access Journals (Sweden)

Antonov Anca Elena

2017-01-01

Full Text Available The paper is aiming to implement the general principles of risk prevention at employer’s level, with respect to occupational risks evaluation, the elimination of risk and accident factors, and information of workers which are using the machinery in the forestry sector. For the use of machinery in the forestry sector in terms of economic performance and a level of maximum safety, it is necessary to ensure the user guides set by the manufacturer in terms of commissioning, use and to provide appropriate safe working operations and interventions and to guarantee the technical and environmental requirements, including appropriate measures and means of protection against accidents and occupational disease. The impact of occupational risks for machinery used in this sector can be reduced through the application of modern principles in conformity assessment and certification and, where appropriate, through technical diagnostics and inspection, taking into account the provisions of the new Machinery Directive 2006/42 / EC which is imposing the obligation of manufacturer to implement conformity assessment procedures in accordance with the methods of assessment and verification of safety at the certification bodies, notified at the European Commission. The paper aims to develop modern technical tools for conformity assessment and verification of this category of machines used in the forestry sector that would provide prerequisite for increasing competitiveness of employers in the market economy. Applying these tools of modern technology for manufacturers and users of this category of machinery provides the necessary conditions for placing on the market of safe products with a appropriate safety level, in the intended using conditions, in order to guarantee the essential requirements for safety and health, technical and environmental conditions, including measures and means of protection. The result of this research is to develop technical tools needed to

The accident analysis of mobile mine machinery in Indian opencast coal mines.

Science.gov (United States)

Kumar, R; Ghosh, A K

2014-01-01

This paper presents the analysis of large mining machinery related accidents in Indian opencast coal mines. The trends of coal production, share of mining methods in production, machinery deployment in open cast mines, size and population of machinery, accidents due to machinery, types and causes of accidents have been analysed from the year 1995 to 2008. The scrutiny of accidents during this period reveals that most of the responsible factors are machine reversal, haul road design, human fault, operator's fault, machine fault, visibility and dump design. Considering the types of machines, namely, dumpers, excavators, dozers and loaders together the maximum number of fatal accidents has been caused by operator's faults and human faults jointly during the period from 1995 to 2008. The novel finding of this analysis is that large machines with state-of-the-art safety system did not reduce the fatal accidents in Indian opencast coal mines.

Densification of chipper harvested SRC using on-farm machinery

Energy Technology Data Exchange (ETDEWEB)

Paulson, M.

2003-07-01

This report gives details of a project to density wood chips using on-farm machinery in order to avoid problems encountered in bulk handling and storage of low density short rotation cultivation (SRC) wood chips - especially as some customers can only accept baled material. Trials using different lengths of chips produced by a standard SRC harvester are described, and the failure to produce acceptable bales is reported. The potential cost of modifying equipment is deemed to make the baling of SRC chips using standard farm machinery currently not viable.

49 CFR 393.130 - What are the rules for securing heavy vehicles, equipment and machinery?

Science.gov (United States)

2010-10-01

... heavy vehicles, equipment and machinery? (a) Applicability. The rules in this section apply to the transportation of heavy vehicles, equipment and machinery which operate on wheels or tracks, such as front end... heavy vehicles, equipment or machinery with crawler tracks or wheels. (1) In addition to the...

Intelligence and the machinery of government: conceptualizing the intelligence community

OpenAIRE

Davies, PHJ

2010-01-01

This article argues that the failure to address intelligence agencies as public organizations part and parcel with the overt machinery of government constitutes a significant lacuna both in the specialist study of intelligence and the broader discipline of public administration studies. The role and status of intelligence institutions as aspects of the machinery of central government is examined, along with the prospects of certain key paradigms in the field for understanding those institutio...

Vibration Feature Extraction and Analysis for Fault Diagnosis of Rotating Machinery-A Literature Survey

OpenAIRE

Saleem Riaz; Hassan Elahi; Kashif Javaid; Tufail Shahzad

2017-01-01

Safety, reliability, efficiency and performance of rotating machinery in all industrial applications are the main concerns. Rotating machines are widely used in various industrial applications. Condition monitoring and fault diagnosis of rotating machinery faults are very important and often complex and labor-intensive. Feature extraction techniques play a vital role for a reliable, effective and efficient feature extraction for the diagnosis of rotating machinery. Therefore, deve...

Properties of phosphorylated thymidylate synthase

DEFF Research Database (Denmark)

Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr

2015-01-01

by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were...... also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent....

High voltage electrification of tractor and agricultural machinery – A review

International Nuclear Information System (INIS)

Moreda, G.P.; Muñoz-García, M.A.; Barreiro, P.

2016-01-01

Highlights: • CO_2 emission reduction calls for energy-efficient mobile work machines. • Energy recovery could be possible in some agricultural machinery operations. • Owning and operating costs of electrified tractor-implement pairs should be analyzed. • Plug-in hybrid electric tractors running along tramlines make sense. • Electrification allows for precise speed control, noise reduction and flexible design. - Abstract: Reduction of both pollutant emissions and fossil fuel dependency is an objective of energy policies worldwide. In many countries, governments promote the use of efficient vehicles like the hybrid electric vehicle. Incorporation of electric drives in tractor and agricultural machinery presents advantages in terms of increased energy efficiency and expanded functionalities. Higher efficiency means reduction in fuel consumption and subsequent decrease in CO_2 emission. New functionalities improve work quality and increase operator comfort. Tractor electrification takes advantage of decoupling loads and drives from the engine, which allows operating the latter at its highest efficiency point. Major advantages of machinery electrification are torque and speed control, noise reduction, and a more flexible design. In this paper, a review of the state-of-the-art of agricultural machinery high voltage electrification is presented.

CCPIT Machinery Exhibition Succeeded in Kuala Lumpur

Institute of Scientific and Technical Information of China (English)

无

2005-01-01

@@ From August 18 to 20, 2005, China Council for the Promotion of International Trade(CCPIT) held China Machinery and Electronics Trade Exhibition, CME 2005 in Kuala Lumpur, the capital of Malaysia on behalf of China, a good job has been done.

Structural insights into the bacterial carbon - phosphorus lyase machinery

DEFF Research Database (Denmark)

Seweryn, Paulina; Van, Lan Bich; Kjeldgaard, Morten

2015-01-01

Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon......–phosphorus (C–P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C–P lyase core complex (PhnG–PhnH–PhnI–PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero...

Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

International Nuclear Information System (INIS)

Smet-Nocca, Caroline; Launay, Hélène; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle

2013-01-01

The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer’s disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the 1 H, 15 N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice.

Science.gov (United States)

Abdallah, Cosette; Lejamtel, Charlène; Benzoubir, Nassima; Battaglia, Serena; Sidahmed-Adrar, Nazha; Desterke, Christophe; Lemasson, Matthieu; Rosenberg, Arielle R; Samuel, Didier; Bréchot, Christian; Pflieger, Delphine; Le Naour, François; Bourgeade, Marie-Françoise

2017-08-22

Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.

46 CFR 167.65-60 - Examination of boilers and machinery by engineer.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Examination of boilers and machinery by engineer. 167.65-60 Section 167.65-60 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL... machinery by engineer. It shall be the duty of an engineer when he assumes charge of the boilers and...

Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

Energy Technology Data Exchange (ETDEWEB)

Smet-Nocca, Caroline, E-mail: caroline.smet@univ-lille1.fr; Launay, Helene; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle, E-mail: isabelle.landrieu@univ-lille1.fr [Universite de Lille-Nord de France, Institut Federatif de Recherches 147, CNRS UMR 8576 (France)

2013-04-15

The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer's disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the {sup 1}H,{sup 15}N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

A comparison of pine and spruce in recovery from winter stress; changes in recovery kinetics, and the abundance and phosphorylation status of photosynthetic proteins during winter.

Science.gov (United States)

Merry, Ryan; Jerrard, Jacob; Frebault, Julia; Verhoeven, Amy

2017-09-01

During winter evergreens maintain a sustained form of thermal energy dissipation that results in reduced photochemical efficiency measured using the chlorophyll fluorescence parameter Fv/Fm. Eastern white pine (Pinus strobus L.) and white spruce [Picea glauca (Moench) Voss] have been shown to differ in their rate of recovery of Fv/Fm from winter stress. The goal of this study was to monitor changes in photosynthetic protein abundance and phosphorylation status during winter recovery that accompany these functional changes. An additional goal was to determine whether light-dependent changes in light harvesting complex II (LHCII) phosphorylation occur during winter conditions. We used a combination of field measurements and recovery experiments to monitor chlorophyll fluorescence and photosynthetic protein content and phosphorylation status. We found that pine recovered three times more slowly than spruce, and that the kinetics of recovery in spruce included a rapid and slow component, while in pine there was only a rapid component to recovery. Both species retained relatively high amounts of the light harvesting protein Lhcb5 (CP26) and the PsbS protein during winter, suggesting a role for these proteins in sustained thermal dissipation. Both species maintained high phosphorylation of LHCII and the D1 protein in darkness during winter. Pine and spruce differed in the kinetics of the dephosphorylation of LHCII and D1 upon warming, suggesting the rate of dephosphorylation of LHCII and D1 may be important in the rapid component of recovery from winter stress. Finally, we demonstrated that light-dependent changes in LHII phosphorylation do not continue to occur on subzero winter days and that needles are maintained in a phosphorylation pattern consistent with the high light conditions to which those needles are exposed. Our results suggest a role for retained phosphorylation of both LHCII and D1 in maintenance of the photosynthetic machinery in a winter conformation

CCPIT Machinery Exhibition Succeeded in Kuala Lumpur

Institute of Scientific and Technical Information of China (English)

无

2005-01-01

　　From August 18 to 20, 2005, China Council for the Promotion of International Trade(CCPIT) held China Machinery and Electronics Trade Exhibition, CME 2005 in Kuala Lumpur, the capital of Malaysia on behalf of China, a good job has been done.……

Web-based Interactive Simulator for Rotating Machinery.

Science.gov (United States)

Sirohi, Vijayalaxmi

1999-01-01

Baroma (Balance of Rotating Machinery), the Web-based educational engineering interactive software for teaching/learning combines didactical and software ergonomical approaches. The software in tutorial form simulates a problem using Visual Interactive Simulation in graphic display, and animation is brought about through graphical user interface…

Directional Wigner-Ville distribution and its application for rotating-machinery condition monitoring

International Nuclear Information System (INIS)

Kim, Dong Wan; Ha, Jae HOng; Shin, Hae Gon; Lee, Yoon Hee; Kim, Young Baik

1996-01-01

Vibration analysis is one of the most powerful tools available for the detection and isolation of incipient faults in mechanical systems. The methods of vibration analysis in use today and under continuous study are broad band vibration monitoring, time domain analysis, and frequency domain analysis. In recent years, great interest has been generated concerning the use of time-frequency representation and its application for a machinery diagnostics and condition monitoring system. The objective of the research described in this paper was to develop a new diagnostic tool for the rotating machinery. This paper introduces a new time-frequency representation, Directional Wigner-Ville Distribution, which analyses the time-frequency structure of the rotating machinery vibration

ERGONOMIC AND ECOLOGICALSAFETY - FACTORSNESSARY TO IMPROVETHE COMPETITIVENESS OF THE DOMESTIC CONSTRUCTION MACHINERY

Directory of Open Access Journals (Sweden)

Volkov Andrey Anatol’evich

2017-03-01

Full Text Available The analysis of output and domestic used construction machinery showed that the most vulnerable link remains a problem of underreporting of ergonomics and environmental safety requirements. Compliance with ergonomic requirements enhances the performance of mechanized operations, leading to a decrease in construction costs. Increased environmental requirements reduce the negative impact on air basin. These results suggest the need to improve the structures of building machines. Made recommendations for a comprehensive solution to the problem. The measures to improve environmental safety, ergonomic design requirements of construction machinery, mechanisms of the optimal development of the domestic construction machinery, through the development and implementation of appropriate measures. Keywords: ergonomics of construction equipment, environmental safety of construction equipment

46 CFR 111.103-1 - Power ventilation systems except machinery space ventilation systems.

Science.gov (United States)

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Power ventilation systems except machinery space ventilation systems. 111.103-1 Section 111.103-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY... Power ventilation systems except machinery space ventilation systems. Each power ventilation system must...

A Method of Rotating Machinery Fault Diagnosis Based on the Close Degree of Information Entropy

Institute of Scientific and Technical Information of China (English)

GENG Jun-bao; HUANG Shu-hong; JIN Jia-shan; CHEN Fei; LIU Wei

2006-01-01

This paper presents a method of rotating machinery fault diagnosis based on the close degree of information entropy. In the view of the information entropy, we introduce four information entropy features of the rotating machinery, which describe the vibration condition of the machinery. The four features are, respectively, denominated as singular spectrum entropy, power spectrum entropy, wavelet space state feature entropy and wavelet power spectrum entropy. The value scopes of the four information entropy features of the rotating machinery in some typical fault conditions are gained by experiments, which can be acted as the standard features of fault diagnosis. According to the principle of the shorter distance between the more similar models, the decision-making method based on the close degree of information entropy is put forward to deal with the recognition of fault patterns. We demonstrate the effectiveness of this approach in an instance involving the fault pattern recognition of some rotating machinery.

Glycogen phosphorylation and Lafora disease.

Science.gov (United States)

Roach, Peter J

2015-12-01

Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fault Diagnosis for Rotating Machinery Using Vibration Measurement Deep Statistical Feature Learning

Directory of Open Access Journals (Sweden)

Chuan Li

2016-06-01

Full Text Available Fault diagnosis is important for the maintenance of rotating machinery. The detection of faults and fault patterns is a challenging part of machinery fault diagnosis. To tackle this problem, a model for deep statistical feature learning from vibration measurements of rotating machinery is presented in this paper. Vibration sensor signals collected from rotating mechanical systems are represented in the time, frequency, and time-frequency domains, each of which is then used to produce a statistical feature set. For learning statistical features, real-value Gaussian-Bernoulli restricted Boltzmann machines (GRBMs are stacked to develop a Gaussian-Bernoulli deep Boltzmann machine (GDBM. The suggested approach is applied as a deep statistical feature learning tool for both gearbox and bearing systems. The fault classification performances in experiments using this approach are 95.17% for the gearbox, and 91.75% for the bearing system. The proposed approach is compared to such standard methods as a support vector machine, GRBM and a combination model. In experiments, the best fault classification rate was detected using the proposed model. The results show that deep learning with statistical feature extraction has an essential improvement potential for diagnosing rotating machinery faults.

Fault Diagnosis for Rotating Machinery Using Vibration Measurement Deep Statistical Feature Learning.

Science.gov (United States)

Li, Chuan; Sánchez, René-Vinicio; Zurita, Grover; Cerrada, Mariela; Cabrera, Diego

2016-06-17

Fault diagnosis is important for the maintenance of rotating machinery. The detection of faults and fault patterns is a challenging part of machinery fault diagnosis. To tackle this problem, a model for deep statistical feature learning from vibration measurements of rotating machinery is presented in this paper. Vibration sensor signals collected from rotating mechanical systems are represented in the time, frequency, and time-frequency domains, each of which is then used to produce a statistical feature set. For learning statistical features, real-value Gaussian-Bernoulli restricted Boltzmann machines (GRBMs) are stacked to develop a Gaussian-Bernoulli deep Boltzmann machine (GDBM). The suggested approach is applied as a deep statistical feature learning tool for both gearbox and bearing systems. The fault classification performances in experiments using this approach are 95.17% for the gearbox, and 91.75% for the bearing system. The proposed approach is compared to such standard methods as a support vector machine, GRBM and a combination model. In experiments, the best fault classification rate was detected using the proposed model. The results show that deep learning with statistical feature extraction has an essential improvement potential for diagnosing rotating machinery faults.

Fibronectin phosphorylation by ecto-protein kinase

International Nuclear Information System (INIS)

Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru

1988-01-01

The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with [γ- 32 ]ATP for 10 min at 37 degree C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with [γ- 32 P]ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation

Performance of machinery in potato production in one growing season

Directory of Open Access Journals (Sweden)

Kun Zhou

2015-12-01

Full Text Available Statistics on the machinery performance are essential for farm managers to make better decisions. In this paper, the performance of all machineries in five sequential operations, namely bed forming, stone separation, planting, spraying and harvesting in the potato production system, were investigated during one growing season. In order to analyse and decompose the recorded GPS data into various time and distance elements for estimation of the machinery performance, an automatic GPS analysis tool was developed. The field efficiency and field capacity were estimated for each operation. Specifically, the measured average field efficiency was 71.3% for bed forming, 68.5% for stone separation, 40.3% for planting, 69.7% for spraying, and 67.4% for harvesting. The measured average field capacities were 1.46 ha/h, 0.53 ha/h, 0.47 ha/h, 10.21 ha/h, 0.51 ha/h, for the bed forming, stone separation, planting, spraying, and harvesting operations, respectively. These results deviate from the corresponding estimations calculated based on norm data from the American Society of Agricultural and Biological Engineers (ASABE. The deviations indicate that norms provided by ASABE cannot be used directly for the prediction of performance of the machinery used in this work. Moreover, the measured data of bed forming and stone separation could be used as supplementary data for the ASABE which does not provide performance norms for these two operations. The gained results can help farm managers to make better management and operational decisions that result in potential improvement in productivity and profitability as well as in potential environmental benefits.

Performance of machinery in potato production in one growing season

Energy Technology Data Exchange (ETDEWEB)

Zhou, K.; Jensen, A.L.; Bochtis, D.D.; Sørensen, C.G.

2015-07-01

Statistics on the machinery performance are essential for farm managers to make better decisions. In this paper, the performance of all machineries in five sequential operations, namely bed forming, stone separation, planting, spraying and harvesting in the potato production system, were investigated during one growing season. In order to analyse and decompose the recorded GPS data into various time and distance elements for estimation of the machinery performance, an automatic GPS analysis tool was developed. The field efficiency and field capacity were estimated for each operation. Specifically, the measured average field efficiency was 71.3% for bed forming, 68.5% for stone separation, 40.3% for planting, 69.7% for spraying, and 67.4% for harvesting. The measured average field capacities were 1.46 ha/h, 0.53 ha/h, 0.47 ha/h, 10.21 ha/h, 0.51 ha/h, for the bed forming, stone separation, planting, spraying, and harvesting operations, respectively. These results deviate from the corresponding estimations calculated based on norm data from the American Society of Agricultural and Biological Engineers (ASABE). The deviations indicate that norms provided by ASABE cannot be used directly for the prediction of performance of the machinery used in this work. Moreover, the measured data of bed forming and stone separation could be used as supplementary data for the ASABE which does not provide performance norms for these two operations. The gained results can help farm managers to make better management and operational decisions that result in potential improvement in productivity and profitability as well as in potential environmental benefits. (Author)

Threonine phosphorylation of rat liver glycogen synthase

International Nuclear Information System (INIS)

Arino, J.; Arro, M.; Guinovart, J.J.

1985-01-01

32 P-labeled glycogen synthase specifically immunoprecipitated from 32 P-phosphate incubated rat hepatocytes contains, in addition to [ 32 P] phosphoserine, significant levels of [ 32 P] phosphothreonine. When the 32 P-immunoprecipitate was cleaved with CNBr, the [ 32 P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 in vitro. After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32 P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the ''in vivo'' phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase

Machinery and labour force requirements for forest chip production in Finland in 2020

Energy Technology Data Exchange (ETDEWEB)

Kaerhae, K.; Strandsroem, M. (Metsaeteho Oy, Helsinki (Finland)), Email: kalle.karha@metsateho.fi, Email: markus.strandstrom@metsateho.fi; Lahtinen, P.; Elo, J. (Poeyry Energy Oy, Espoo (Finland)), Email: perttu.lahtinen@poyry.com, Email: juha.elo@poyry.com

2009-07-01

The research carried out by Metsaeteho Oy and Poeyry Energy Oy estimated how much machinery and labour would be needed for large-scale forest chip production if the use of forest chips increases extensively in Finland during the coming decade. If the production and consumption of forest chips are 25 to 30 TWh in Finland 2020, then 1,900 to 2,200 machinery units, i.e. machines and trucks, would be needed. This would mean total investments in production machinery of 530 to 630 million euro (VAT 0 %). The labour demand would be 3,400 to 4,000 machine operators and drivers, and 4,200 to 5,100 labour years including indirect labour. Respectively, if the production and consumption of forest chips is 15 to 20 TWh in Finland in 2020, then the production machinery requirement would be 1,100 to 1,500 machines and trucks. The total machinery investment cost would be 320 to 420 million euro (VAT 0 %) and the calculated labour demand 2,000 to 2,700 machine operators and drivers (2,500 to 3,400 labour years). The results of the study indicated that forest chip production resources will be a major bottleneck in reaching the consumption target of 12 million m3, i.e. around 24 TWh of forest chips in Finland by 2020. (orig.)

Spatiotemporal Regulation of Nuclear Transport Machinery and Microtubule Organization

Science.gov (United States)

Okada, Naoyuki; Sato, Masamitsu

2015-01-01

Spindle microtubules capture and segregate chromosomes and, therefore, their assembly is an essential event in mitosis. To carry out their mission, many key players for microtubule formation need to be strictly orchestrated. Particularly, proteins that assemble the spindle need to be translocated at appropriate sites during mitosis. A small GTPase (hydrolase enzyme of guanosine triphosphate), Ran, controls this translocation. Ran plays many roles in many cellular events: nucleocytoplasmic shuttling through the nuclear envelope, assembly of the mitotic spindle, and reorganization of the nuclear envelope at the mitotic exit. Although these events are seemingly distinct, recent studies demonstrate that the mechanisms underlying these phenomena are substantially the same as explained by molecular interplay of the master regulator Ran, the transport factor importin, and its cargo proteins. Our review focuses on how the transport machinery regulates mitotic progression of cells. We summarize translocation mechanisms governed by Ran and its regulatory proteins, and particularly focus on Ran-GTP targets in fission yeast that promote spindle formation. We also discuss the coordination of the spatial and temporal regulation of proteins from the viewpoint of transport machinery. We propose that the transport machinery is an essential key that couples the spatial and temporal events in cells. PMID:26308057

Research on bearing fault diagnosis of large machinery based on mathematical morphology

Science.gov (United States)

Wang, Yu

2018-04-01

To study the automatic diagnosis of large machinery fault based on support vector machine, combining the four common faults of the large machinery, the support vector machine is used to classify and identify the fault. The extracted feature vectors are entered. The feature vector is trained and identified by multi - classification method. The optimal parameters of the support vector machine are searched by trial and error method and cross validation method. Then, the support vector machine is compared with BP neural network. The results show that the support vector machines are short in time and high in classification accuracy. It is more suitable for the research of fault diagnosis in large machinery. Therefore, it can be concluded that the training speed of support vector machines (SVM) is fast and the performance is good.

Phosphorylation of eukaryotic aminoacyl-tRNA synthetases

International Nuclear Information System (INIS)

Pendergast, A.M.

1986-01-01

The phosphorylation of the highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes was examined. The synthetase complex contained, in addition to eight aminoacyl-tRNA synthetases, three unidentified proteins and was free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP resulted in the phosphorylation of four synthetases, the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I altered binding to tRNA-Sepharose such that the phosphorylated complex eluted at 190 mM NaCl instead of the 275 mM salt observed for the nonphosphorylated form. Phosphorylation by casein kinase I resulted in a significant inhibition of aminoacylation with the four synthetases; the activities of the nonphosphorylated synthetases were unchanged. One of the unidentified proteins in the complex (M/sub r/ 37,000) was also an excellent substrate for casein kinase I. A comparison of the properties and two-dimensional phosphopeptide pattern of this protein with that of casein kinase I suggest that the 37,000 dalton protein in the synthetase complex is an inactive form of casein kinase I. Two other protein kinases were shown to phosphorylate aminoacyl-tRNA synthetases in the complex. The phosphorylation of threonyl-tRNA synthetase was also investigated. Five aminoacyl-tRNA synthetases in the high molecular weight complex were shown to be phosphorylated in rabbit reticulocytes following labeling with ( 32 P)orthophosphate

Application of Choi—Williams Reduced Interference Time Frequency Distribution to Machinery Diagnostics

Directory of Open Access Journals (Sweden)

Howard A. Gaberson

1995-01-01

Full Text Available This article discusses time frequency analysis of machinery diagnostic vibration signals. The short time Fourier transform, the Wigner, and the Choi–Williams distributions are explained and illustrated with test cases. Examples of Choi—Williams analyses of machinery vibration signals are presented. The analyses detect discontinuities in the signals and their timing, amplitude and frequency modulation, and the presence of different components in a vibration signal.

An Association of Unique microRNA Turnover Machinery with Prostate Cancer Progression

Science.gov (United States)

2017-10-01

targeting of critical androgen receptor -604 coregulator interactions in prostate cancer . Nature communications 4, 1923, 605 doi:10.1038/ncomms2912 (2013...AWARD NUMBER: W81XWH-16-1-0474 TITLE: An Association of Unique microRNA Turnover Machinery with Prostate Cancer Progression PRINCIPAL INVESTIGATOR...14 Sep 2017 4. Title An Association of Unique microRNA Turnover Machinery with Prostate Cancer Progression 5a. CONTRACT NUMBER 5b. GRANT NUMBER

Phosphorylation of human skeletal muscle myosin

International Nuclear Information System (INIS)

Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

1986-01-01

Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30 0 C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with ( 30 P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ

Molecular building blocks and their architecture in biologically/environmentally compatible soft matter chemical machinery.

Science.gov (United States)

Toyota, Taro; Banno, Taisuke; Nitta, Sachiko; Takinoue, Masahiro; Nomoto, Tomonori; Natsume, Yuno; Matsumura, Shuichi; Fujinami, Masanori

2014-01-01

This review briefly summarizes recent developments in the construction of biologically/environmentally compatible chemical machinery composed of soft matter. Since environmental and living systems are open systems, chemical machinery must continuously fulfill its functions not only through the influx and generation of molecules but also via the degradation and dissipation of molecules. If the degradation or dissipation of soft matter molecular building blocks and biomaterial molecules/polymers can be achieved, soft matter particles composed of them can be used to realize chemical machinery such as selfpropelled droplets, drug delivery carriers, tissue regeneration scaffolds, protocell models, cell-/tissuemarkers, and molecular computing systems.

The RNAi Inheritance Machinery of Caenorhabditis elegans.

Science.gov (United States)

Spracklin, George; Fields, Brandon; Wan, Gang; Becker, Diveena; Wallig, Ashley; Shukla, Aditi; Kennedy, Scott

2017-07-01

Gene silencing mediated by dsRNA (RNAi) can persist for multiple generations in Caenorhabditis elegans (termed RNAi inheritance). Here we describe the results of a forward genetic screen in C. elegans that has identified six factors required for RNAi inheritance: GLH-1/VASA, PUP-1/CDE-1, MORC-1, SET-32, and two novel nematode-specific factors that we term here (heritable RNAi defective) HRDE-2 and HRDE-4 The new RNAi inheritance factors exhibit mortal germline (Mrt) phenotypes, which we show is likely caused by epigenetic deregulation in germ cells. We also show that HRDE-2 contributes to RNAi inheritance by facilitating the binding of small RNAs to the inheritance Argonaute (Ago) HRDE-1 Together, our results identify additional components of the RNAi inheritance machinery whose conservation provides insights into the molecular mechanism of RNAi inheritance, further our understanding of how the RNAi inheritance machinery promotes germline immortality, and show that HRDE-2 couples the inheritance Ago HRDE-1 with the small RNAs it needs to direct RNAi inheritance and germline immortality. Copyright © 2017 by the Genetics Society of America.

beta2-adaptin is constitutively de-phosphorylated by serine/threonine protein phosphatase PP2A and phosphorylated by a staurosporine-sensitive kinase

DEFF Research Database (Denmark)

Lauritsen, Jens Peter Holst; Menné, C; Kastrup, J

2000-01-01

Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2-ada...... the hypothesis that phosphorylation/de-phosphorylation of coat proteins plays a regulatory role in the assembly/disassembly cycle of clathrin-coated vesicles.......Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2......-adaptin undergoes cycles of phosphorylation/de-phosphorylation in intact cells. Thus, beta2-adaptin was constitutively de-phosphorylated by serine/threonine protein phosphatase 2A and phosphorylated by a staurosporine-sensitive kinase in vivo. Confocal laser scanning microscopy demonstrated...

SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

Energy Technology Data Exchange (ETDEWEB)

JOHN C WALKER

2011-11-01

Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

Machinery health prognostics: A systematic review from data acquisition to RUL prediction

Science.gov (United States)

Lei, Yaguo; Li, Naipeng; Guo, Liang; Li, Ningbo; Yan, Tao; Lin, Jing

2018-05-01

Machinery prognostics is one of the major tasks in condition based maintenance (CBM), which aims to predict the remaining useful life (RUL) of machinery based on condition information. A machinery prognostic program generally consists of four technical processes, i.e., data acquisition, health indicator (HI) construction, health stage (HS) division, and RUL prediction. Over recent years, a significant amount of research work has been undertaken in each of the four processes. And much literature has made an excellent overview on the last process, i.e., RUL prediction. However, there has not been a systematic review that covers the four technical processes comprehensively. To fill this gap, this paper provides a review on machinery prognostics following its whole program, i.e., from data acquisition to RUL prediction. First, in data acquisition, several prognostic datasets widely used in academic literature are introduced systematically. Then, commonly used HI construction approaches and metrics are discussed. After that, the HS division process is summarized by introducing its major tasks and existing approaches. Afterwards, the advancements of RUL prediction are reviewed including the popular approaches and metrics. Finally, the paper provides discussions on current situation, upcoming challenges as well as possible future trends for researchers in this field.

Availability analysis of selected mining machinery

Directory of Open Access Journals (Sweden)

Brodny Jarosław

2017-06-01

Full Text Available Underground extraction of coal is characterized by high variability of mining and geological conditions in which it is conducted. Despite ever more effective methods and tools, used to identify the factors influencing this process, mining machinery, used in mining underground, work in difficult and not always foreseeable conditions, which means that these machines should be very universal and reliable. Additionally, a big competition, occurring on the coal market, causes that it is necessary to take action in order to reduce the cost of its production, e.g. by increasing the efficiency of utilization machines. To meet this objective it should be pro-ceed with analysis presented in this paper. The analysis concerns to availability of utilization selected mining machinery, conducted using the model of OEE, which is a tool for quantitative estimate strategy TPM. In this article we considered the machines being part of the mechanized longwall complex and the basis of analysis was the data recording by the industrial automation system. Using this data set we evaluated the availability of studied machines and the structure of registered breaks in their work. The results should be an important source of information for maintenance staff and management of mining plants, needed to improve the economic efficiency of underground mining.

Comparison of Management-Operational Efficiency of Agricultural Machinery Operating Systems (Case Study Alborz Province

Directory of Open Access Journals (Sweden)

A Omidi

2017-10-01

Full Text Available Introduction Measuring the efficiency of operating systems in comparison with the methods of comparing the performance of systems explains the various dimensions of issues such as, the lack of full use of agricultural machinery capacity, improper selection of machine, incorrect use of machinery, ownership, etc.. Any improvement in operating system conditions reduces costs,, consumption of inputs, increases the efficiency of production factors and consequently reduces the price and increases agricultural profitability. The main objective of this research is to compare the operational-management efficiency of operating systems in Alborz province and comparison of managerial and operational efficiency of agricultural machinery farming systems by calculating the efficiency of its major components in agricultural machinery farming systems including efficiency, social, economic, technical-operational and managerial and ranking them in order to understand the optimal model of agricultural machinery systems. Materials and Methods This research is a survey study.The study population was beneficiaries of agricultural machinery in the Alborz province which in the multi-stage random sample was determined. Alborz province has 31,438 agricultural operations, of which 543 are exploited agricultural machinery. Cochran formula was used to determine sample size. Since, Cronbach's alpha coefficient greater than 0.7 was obtained by questionnaire, the reliability of the questionnaires was assessed as desirable. To calculate the efficiency the component data were extracted from 4 specialized questionnaires after the initial examination and encoding, then they were analyzed using the software SPSS, MCDM Engine. TOPSIS techniques were used for ranking managerial performance operating system for operating agricultural machinery Alborz province. Results and Discussion The results showed that social efficiency of dedicated-professional operation with an average of 6.6 had

Mapping of p140Cap phosphorylation sites

DEFF Research Database (Denmark)

Repetto, Daniele; Aramu, Simona; Boeri Erba, Elisabetta

2013-01-01

phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine...... residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant...

In the Name of the Father and of the Revolution: The Sacred and the Effects of the Master Signifier on Subjectivity in War Contexts

Directory of Open Access Journals (Sweden)

Cristian Jesús Palma Florián

2018-01-01

Full Text Available As a symbolic function, the Ideal of the Revolution becomes the central signifier in the subjectivity of its militants, and creates its own Names of the Father, its own master signifiers that alienate the combatants and generate effects at the level of "the sacred" in the symbolic, imaginary, and real registers, in both the public setting of the collective and the private one of the subject. The objective of the article is to understand the deployment of the ideal that drives the revolutionary cause, the ways it operates in the collective and singular setting, and the effects it causes on subjectivity. Finally, it examines the process of downfall of the ideal and some of its dilemmas as a preamble of the subject's exit from war.

Protein-Tyrosine Phosphorylation in Bacillus subtilis

DEFF Research Database (Denmark)

Mijakovic, Ivan; Petranovic, Dina; Bottini, N.

2005-01-01

phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge...... on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network...

[Effects of heavy machinery operation on the structural characters of cultivated soils in black soil region of Northeast China].

Science.gov (United States)

Wang, En-Heng; Chai, Ya-Fan; Chen, Xiang-Wei

2008-02-01

With the cultivated soils in black soil region of Northeast China as test objects, this paper measured their structural characters such as soil strength, bulk density, and non-capillary porosity/capillary porosity (NCP/CP) ratio before and after heavy and medium-sized machinery operation, aimed to study the effects of machinery operation on the physical properties of test soils. The results showed that after machinery operation, there existed three distinct layers from top to bottom in the soil profiles, i.e., plowed layer, cumulative compacted layer, and non-affected layer, according to the changes of soil strength. Under medium-sized machinery operation, these three layers were shallower, and there was a new plow pan at the depth between 17.5 and 30 cm. Heavy machinery operation had significant positive effects on the improvement of topsoil structure (P heavy machinery, the bulk density of topsoil decreased by 7.2% and 3.5%, respectively, and NCP/CP increased by 556.6% after subsoiling, which would benefit water infiltration, reinforce water storage, and weaken the threat of soil erosion. The main action of heavy machinery operation was soil loosening, while that of medium-sized machinery operation was soil compacting.

Deciphering the role of the signal- and Sty1 kinase-dependent phosphorylation of the stress-responsive transcription factor Atf1 on gene activation.

Science.gov (United States)

Salat-Canela, Clàudia; Paulo, Esther; Sánchez-Mir, Laura; Carmona, Mercè; Ayté, José; Oliva, Baldo; Hidalgo, Elena

2017-08-18

Adaptation to stress triggers the most dramatic shift in gene expression in fission yeast ( Schizosaccharomyces pombe ), and this response is driven by signaling via the MAPK Sty1. Upon activation, Sty1 accumulates in the nucleus and stimulates expression of hundreds of genes via the nuclear transcription factor Atf1, including expression of atf1 itself. However, the role of stress-induced, Sty1-mediated Atf1 phosphorylation in transcriptional activation is unclear. To this end, we expressed Atf1 phosphorylation mutants from a constitutive promoter to uncouple Atf1 activity from endogenous, stress-activated Atf1 expression. We found that cells expressing a nonphosphorylatable Atf1 variant are sensitive to oxidative stress because of impaired transcription of a subset of stress genes whose expression is also controlled by another transcription factor, Pap1. Furthermore, cells expressing a phospho-mimicking Atf1 mutant display enhanced stress resistance, and although expression of the Pap1-dependent genes still relied on stress induction, another subset of stress-responsive genes was constitutively expressed in these cells. We also observed that, in cells expressing the phospho-mimicking Atf1 mutant, the presence of Sty1 was completely dispensable, with all stress defects of Sty1-deficient cells being suppressed by expression of the Atf1 mutant. We further demonstrated that Sty1-mediated Atf1 phosphorylation does not stimulate binding of Atf1 to DNA but, rather, establishes a platform of interactions with the basal transcriptional machinery to facilitate transcription initiation. In summary, our results provide evidence that Atf1 phosphorylation by the MAPK Sty1 is required for oxidative stress responses in fission yeast cells by promoting transcription initiation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

On the reversed Brayton cycle with high speed machinery

Energy Technology Data Exchange (ETDEWEB)

Backman, J.

1996-12-31

This work was carried out in the laboratory of Fluid Dynamics, at Lappeenranta University of Technology during the years 1991-1996. The research was a part of larger high speed technology development research. First, there was the idea of making high speed machinery applications with the Brayton cycle. There was a clear need to deepen the knowledge of the cycle itself and to make a new approach in the field of the research. Also, the removal of water from the humid air seemed very interesting. The goal of this work was to study methods of designing high speed machinery for the reversed Brayton cycle, from theoretical principles to practical applications. The reversed Brayton cycle can be employed as an air dryer, a heat pump or a refrigerating machine. In this research the use of humid air as a working fluid has an environmental advantage, as well. A new calculation method for the Brayton cycle is developed. In this method especially the expansion process in the turbine is important because of the condensation of the water vapour in the humid air. This physical phenomena can have significant effects on the level of performance of the application. Also, the influence of calculating the process with actual, achievable process equipment efficiencies is essential for the development of future machinery. The above theoretical calculations are confirmed with two different laboratory prototypes. (53 refs.)

On the reversed Brayton cycle with high speed machinery

Energy Technology Data Exchange (ETDEWEB)

Backman, J

1997-12-31

This work was carried out in the laboratory of Fluid Dynamics, at Lappeenranta University of Technology during the years 1991-1996. The research was a part of larger high speed technology development research. First, there was the idea of making high speed machinery applications with the Brayton cycle. There was a clear need to deepen the knowledge of the cycle itself and to make a new approach in the field of the research. Also, the removal of water from the humid air seemed very interesting. The goal of this work was to study methods of designing high speed machinery for the reversed Brayton cycle, from theoretical principles to practical applications. The reversed Brayton cycle can be employed as an air dryer, a heat pump or a refrigerating machine. In this research the use of humid air as a working fluid has an environmental advantage, as well. A new calculation method for the Brayton cycle is developed. In this method especially the expansion process in the turbine is important because of the condensation of the water vapour in the humid air. This physical phenomena can have significant effects on the level of performance of the application. Also, the influence of calculating the process with actual, achievable process equipment efficiencies is essential for the development of future machinery. The above theoretical calculations are confirmed with two different laboratory prototypes. (53 refs.)

Altered phosphorylation of rhodopsin in retinal dystrophic Irish Setters

International Nuclear Information System (INIS)

Cunnick, J.; Takemoto, D.J.; Takemoto, L.J.

1986-01-01

The carboxyl-terminus of rhodopsin in retinal dystrophic (rd) Irish Setters is altered near a possible phosphorylation site. To determine if this alteration affects ATP-mediated phosphorylation they compared the phosphorylation of rhodopsin from rd affected Irish Setters and normal unaffected dogs. Retinas from 8-week-old Irish Setters were phosphorylated with γ- 32 P-ATP and separated on SDS-PAGE. Compared to unaffected normal retinas, equalized for rhodopsin content, phosphorylation of rd rhodopsin was drastically reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Inhibition also occurred when bovine retinas were mixed with rd retinas. The rd-mediated inhibition of phosphorylation was prevented by including 1mM NaF in the reaction mixture. Likewise, 1mM NaF restored phosphorylation of rd rhodopsin to normal levels. Phosphopeptide maps of rd and normal rhodopsin were identical and indicated 5 phosphopeptides present in each. Results suggest that one cause of the depressed rd rhodopsin phosphorylation is an increased phosphatase activity

Tyrosine phosphorylation of Grb14 by Tie2

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Dumont Daniel J

2010-10-01

Full Text Available Abstract Background Growth factor receptor bound (Grb proteins 7, 10 and 14 are a family of structurally related multi-domain adaptor proteins involved in a variety of biological processes. Grb7, 10 and 14 are known to become serine and/or threonine phosphorylated in response to growth factor (GF stimulation. Grb7 and 10 have also been shown to become tyrosine phosphorylated under certain conditions. Under experimental conditions Grb7 is tyrosine phosphorylated by the Tie2/Tie-2/Tek angiogenic receptor tyrosine kinase (RTK. Furthermore, Grb14 has also been shown to interact with Tie2, however tyrosine phosphorylation of this Grb family member has yet to be reported. Results Here we report for the first time tyrosine phosphorylation of Grb14. This phosphorylation requires a kinase competent Tie2 as well as intact tyrosines 1100 and 1106 (Y1100 and Y1106 on the receptor. Furthermore, a complete SH2 domain on Grb14 is required for Grb14 tyrosine phosphorylation by Tie2. Grb14 was also able to become tyrosine phosphorylated in primary endothelial cells when treated with a soluble and potent variant of the Tie2 ligand, cartilage oligomeric matrix protein (COMP Ang1. Conclusion Our results show that Grb14, like its family members Grb7 and Grb10, is able to be tyrosine phosphorylated. Furthermore, our data indicate a role for Grb14 in endothelial signaling downstream of the Tie2 receptor.

Phosphorylation prevents C/EBPβ from the calpain-dependent degradation

International Nuclear Information System (INIS)

Zhang, Yuan-yuan; Li, Shu-fen; Qian, Shu-wen; Zhang, You-you; Liu, Yuan; Tang, Qi-Qun; Li, Xi

2012-01-01

Highlights: ► Phosphorylation protected C/EBPβ from μ-calpain-mediated proteolysis in vitro. ► Phosphorylation mimic C/EBPβ was insensitive to calpain accelerator and inhibitor. ► Phosphorylation on Thr 188 contributed more to the stabilization of C/EBPβ. -- Abstract: CCAAT/enhancer-binding protein (C/EBP) β plays an important role in proliferation and differentiation of 3T3-L1 preadipocytes. C/EBPβ is sequentially phosphorylated during the 3T3-L1 adipocyte differentiation program, first by MAPK/Cyclin A/cdk2 on Thr 188 and subsequently by GSK3β on Ser 184 or Thr 179 . Dual phosphorylation is critical for the gain of DNA binding activity of C/EBPβ. In this manuscript, we found that phosphorylation also contributed to the stability of C/EBPβ. Both ex vivo and in vitro experiments showed that phosphorylation by MAPK/Cyclin A/cdk2 and GSK3β protected C/EBPβ from μ-calpain-mediated proteolysis, while phosphorylation on Thr 188 by MAPK/Cyclin A/cdk2 contributed more to the stabilization of C/EBPβ, Further studies indicated that phosphorylation mimic C/EBPβ was insensitive to both calpain accelerator and calpain inhibitor. Thus, phosphorylation might contribute to the stability as well as the gain of DNA binding activity of C/EBPβ.

PTP1B targets the endosomal sorting machinery

DEFF Research Database (Denmark)

Stuible, Matthew; Abella, Jasmine V; Feldhammer, Matthew

2010-01-01

Dephosphorylation and endocytic down-regulation are distinct processes that together control the signaling output of a variety of receptor tyrosine kinases (RTKs). PTP1B can directly dephosphorylate several RTKs, but it can also promote activation of downstream pathways through largely unknown...... mechanisms. These positive signaling functions likely contribute to the tumor-promoting effect of PTP1B in mouse cancer models. Here, we have identified STAM2, an endosomal protein involved in sorting activated RTKs for lysosomal degradation, as a substrate of PTP1B. PTP1B interacts with STAM2 at defined...... phosphotyrosine sites, and knockdown of PTP1B expression augments STAM2 phosphorylation. Intriguingly, manipulating the expression and phosphorylation state of STAM2 did not have a general effect on epidermal growth factor (EGF)-induced EGF receptor trafficking, degradation, or signaling. Instead, phosphorylated...

Machinery fault diagnosis using joint global and local/nonlocal discriminant analysis with selective ensemble learning

Science.gov (United States)

Yu, Jianbo

2016-11-01

The vibration signals of faulty machine are generally non-stationary and nonlinear under those complicated working conditions. Thus, it is a big challenge to extract and select the effective features from vibration signals for machinery fault diagnosis. This paper proposes a new manifold learning algorithm, joint global and local/nonlocal discriminant analysis (GLNDA), which aims to extract effective intrinsic geometrical information from the given vibration data. Comparisons with other regular methods, principal component analysis (PCA), local preserving projection (LPP), linear discriminant analysis (LDA) and local LDA (LLDA), illustrate the superiority of GLNDA in machinery fault diagnosis. Based on the extracted information by GLNDA, a GLNDA-based Fisher discriminant rule (FDR) is put forward and applied to machinery fault diagnosis without additional recognizer construction procedure. By importing Bagging into GLNDA score-based feature selection and FDR, a novel manifold ensemble method (selective GLNDA ensemble, SE-GLNDA) is investigated for machinery fault diagnosis. The motivation for developing ensemble of manifold learning components is that it can achieve higher accuracy and applicability than single component in machinery fault diagnosis. The effectiveness of the SE-GLNDA-based fault diagnosis method has been verified by experimental results from bearing full life testers.

The phototransduction machinery in the rod outer segment has a strong efficacy gradient

KAUST Repository

Mazzolini, Monica

2015-05-04

Rod photoreceptors consist of an outer segment (OS) and an inner segment. Inside the OS a biochemical machinery transforms the rhodopsin photoisomerization into electrical signal. This machinery has been treated as and is thought to be homogenous with marginal inhomogeneities. To verify this assumption, we developed a methodology based on special tapered optical fibers (TOFs) to deliver highly localized light stimulations. By using these TOFs, specific regions of the rod OS could be stimulated with spots of light highly confined in space. As the TOF is moved from the OS base toward its tip, the amplitude of saturating and single photon responses decreases, demonstrating that the efficacy of the transduction machinery is not uniform and is 5-10 times higher at the base than at the tip. This gradient of efficacy of the transduction machinery is attributed to a progressive depletion of the phosphodiesterase along the rod OS. Moreover we demonstrate that, using restricted spots of light, the duration of the photoresponse along the OS does not increase linearly with the light intensity as with diffuse light. © 2015, National Academy of Sciences. All rights reserved.

Non-machinery dialysis that achieves blood purification therapy without using full-scale dialysis machines.

Science.gov (United States)

Abe, Takaya; Onoda, Mistutaka; Matsuura, Tomohiko; Sugimura, Jun; Obara, Wataru; Sato, Toshiya; Takahashi, Mihoko; Chiba, Kenta; Abe, Tomiya

2017-09-01

An electrical or water supply and a blood purification machine are required for renal replacement therapy. There is a possibility that acute kidney injury can occur in large numbers and on a wide scale in the case of a massive earthquake, and there is the potential risk that the current supply will be unable to cope with acute kidney injury cases. However, non-machinery dialysis requires exclusive circuits and has the characteristic of not requiring the full-scale dialysis machines. We performed perfusion experiments that used non-machinery dialysis and recent blood purification machines in 30-min intervals, and the effectiveness of non-machinery dialysis was evaluated by the assessing the removal efficiency of potassium, which causes lethal arrhythmia during acute kidney injury. The non-machinery dialysis potassium removal rate was at the same level as continuous blood purification machines with a dialysate flow rate of 5 L/h after 15 min and continuous blood purification machines with a dialysate flow rate of 3 L/h after 30 min. Non-machinery dialysis required an exclusive dialysate circuit, the frequent need to replace bags, and new dialysate exchanged once every 30 min. However, it can be seen as an effective renal replacement therapy for crush-related acute kidney injury patients, even in locations or facilities not having the full-scale dialysis machines.

Acoustic Camera as a Fool for Identifying Machinery and Equipment Failures

Directory of Open Access Journals (Sweden)

Ľudmila Pavlikova

2018-03-01

Full Text Available Sound and noise are as old as humanity itself. They have accompanied civilization, evolution, and development for centuries. Music and speech represent not only the key elements of human life but also unpleasant feelings of noise that have always been an integral part of human existence. As industrial development has required more energy, powerful machinery, and equipment, there have been still noisier machines. Traffic has grown quickly due to the number and speed of vehicles. For that reason, an acoustic camera is used for the dynamic visualization of machinery and equipment noise as it analyses the sources of noise in details. Subsequently, qualified measures are introduced based on the results of the analysis. The paper considers launching another application. According to the proposed methodology, its use in identifying machinery and equipment failures and their maintenance is proved. The experiment was performed on a four-wheel lawn mower. The primary focus was on the identification of failures using an acoustic camera. The previous method allowed to quickly, precisely and efficiently identifying the failures in two out of five tested machines.

Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

Science.gov (United States)

Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan

2016-02-15

Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated

Propofol directly increases tau phosphorylation.

Directory of Open Access Journals (Sweden)

Robert A Whittington

2011-01-01

Full Text Available In Alzheimer's disease (AD and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of

PhosphoBase: a database of phosphorylation sites

DEFF Research Database (Denmark)

Blom, Nikolaj; Kreegipuu, Andres; Brunak, Søren

1998-01-01

PhosphoBase is a database of experimentally verified phosphorylation sites. Version 1.0 contains 156 entries and 398 experimentally determined phosphorylation sites. Entries are compiled and revised from the literature and from major protein sequence databases such as SwissProt and PIR. The entries...... provide information about the phosphoprotein and the exact position of its phosphorylation sites. Furthermore, part of the entries contain information about kinetic data obtained from enzyme assays on specific peptides. To illustrate the use of data extracted from PhosphoBase we present a sequence logo...... displaying the overall conservation of positions around serines phosphorylated by protein kinase A (PKA). PhosphoBase is available on the WWW at http://www.cbs.dtu.dk/databases/PhosphoBase/....

Fluorescence excitation-emission matrix spectroscopy for degradation monitoring of machinery lubricants

Science.gov (United States)

Sosnovski, Oleg; Suresh, Pooja; Dudelzak, Alexander E.; Green, Benjamin

2018-02-01

Lubrication oil is a vital component of heavy rotating machinery defining the machine's health, operational safety and effectiveness. Recently, the focus has been on developing sensors that provide real-time/online monitoring of oil condition/lubricity. Industrial practices and standards for assessing oil condition involve various analytical methods. Most these techniques are unsuitable for online applications. The paper presents the results of studying degradation of antioxidant additives in machinery lubricants using Fluorescence Excitation-Emission Matrix (EEM) Spectroscopy and Machine Learning techniques. EEM Spectroscopy is capable of rapid and even standoff sensing; it is potentially applicable to real-time online monitoring.

Safeness of radiological machinery

International Nuclear Information System (INIS)

Yokoyama, Shun

1979-01-01

The human factors affecting the safeness of radiological machinery, which are often very big and complicated machines, are described from the stand point of handling. 20 to 50% of the troubles on equipments seem to be caused by men. This percentage will become even higher in highly developed equipments. Human factors have a great influence on the safeness of radiological equipments. As the human factors, there are sensory factors and knowledge factors as well as psychological factors, and the combination of these factors causes mishandling and danger. Medical services at present are divided in various areas, and consist of the teamwork of the people in various professions. Good human relationship, education and control are highly required to secure the safeness. (Kobatake, H.)

Welding technologies for nuclear machinery and equipment

International Nuclear Information System (INIS)

Kobayashi, Masahiro; Yokono, Tomomi.

1991-01-01

The main welding methods applied to nuclear machinery and equipment are shielded metal arc welding, submerged arc welding, MAG welding and TIG welding. But in the last 10 years, in order to improve the reliability required for the welding of nuclear machinery and equipment, the welding technologies aiming at the reduction of heat input, the decrease of the number of welding pass and the automatic control of welding factors have been applied for the main purpose of bettering the quality and excluding human errors. The merits and the technology of narrow gap, pulsed MAG welding and melt-through welding are explained. As the automation of TIG welding, image processing type narrow gap, hot wire TIG welding and remote control type automatic TIG welding are described. For the longitudinal welding of active metal sheet products, plasma key-hole welding is applied. Since the concentration of its arc is good, high speed welding with low heat input can be done. For the stainless steel cladding by welding, electroslag welding has become to be employed in place of conventional submerged arc welding. Arc is not generated in the electroslag welding, and the penetration into base metal is small. (K.I.)

Rotating Machinery Predictive Maintenance Through Expert System

Directory of Open Access Journals (Sweden)

M. Sarath Kumar

2000-01-01

Full Text Available Modern rotating machines such as turbomachines, either produce or absorb huge amount of power. Some of the common applications are: steam turbine-generator and gas turbine-compressor-generator trains produce power and machines, such as pumps, centrifugal compressors, motors, generators, machine tool spindles, etc., are being used in industrial applications. Condition-based maintenance of rotating machinery is a common practice where the machine's condition is monitored constantly, so that timely maintenance can be done. Since modern machines are complex and the amount of data to be interpreted is huge, we need precise and fast methods in order to arrive at the best recommendations to prevent catastrophic failure and to prolong the life of the equipment. In the present work using vibration characteristics of a rotor-bearing system, the condition of a rotating machinery (electrical rotor is predicted using an off-line expert system. The analysis of the problem is carried out in an Object Oriented Programming (OOP framework using the finite element method. The expert system which is also developed in an OOP paradigm gives the type of the malfunctions, suggestions and recommendations. The system is implemented in C++.

SH3 domain tyrosine phosphorylation--sites, role and evolution.

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Zuzana Tatárová

Full Text Available BACKGROUND: SH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains. RESULTS: To explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c-Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F. This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains. CONCLUSIONS: While tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.

46 CFR 35.25-1 - Examination of boilers and machinery by engineer-T/ALL.

Science.gov (United States)

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Examination of boilers and machinery by engineer-T/ALL... Engine Department § 35.25-1 Examination of boilers and machinery by engineer—T/ALL. It shall be the duty of an engineer when assuming charge of the boilers to examine the same forthwith and thoroughly. If...

Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins

DEFF Research Database (Denmark)

Rao, R Shyama Prasad; Møller, Ian Max

2012-01-01

in proteins is currently lacking. We have therefore analyzed the occurrence and occupancy of phosphorylated sites (~ 100,281) in a large set of eukaryotic proteins (~ 22,995). Phosphorylation probability was found to be much higher in both the  termini of protein sequences and this is much pronounced...... maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins.......Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites...

Machinery prognostics and prognosis oriented maintenance management

CERN Document Server

Yan, Jihong

2014-01-01

This book gives a complete presentatin of the basic essentials of machinery prognostics and prognosis oriented maintenance management, and takes a look at the cutting-edge discipline of intelligent failure prognosis technologies for condition-based maintenance.  Latest research results and application methods are introduced for signal processing, reliability moelling, deterioration evaluation, residual life prediction and maintenance-optimization as well as applications of these methods.

An island in a small black doll. The path of the signifier Gorogó in Ana María Matute’s Primera memoria

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Emilio Ceruti

2018-05-01

Full Text Available When we project an emotional charge onto an object, this turns into a thing that refers to ourselves, and allows the subject to know himself/herself better. In this study, the relationship between a black doll called Gorogó and the characters of Ana María Matute’s Primera Memoria will be analyzed in order to glimpse the signified that the signifier Gorogó acquires through its displacements. Following Lacan’s postulates, it is possible to determine three positions of the doll. When it remains hidden, it confers a certain protection to the protagonist. When it is revealed and enters in a relationship with the other characters, it causes an odd sensation (unheimlich, for being a thing that had to be kept unseen and that has been revealed. At the end, Gorogó stands out for his absence and reveals the temporal dimension of things, namely, the protagonist’s growth and the internal conflict that this entails.

Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

Science.gov (United States)

Ohta, Shinya; Kimura, Michiko; Takagi, Shunsuke; Toramoto, Iyo; Ishihama, Yasushi

2016-09-02

During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

Optimization Model for Machinery Selection of Multi-Crop Farms in Elsuki Agricultural Scheme

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Mysara Ahmed Mohamed

2017-07-01

Full Text Available The optimization machinery model was developed to aid decision-makers and farm machinery managers in determining the optimal number of tractors, scheduling the agricultural operation and minimizing machinery total costs. For purpose of model verification, validation and application input data was collected from primary & secondary sources from Elsuki agricultural scheme for two seasons namely 2011-2012 and 2013-2014. Model verification was made by comparing the numbers of tractors of Elsuki agricultural scheme for season 2011-2012 with those estimated by the model. The model succeeded in reducing the number of tractors and operation total cost by 23%. The effect of optimization model on elements of direct cost saving indicated that the highest cost saving is reached with depreciation, repair and maintenance (23% and the minimum cost saving is attained with fuel cost (22%. Sensitivity analysis in terms of change in model input for each of cultivated area and total costs of operations showing that: Increasing the operation total cost by 10% decreased the total number of tractors after optimization by 23% and total cost of operations was also decreased by 23%. Increasing the cultivated area by 10%, decreased the total number of tractors after optimization by(12% and total cost of operations was also decreased by 12% (16669206 SDG(1111280 $ to 14636376 SDG(975758 $. For the case of multiple input effect of the area and operation total cost resulted in decrease maximum number of tractors by 12%, and the total cost of operations also decreased by 12%. It is recommended to apply the optimization model as pre-requisite for improving machinery management during implementation of machinery scheduling.

Machinery for Forest Chip Production in Finland in 2007 and in the Future

Energy Technology Data Exchange (ETDEWEB)

Kaerhae, Kalle (Metsaeteho Oy, P.O. Box 101, FI-00171 Helsinki (Finland))

2008-10-15

Metsaeteho Oy's study consisted of a survey of the production machinery for forest chips used by energy plants in 2007. The major forest chip suppliers in Finland were involved in the study. In addition, the machinery and equipment stocked by the manufacturers and vendors of energy wood harvester heads, stump lifting devices, and chippers were also surveyed. The study provided also an estimate of future machinery requirements for forest chip production in Finland. The study estimated that a total of 1,100 machine and truck units were employed in the production of forest chips for energy plants in 2007. A total of 770 machine and truck units were contracted for the major forest chip suppliers in 2007. Increasing forest chip consumption will considerable increase the demand for additional forest chip production resources in the future. If the consumption of forest chips by energy plants in 2015 reaches 15 TWh, i.e. about 7.5 mill. m3, then the forest machine and truck requirement will be over 1,700 units. The corresponding machinery requirement at an energy plant with a forest chip consumption of 25 TWh (approx. 12.5 mill. m3), will be close to 2,300 machine and truck units

Ribosome-dependent ATPase interacts with conserved membrane protein in Escherichia coli to modulate protein synthesis and oxidative phosphorylation.

Directory of Open Access Journals (Sweden)

Mohan Babu

Full Text Available Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.

Power electronics and electric machinery challenges and opportunities in electric and hybrid vehicles

Energy Technology Data Exchange (ETDEWEB)

Adams, D.J.; Hsu, J.S.; Young, R.W. [Oak Ridge National Lab., TN (United States); Peng, F.Z. [Univ. of Tennessee, Knoxville, TN (United States)

1997-06-01

The development of power electronics and electric machinery presents significant challenges to the advancement of electric and hybrid vehicles. Electronic components and systems development for vehicle applications have progressed from the replacement of mechanical systems to the availability of features that can only be realized through interacting electronic controls and devices. Near-term applications of power electronics in vehicles will enable integrated powertrain controls, integrated chassis system controls, and navigation and communications systems. Future applications of optimized electric machinery will enable highly efficient and lightweight systems. This paper will explore the areas where research and development is required to ensure the continued development of power electronics and electric machines to meet the rigorous demands of automotive applications. Additionally, recent advances in automotive related power electronics and electric machinery at Oak Ridge National Laboratory will be explained. 3 refs., 5 figs.

The experimental study on efficiency improvement of turbo machinery supported with magnetic bearings

International Nuclear Information System (INIS)

Park, In Hwang; Park, Young Ho; Han, Dong Chul

2007-01-01

To implement a conventional electromagnetic bearing in small turbo machinery, it has problems such as load capacity and size. Therefore, in this paper, these problems are resolved by using a permanent magnet biased electromagnetic bearing as a thrust bearing of small turbo machinery. Because the flux path of the bearing is designed by reluctance path modulation using an electromagnet and a permanent magnet, the bearing improves upon non-linearity, power consumption, size and load capacity of a conventional electromagnetic bearing. Test rotating the shaft over 500,000DN were carried out to verify the performance of the proposed small turbo machinery. In addition, the relationships between mass flow rate and pressure rise were measured as changing the tip clearance to verify the feasibility of efficiency improvement and active surge control and these results were compared with theoretical results

Membrane phosphorylation and nerve cell function

International Nuclear Information System (INIS)

Baer, P.R.

1982-01-01

This thesis deals with the phosphorylation of membrane components. In part I a series of experiments is described using the hippocampal slice as a model system. In part II a different model system - cultured hybrid cells - is used to study protein and lipid phosphorylation, influenced by incubation with neuropeptides. In part III in vivo and in vitro studies are combined to study protein phosphorylation after neuroanatomical lesions. In a section of part II (Page 81-90) labelling experiments of the membrane inositol-phospholipids are described. 32 P-ATP was used to label phospholipids in intact hybrid cells, and short incubations were found to be the most favourable. (C.F.)

Importance of tyrosine phosphorylation in receptor kinase complexes.

Science.gov (United States)

Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

2015-05-01

Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tyrosine Phosphorylation in Toll-Like Receptor Signaling

Science.gov (United States)

Chattopadhyay, Saurabh; Sen, Ganes C.

2014-01-01

There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge on the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways. PMID:25022196

Systematic inference of functional phosphorylation events in yeast metabolism.

Science.gov (United States)

Chen, Yu; Wang, Yonghong; Nielsen, Jens

2017-07-01

Protein phosphorylation is a post-translational modification that affects proteins by changing their structure and conformation in a rapid and reversible way, and it is an important mechanism for metabolic regulation in cells. Phosphoproteomics enables high-throughput identification of phosphorylation events on metabolic enzymes, but identifying functional phosphorylation events still requires more detailed biochemical characterization. Therefore, development of computational methods for investigating unknown functions of a large number of phosphorylation events identified by phosphoproteomics has received increased attention. We developed a mathematical framework that describes the relationship between phosphorylation level of a metabolic enzyme and the corresponding flux through the enzyme. Using this framework, it is possible to quantitatively estimate contribution of phosphorylation events to flux changes. We showed that phosphorylation regulation analysis, combined with a systematic workflow and correlation analysis, can be used for inference of functional phosphorylation events in steady and dynamic conditions, respectively. Using this analysis, we assigned functionality to phosphorylation events of 17 metabolic enzymes in the yeast Saccharomyces cerevisiae , among which 10 are novel. Phosphorylation regulation analysis cannot only be extended for inference of other functional post-translational modifications but also be a promising scaffold for multi-omics data integration in systems biology. Matlab codes for flux balance analysis in this study are available in Supplementary material. yhwang@ecust.edu.cn or nielsenj@chalmers.se. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Protein phosphorylation during coconut zygotic embryo development

International Nuclear Information System (INIS)

Islas-Flores, I.; Oropeza, C.; Hernandez-Sotomayor, S.M.T.

1998-01-01

Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [gamma-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species

Identification of the protein kinase C phosphorylation site in neuromodulin

International Nuclear Information System (INIS)

Apel, E.D.; Byford, M.F.; Au, D.; Walsh, K.A.; Storm, D.R.

1990-01-01

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin and the authors have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar K m values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [γ- 32 P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32 P-labeled tryptic peptide was generated from phosphorylated neuromodulin. They conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmoculin to neuromodulin. The proximity of serine-41 to the calmodulin binding domain in neuromodulin very likely explains the effect of phosphorylation on the affinity of neuromodulin for calmodulin

Detection and characterization of 3D-signature phosphorylation site motifs and their contribution towards improved phosphorylation site prediction in proteins

Directory of Open Access Journals (Sweden)

Selbig Joachim

2009-04-01

Full Text Available Abstract Background Phosphorylation of proteins plays a crucial role in the regulation and activation of metabolic and signaling pathways and constitutes an important target for pharmaceutical intervention. Central to the phosphorylation process is the recognition of specific target sites by protein kinases followed by the covalent attachment of phosphate groups to the amino acids serine, threonine, or tyrosine. The experimental identification as well as computational prediction of phosphorylation sites (P-sites has proved to be a challenging problem. Computational methods have focused primarily on extracting predictive features from the local, one-dimensional sequence information surrounding phosphorylation sites. Results We characterized the spatial context of phosphorylation sites and assessed its usability for improved phosphorylation site predictions. We identified 750 non-redundant, experimentally verified sites with three-dimensional (3D structural information available in the protein data bank (PDB and grouped them according to their respective kinase family. We studied the spatial distribution of amino acids around phosphorserines, phosphothreonines, and phosphotyrosines to extract signature 3D-profiles. Characteristic spatial distributions of amino acid residue types around phosphorylation sites were indeed discernable, especially when kinase-family-specific target sites were analyzed. To test the added value of using spatial information for the computational prediction of phosphorylation sites, Support Vector Machines were applied using both sequence as well as structural information. When compared to sequence-only based prediction methods, a small but consistent performance improvement was obtained when the prediction was informed by 3D-context information. Conclusion While local one-dimensional amino acid sequence information was observed to harbor most of the discriminatory power, spatial context information was identified as

Fluid machinery application, selection, and design

CERN Document Server

Wright, Terry

2013-01-01

Published nearly a decade ago, Fluid Machinery: Performance, Analysis, and Design quickly became popular with students, professors, and professionals because of its comprehensive and comprehensible introduction to the fluid mechanics of turbomachinery. Renamed to reflect its wider scope and reorganized content, this second edition provides a more logical flow of information that will enhance understanding. In particular, it presents a consistent notation within and across chapters, updating material when appropriate. Although the authors do account for the astounding growth in the field of com

The kinematics of machinery outlines of a theory of machines

CERN Document Server

Reuleaux, Franz

2012-01-01

A classic on the kinematics of machinery, this volume was written by the Father of Kinematics. Reuleaux writes with authority and precision, developing the subject from its fundamentals. 450 figures. 1876 edition.

Neurofilament subunit (NFL) head domain phosphorylation regulates axonal transport of neurofilaments.

LENUS (Irish Health Repository)

Yates, Darran M

2009-04-01

Neurofilaments are the intermediate filaments of neurons and are synthesised in neuronal cell bodies and then transported through axons. Neurofilament light chain (NFL) is a principal component of neurofilaments, and phosphorylation of NFL head domain is believed to regulate the assembly of neurofilaments. However, the role that NFL phosphorylation has on transport of neurofilaments is poorly understood. To address this issue, we monitored axonal transport of phosphorylation mutants of NFL. We mutated four known phosphorylation sites in NFL head domain to either preclude phosphorylation, or mimic permanent phosphorylation. Mutation to preclude phosphorylation had no effect on transport but mutation of three sites to mimic permanent phosphorylation inhibited transport. Mutation of all four sites together to mimic permanent phosphorylation proved especially potent at inhibiting transport and also disrupted neurofilament assembly. Our results suggest that NFL head domain phosphorylation is a regulator of neurofilament axonal transport.

Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly

Directory of Open Access Journals (Sweden)

Danming Tang

2012-09-01

GRASP65 phosphorylation during mitosis and dephosphorylation after mitosis are required for Golgi disassembly and reassembly during the cell cycle. At least eight phosphorylation sites on GRASP65 have been identified, but whether they are modified in a coordinated fashion during mitosis is so far unknown. In this study, we raised phospho-specific antibodies that recognize phosphorylated T220/T224, S277 and S376 residues of GRASP65, respectively. Biochemical analysis showed that cdc2 phosphorylates all three sites, while plk1 enhances the phosphorylation. Microscopic studies using these antibodies for double and triple labeling demonstrate sequential phosphorylation and dephosphorylation during the cell cycle. S277 and S376 are phosphorylated from late G2 phase through metaphase until telophase when the new Golgi is reassembled. T220/224 is not modified until prophase, but is highly modified from prometaphase to anaphase. In metaphase, phospho-T220/224 signal localizes on both Golgi haze and mitotic Golgi clusters that represent dispersed Golgi vesicles and Golgi remnants, respectively, while phospho-S277 and S376 labeling is more concentrated on mitotic Golgi clusters. Expression of a phosphorylation-resistant GRASP65 mutant T220A/T224A inhibited mitotic Golgi fragmentation to a much larger extent than the expression of the S277A and S376A mutants. In cytokinesis, T220/224 dephosphorylation occurs prior to that of S277, but after S376. This study provides evidence that GRASP65 is sequentially phosphorylated and dephosphorylated during mitosis at different sites to orchestrate Golgi disassembly and reassembly during cell division, with phosphorylation of the T220/224 site being most critical in the process.

Myosin light chain kinase phosphorylation in tracheal smooth muscle

International Nuclear Information System (INIS)

Stull, J.T.; Hsu, L.C.; Tansey, M.G.; Kamm, K.E.

1990-01-01

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32 P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+

The Design of High Reliability Magnetic Bearing Systems for Helium Cooled Reactor Machinery

International Nuclear Information System (INIS)

Swann, M.; Davies, N.; Jayawant, R.; Leung, R.; Shultz, R.; Gao, R.; Guo, Z.

2014-01-01

The requirements for magnetic bearing equipped machinery used in high temperature, helium cooled, graphite moderated reactor applications present a set of design considerations that are unlike most other applications of magnetic bearing technology in large industrial rotating equipment, for example as used in the oil and gas or other power generation applications. In particular, the bearings are typically immersed directly in the process gas in order to take advantage of the design simplicity that comes about from the elimination of ancillary lubrication and cooling systems for bearings and seals. Such duty means that the bearings will usually see high temperatures and pressures in service and will also typically be subject to graphite particulate and attendant radioactive contamination over time. In addition, unlike most industrial applications, seismic loading events become of paramount importance for the magnetic bearings system, both for actuators and controls. The auxiliary bearing design requirements, in particular, become especially demanding when one considers that the whole mechanical structure of the magnetic bearing system is located inside an inaccessible pressure vessel that should be rarely, if ever, disassembled over the service life of the power plant. Lastly, many machinery designs for gas cooled nuclear power plants utilize vertical orientation. This circumstance presents its own unique requirements for the machinery dynamics and bearing loads. Based on the authors’ experience with machine design and supply on several helium cooled reactor projects including Ft. St. Vrain (US), GT-MHR (Russia), PBMR (South Africa), GTHTR (Japan), and most recently HTR-PM (China), this paper addresses many of the design considerations for such machinery and how the application of magnetic bearings directly affects machinery reliability and availability, operability, and maintainability. Remote inspection and diagnostics are a key focus of this paper. (author)

Phosphorylation of the Epstein-Barr virus nuclear antigen 2

DEFF Research Database (Denmark)

Grässer, F A; Göttel, S; Haiss, P

1992-01-01

A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK......-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic...

Managing the exploitation life of the mining machinery for an unlimited duration of time

Directory of Open Access Journals (Sweden)

Vujić Slobodan

2011-01-01

Full Text Available The problem of determining the optimum exploitation life of machinery, namely, the optimum time for machinery and equipment replacement, represents a complex and highly responsible engineering task. Taking into consideration the situation prevailing at coal pit mines in Serbia, the tasks of this rank are very complex and difficult. To make a decision on the replacement of capital equipment and machinery, such as bucket wheel excavators within the mentioned systems, implies a management task of utmost responsibility. It requires high professional and analytical knowledge as well as reliable arguments, based on a multidisciplinary professional approach. In this paper, the authors present their views on the problem of establishing the optimum exploitation life of bucket wheel excavators, offering an algorithm, based on dynamic programming, as a solution.

Noise test system of rotating machinery in nuclear power station based on microphone array

Science.gov (United States)

Chang, Xincai; Guan, Jishi; Qi, Liangcai

2017-12-01

Rotating machinery plays an important role in all walks of life. Once the equipment fails, equipment maintenance and shutdown will cause great social harm and economic losses. Equipment safety operations at nuclear power stations have always been of top priority. It is prone to noise when the equipment is out of order or aging. Failure to find or develop equipment at the initial stage of equipment failure or ageing will pose a serious threat to the safety of the plant’s equipment. In this paper, sound imaging diagnosis technology is applied as a supplementary method to the condition monitoring and diagnosis system of rotating machinery in nuclear power stations. It provides a powerful guarantee for the condition monitoring and fault diagnosis of rotating machinery in nuclear power stations.

Interaction of butylated hydroxyanisole with mitochondrial oxidative phosphorylation.

Science.gov (United States)

Fusi, F; Sgaragli, G; Murphy, M P

1992-03-17

The antioxidant, butylated hydroxyanisole (BHA), has a number of effects on mitochondrial oxidative phosphorylation. In this study we apply the novel approach developed by Brand (Brand MD, Biochim Biophys Acta 1018: 128-133, 1990) to investigate the site of action of BHA on oxidative phosphorylation in rat liver mitochondria. Using this approach we show that BHA increases the proton leak through the mitochondrial inner membrane and that it also inhibits the delta p (proton motive force across the mitochondrial inner membrane) generating system, but has no effect on the phosphorylation system. This demonstrates that compounds having pleiotypic effects on mitochondrial oxidative phosphorylation in vitro can be analysed and their many effects distinguished. This approach is of general use in analysing many other compounds of pharmacological interest which interact with mitochondria. The implications of these results for the mechanism of interaction of BHA with mitochondrial oxidative phosphorylation are discussed.

Present Situation and Activities of ISO 14000 series in Heavy Machinery Industries; Jukikai meka no taiou jokyo

Energy Technology Data Exchange (ETDEWEB)

Tomisawa, Yukio. [Ishikawajima-Harima Heavy Industries Corp., Tokyo (Japan). Research Institute

1999-01-15

This paper shows the outline of the present situation and activities of ISO14000 series, environmental management system in heavy machinery industries. Today, main heavy machinery industries have already certified to ISO 14001 until this summer. The aim of the introduction of ISO 14001 are to establish the system improvement continuously by PDCA, to have essential elements in international trade and to keep up with the risk management. Some example of conducted ISO 14001 in heavy machinery industries are shown. The trend of heavy machinery industries will be able to predict to three key words, internationalization, network and opening information. The new role expected to environmental material engineer are indicated to research and develop ecomaterial by using LCA and to support the recycling, waste treatment and other technical basis of the environmental management system. (author)

Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein.

Science.gov (United States)

Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

2009-11-30

Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions.

Phosphorylation prevents C/EBP{beta} from the calpain-dependent degradation

Energy Technology Data Exchange (ETDEWEB)

Zhang, Yuan-yuan; Li, Shu-fen; Qian, Shu-wen; Zhang, You-you; Liu, Yuan; Tang, Qi-Qun; Li, Xi, E-mail: lixi@shmu.edu.cn

2012-03-16

Highlights: Black-Right-Pointing-Pointer Phosphorylation protected C/EBP{beta} from {mu}-calpain-mediated proteolysis in vitro. Black-Right-Pointing-Pointer Phosphorylation mimic C/EBP{beta} was insensitive to calpain accelerator and inhibitor. Black-Right-Pointing-Pointer Phosphorylation on Thr{sub 188} contributed more to the stabilization of C/EBP{beta}. -- Abstract: CCAAT/enhancer-binding protein (C/EBP) {beta} plays an important role in proliferation and differentiation of 3T3-L1 preadipocytes. C/EBP{beta} is sequentially phosphorylated during the 3T3-L1 adipocyte differentiation program, first by MAPK/Cyclin A/cdk2 on Thr{sub 188} and subsequently by GSK3{beta} on Ser{sub 184} or Thr{sub 179}. Dual phosphorylation is critical for the gain of DNA binding activity of C/EBP{beta}. In this manuscript, we found that phosphorylation also contributed to the stability of C/EBP{beta}. Both ex vivo and in vitro experiments showed that phosphorylation by MAPK/Cyclin A/cdk2 and GSK3{beta} protected C/EBP{beta} from {mu}-calpain-mediated proteolysis, while phosphorylation on Thr{sub 188} by MAPK/Cyclin A/cdk2 contributed more to the stabilization of C/EBP{beta}, Further studies indicated that phosphorylation mimic C/EBP{beta} was insensitive to both calpain accelerator and calpain inhibitor. Thus, phosphorylation might contribute to the stability as well as the gain of DNA binding activity of C/EBP{beta}.

Proteasome phosphorylation regulates cocaine-induced sensitization.

Science.gov (United States)

Gonzales, Frankie R; Howell, Kristin K; Dozier, Lara E; Anagnostaras, Stephan G; Patrick, Gentry N

2018-04-01

Repeated exposure to cocaine produces structural and functional modifications at synapses from neurons in several brain regions including the nucleus accumbens. These changes are thought to underlie cocaine-induced sensitization. The ubiquitin proteasome system plays a crucial role in the remodeling of synapses and has recently been implicated in addiction-related behavior. The ATPase Rpt6 subunit of the 26S proteasome is phosphorylated by Ca 2+ /calmodulin-dependent protein kinases II alpha at ser120 which is thought to regulate proteasome activity and distribution in neurons. Here, we demonstrate that Rpt6 phosphorylation is involved in cocaine-induced locomotor sensitization. Cocaine concomitantly increases proteasome activity and Rpt6 S120 phosphorylation in cultured neurons and in various brain regions of wild type mice including the nucleus accumbens and prefrontal cortex. In contrast, cocaine does not increase proteasome activity in Rpt6 phospho-mimetic (ser120Asp) mice. Strikingly, we found a complete absence of cocaine-induced locomotor sensitization in the Rpt6 ser120Asp mice. Together, these findings suggest a critical role for Rpt6 phosphorylation and proteasome function in the regulation cocaine-induced behavioral plasticity. Copyright © 2017 Elsevier Inc. All rights reserved.

A grammar inference approach for predicting kinase specific phosphorylation sites.

Science.gov (United States)

Datta, Sutapa; Mukhopadhyay, Subhasis

2015-01-01

Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner.

A Grammar Inference Approach for Predicting Kinase Specific Phosphorylation Sites

Science.gov (United States)

Datta, Sutapa; Mukhopadhyay, Subhasis

2015-01-01

Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner. PMID:25886273

O-GlcNAc modification: why so intimately associated with phosphorylation?

Directory of Open Access Journals (Sweden)

Ande Sudharsana R

2011-01-01

Full Text Available Abstract Post-translational modification of proteins at serine and threonine side chains by β-N-acetylglucosamine (O-GlcNAc mediated by the enzyme β-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68% than its normal occurrence (~2% alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.

A Modelling Approach to Multibody Dynamics of Fluid Power Machinery with Hydrodynamic Lubrication

DEFF Research Database (Denmark)

Johansen, Per; Rømer, Daniel; Andersen, Torben Ole

2013-01-01

The efficiency potential of the digital displacement technology and the increasing interest in hydraulic transmissions in wind and wave energy applications has created an incentive for development of high efficiency fluid power machinery. Modelling and analysis of fluid power machinery loss...... mechanisms is necessary in order to accommodate this demand. At present fully coupled thermo-elastic models for various tribological interfaces has been presented. However, in order to analyse the interaction between tribological interfaces in fluid power pumps and motors, these interface models needs...

Production method of hydrogen jet plasma process in hydro machinery

International Nuclear Information System (INIS)

Amini, F.

2007-01-01

The purpose of present paper is to the process of plasma formation in hydro machinery when a hydro turbine operates at various conditions and load rejection. By investigation the power, shock pressure , and impact effects of hydro machinery, it is revealed that energy and hydrogen are generated by the plasma process. The investigation on several turbines of various hydro power plants reveals that cold fusion process in hydro machinery generates hydrogen. The hypothesis concerning the participation of alkaline metals in river water and the atomic nuclei of the runner blade material in the formation of hydrogen are considered. It is possible to assume hydrogen, deuterium, helium, and tritium atoms (based on Dr. Mizuno and Dr. Kanarev theories) that are formed, diffuse into cavitation bubbles. The plasma is generated during the collapse of the bubble; thus, the quantity of burnt hydrogen determine the volume of generating hydrogen and the impact force caused by hydrogen explosion (noise).There are five main notions, which can determine hydrogen and plasma process: (1) turbine power effect, (2) high shock pressure, (3) crack on turbine parts, (4) impacts effects and (4) the lift of rotating parts. The frequency of the excitation lies in a range from 0.786 to 1.095 Hz.In future, it may be possible to design hydro turbines based on the plasma process that generates hydrogen; or there may exist turbines that rotate with a mixture of hydrogen explosion and water energies

Rotating machinery surveillance system reduces plant downtime and radiation exposure

International Nuclear Information System (INIS)

Bohanick, J.S.; Robinson, J.C.; Allen, J.W.

1988-01-01

A rotating machinery surveillance system (RMSS) was permanently installed at Grand Gulf nuclear station (GGNS) as part of a program sponsored by the US Department of Energy whose goal was to reduce radiation exposure to power plant personnel resulting from the inspection, maintenance, and repair of rotating machinery. The RMSS was installed at GGNS in 1983 to continuously monitor 173 analog vibration signals from proximity probes mounted on 26 machine trains and ∼450 process data points via a computer data link. Vibration frequency spectra, i.e., the vibration amplitude versus frequency of vibration, and various characterizations of these spectra are the fundamental data collected by the RMSS for performing machinery diagnostics. The RMSS collects vibration frequency spectra on a daily basis for all the monitored rotating equipment and automatically stores the collected spectra for review by the vibration engineer. Vibration spectra automatically stored by the RMSS fall into categories that include the last normal, alarm, minimum and maximum, past three-day data set, baseline, current, and user-saved spectra. During first and second fuel-cycle operation at GGNS, several significant vibration problems were detected by the RMSS. Two of these are presented in this paper: recirculation pumps and turbine-generator bearing degradation. The total reduction in personnel radiation exposure at GGNS from 1985 to 1987 due to the presence of the RMSS was estimated to be in the range from 49 to 54 person-rem

Protein phosphorylation systems in postmortem human brain

International Nuclear Information System (INIS)

Walaas, S.I.; Perdahl-Wallace, E.; Winblad, B.; Greengard, P.

1989-01-01

Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders

Novel Role of Src in Priming Pyk2 Phosphorylation.

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Ming Zhao

Full Text Available Proline-rich tyrosine kinase 2 (Pyk2 is a member of the focal adhesion kinase (FAK family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.

Prisoners signify: a political discourse analysis of mental illness in a prison control unit.

Science.gov (United States)

Cloyes, Kristin Gates

2007-09-01

Increasingly, US prisoners diagnosed with mental illness are housed in control units, the most restrictive form of confinement in the US prison system. This situation has led to intense debate over the legal, ethical and clinical status of mental illness. This is a semiotic struggle with profound effects, yet most related work treats mental illness as a neutral, individual variable. Few analyses locate mental illness within a larger sociopolitical context. Fewer still focus on discursive practice. None critically analyze the accounts of control unit prisoners, who talk about extreme marginality and risk for victimization. This paper has two aims: (i) to develop a systematic method of analysis that accounts for signification as discourse-in-action; and (ii) to show how prisoners' signification of mental illness articulates agency through and against marginalizing discourse. Political discourse analysis demonstrates how control unit prisoners with psychiatric diagnoses signify mental illness, and articulate safer identifications in the process.

46 CFR 182.460 - Ventilation of spaces containing machinery powered by, or fuel tanks for, gasoline.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Ventilation of spaces containing machinery powered by, or fuel tanks for, gasoline. 182.460 Section 182.460 Shipping COAST GUARD, DEPARTMENT OF HOMELAND..., gasoline. (a) A space containing machinery powered by, or fuel tanks for, gasoline must have a ventilation...

Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii

Directory of Open Access Journals (Sweden)

Ayswarya Ravi

2018-02-01

Full Text Available Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN, a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a “plant-like” algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT and Gallium-immobilized metal affinity chromatography (Ga-IMAC were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN.

Multiple Cylinder Free-Piston Stirling Machinery

Science.gov (United States)

Berchowitz, David M.; Kwon, Yong-Rak

In order to improve the specific power of piston-cylinder type machinery, there is a point in capacity or power where an advantage accrues with increasing number of piston-cylinder assemblies. In the case of Stirling machinery where primary energy is transferred across the casing wall of the machine, this consideration is even more important. This is due primarily to the difference in scaling of basic power and the required heat transfer. Heat transfer is found to be progressively limited as the size of the machine increases. Multiple cylinder machines tend to preserve the surface area to volume ratio at more favorable levels. In addition, the spring effect of the working gas in the so-called alpha configuration is often sufficient to provide a high frequency resonance point that improves the specific power. There are a number of possible multiple cylinder configurations. The simplest is an opposed pair of piston-displacer machines (beta configuration). A three-cylinder machine requires stepped pistons to obtain proper volume phase relationships. Four to six cylinder configurations are also possible. A small demonstrator inline four cylinder alpha machine has been built to demonstrate both cooling operation and power generation. Data from this machine verifies theoretical expectations and is used to extrapolate the performance of future machines. Vibration levels are discussed and it is argued that some multiple cylinder machines have no linear component to the casing vibration but may have a nutating couple. Example applications are discussed ranging from general purpose coolers, computer cooling, exhaust heat power extraction and some high power engines.

Cisplatinum and Taxol Induce Different Patterns of p53 Phosphorylation

Directory of Open Access Journals (Sweden)

Giovanna Damia

2001-01-01

Full Text Available Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20, only DDP treatment induced p53 phosphorylation at serine 15 (Ser15. Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM cell lines, the role of ATM in druginduced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.

Regulation of protein phosphorylation in oat mitochondria

International Nuclear Information System (INIS)

Pike, C.; Kopeck, K.; Sceppa, E.

1989-01-01

We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with 32 P-γ-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of 35 S-adenosine thiotriphosphate

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

Science.gov (United States)

Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

2017-05-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

INTEGRAL ASSESSMENT OF EFFICIENCY OF A FLEET OF AGRICULTURAL MACHINERY AND TRACTORS

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V. M. Korotchenya

2015-01-01

Full Text Available An indicator for an integral assessment of efficiency of a fleet of agricultural machinery and tractors is proposed. Its calculating consists in multiplying partial efficiencies together: technical and price efficiency of agricultural production, and eco-efficiency of a fleet of agricultural machinery and tractors. Using an axiomatic method, the concept of efficiency of fleet was studied within broader category of productivity and in wider scales - within agriculture. The most general and obvious statements according to which efficiency of machine and tractor fleet is considered as indirectly settlement size counted proceeding from efficiency of agricultural production, were accepted as axioms. In general, efficiency is a ratio between productivity of an object in question and that of the theoretical ideal or an object with maximum productivity efficiency of which is accepted equal to unity or 100%. Technical efficiency of agricultural production characterizes the ability of an agricultural sector to produce the technically maximum output of agricultural produce obtained from the available resources (land, labor, machines, etc.. Price efficiency evaluates the ability of an agricultural sector to produce output of agricultural produce with regard to employing the optimal mix of resources given their prices. Because the theoretical ideal does not exist, efficiency of an agricultural sector, e.g. in Russia and of its fleet of agricultural machinery and tractors, is measured against agriculture of another country with maximum productivity efficiency of which is accepted as equal to unity or 100 percent. The calculation can be made on the basis of the Data Envelopment Analysis method. It is possible to calculate price and economic efficiency of Russian agriculture (fleet of agricultural machinery and tractors due to use its domestic prices.

PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2

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Laura Civiero

2017-12-01

Full Text Available Mutations in Leucine-rich repeat kinase 2 (LRRK2 are associated with Parkinson's disease (PD and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1 Activated Kinase 6 (PAK6. Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.

Control Systems of Rubber Dryer Machinery Components Using Programmable Logic Control (PLC)

Science.gov (United States)

Hendra; Yulianto, A. S.; Indriani, A.; Hernadewita; Hermiyetti

2018-02-01

Application of programmable logic control (PLC) is widely used on the control systems in the many field engineering such as automotive, aviation, food processing and other industries [1-2]. PLC is simply program to control many automatic activity, easy to use, flexible and others. PLC using the ladder program to solve and regulated the control system component. In previous research, PLC was used for control system of rotary dryer machine. In this paper PLC are used for control system of motion component in the rubber dryer machinery. Component of rubber dryer machine is motors, gearbox, sprocket, heater, drying chamber and bearing. Principle working of rubber dryer machinery is wet rubber moving into the drying chamber by sprocket. Sprocket is driven by motors that conducted by PLC to moving and set of wet rubber on the drying chamber. Drying system uses greenhouse effect by making hanger dryer design in the form of line path. In this paper focused on motion control system motors and sensors drying rubber using PLC. The results show that control system of rubber dryer machinery can work in accordance control input and the time required to dry the rubber.

Src kinase regulation by phosphorylation and dephosphorylation

International Nuclear Information System (INIS)

Roskoski, Robert

2005-01-01

Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPε, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined

Phototropism and Protein Phosphorylation in Higher Plants: Unilateral Blue Light Irradiation Generates a Directional Gradient of Protein Phosphorylation Across the Oat Coleoptile

International Nuclear Information System (INIS)

Salomon, M.; Zacherl, M.; Rüdiger, W.

1997-01-01

Blue light induces the phosphorylation of a 116 kDa oat protein found in plasma membrane preparations from coleoptile tips. We developed a very sensitive in vitro method that allowed us to determine the tissue distribution of protein phosphorylation after applying unilateral and bilateral blue light pulses in vivo. We found that following unilateral in vivo irradiation the degree in phosphorylation of the 116 kDa protein is significantly higher at the irradiated than at the shaded side of the coleoptile tip. This asymmetry can be considered as previously missing criterion that protein phosphorylation represents an early event within the transduction chain for phototropism. (author)

Vibration Feature Extraction and Analysis for Fault Diagnosis of Rotating Machinery-A Literature Survey

Directory of Open Access Journals (Sweden)

Saleem Riaz

2017-02-01

Full Text Available Safety, reliability, efficiency and performance of rotating machinery in all industrial applications are the main concerns. Rotating machines are widely used in various industrial applications. Condition monitoring and fault diagnosis of rotating machinery faults are very important and often complex and labor-intensive. Feature extraction techniques play a vital role for a reliable, effective and efficient feature extraction for the diagnosis of rotating machinery. Therefore, developing effective bearing fault diagnostic method using different fault features at different steps becomes more attractive. Bearings are widely used in medical applications, food processing industries, semi-conductor industries, paper making industries and aircraft components. This paper review has demonstrated that the latest reviews applied to rotating machinery on the available a variety of vibration feature extraction. Generally literature is classified into two main groups: frequency domain, time frequency analysis. However, fault detection and diagnosis of rotating machine vibration signal processing methods to present their own limitations. In practice, most healthy ingredients faulty vibration signal from background noise and mechanical vibration signals are buried. This paper also reviews that how the advanced signal processing methods, empirical mode decomposition and interference cancellation algorithm has been investigated and developed. The condition for rotating machines based rehabilitation, prevent failures increase the availability and reduce the cost of maintenance is becoming necessary too. Rotating machine fault detection and diagnostics in developing algorithms signal processing based on a key problem is the fault feature extraction or quantification. Currently, vibration signal, fault detection and diagnosis of rotating machinery based techniques most widely used techniques. Furthermore, the researchers are widely interested to make automatic

Amino acid chirality breaking by N-phosphorylation

International Nuclear Information System (INIS)

Zhao Yufen; Yan Qingjin.

1995-01-01

The chirality breaking of amino acid is a focus issue in the origin of life. For chemists, there are some interesting chemical approaches to solve the symmetry breaking problem. Our previous experiments indicated that when amino acids were phosphorylated, there were many bio-mimic reactions happened. In this paper, it was found that there had significant difference between the N-phosphoryl L- and D- amino acids such as serine and threonine. The optical rotation tracing experiments of the racemic N-phosphoamino acids also showed the similar results. The chirality breaking of amino acids by N-phosphorylation was a novel phenomena. (author). 3 refs, 1 fig. Abstract only

Part-time farmers and accidents with agricultural machinery: a moderated mediated model on the role played by frequency of use and unsafe beliefs.

Science.gov (United States)

Caffaro, Federica; Roccato, Michele; Micheletti Cremasco, Margherita; Cavallo, Eugenio

2018-01-25

We aimed at testing a model of the direct and indirect effects of being a part-time farmer on the probability of being involved in an agricultural machinery-related accident, considering the role played by unsafe beliefs and the frequency of use of machinery. Two-hundred and fifty-two Italian men, regular users of agricultural machinery (age: Mean = 45.1 years, standard Deviation = 17.5), were administered a paper-and-pencil questionnaire addressing their relation with work, unsafe beliefs, and previous experience of machinery-related accidents. Being a part-time farmer showed a positive association with unsafe beliefs only among occasional machinery users. Unsafe beliefs in turn showed a positive association with accidents. The study gave a novel contribution to the knowledge of the chain of events connecting part-time farmers with machinery-related accidents. Preventive training interventions targeting part-timer farmers using agricultural machinery just occasionally should be developed.

2006 China Machinery and Electronical Products Trade Fair:Ample Fruits Shown

Institute of Scientific and Technical Information of China (English)

无

2006-01-01

@@ 2006 Malaysia China Machinery and Electronical Products Trade Fair, organized by China Council for the Promotion of International Trade(CCPIT), was held in August in Prince World Trade Center(PWTC), Kuala Lumpur.

Research on prediction of agricultural machinery total power based on grey model optimized by genetic algorithm

Science.gov (United States)

Xie, Yan; Li, Mu; Zhou, Jin; Zheng, Chang-zheng

2009-07-01

Agricultural machinery total power is an important index to reflex and evaluate the level of agricultural mechanization. It is the power source of agricultural production, and is the main factors to enhance the comprehensive agricultural production capacity expand production scale and increase the income of the farmers. Its demand is affected by natural, economic, technological and social and other "grey" factors. Therefore, grey system theory can be used to analyze the development of agricultural machinery total power. A method based on genetic algorithm optimizing grey modeling process is introduced in this paper. This method makes full use of the advantages of the grey prediction model and characteristics of genetic algorithm to find global optimization. So the prediction model is more accurate. According to data from a province, the GM (1, 1) model for predicting agricultural machinery total power was given based on the grey system theories and genetic algorithm. The result indicates that the model can be used as agricultural machinery total power an effective tool for prediction.

Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced.

Science.gov (United States)

Onnockx, Sheela; Xie, Jingwei; Degraef, Chantal; Erneux, Christophe; Pirson, Isabelle

2009-09-10

Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.

Implications of material selection on the design of packaging machinery.

Science.gov (United States)

Merritt, J P

2009-01-01

Material selection has significant implications on the design and cost of horizontal-form-fill-seal packaging machinery. To avoid excessive costs, machine redesigns and project delays, material selection must be reconciled early in the project and revisited throughout the construction of the machine.

Scattering transform and LSPTSVM based fault diagnosis of rotating machinery

Science.gov (United States)

Ma, Shangjun; Cheng, Bo; Shang, Zhaowei; Liu, Geng

2018-05-01

This paper proposes an algorithm for fault diagnosis of rotating machinery to overcome the shortcomings of classical techniques which are noise sensitive in feature extraction and time consuming for training. Based on the scattering transform and the least squares recursive projection twin support vector machine (LSPTSVM), the method has the advantages of high efficiency and insensitivity for noise signal. Using the energy of the scattering coefficients in each sub-band, the features of the vibration signals are obtained. Then, an LSPTSVM classifier is used for fault diagnosis. The new method is compared with other common methods including the proximal support vector machine, the standard support vector machine and multi-scale theory by using fault data for two systems, a motor bearing and a gear box. The results show that the new method proposed in this study is more effective for fault diagnosis of rotating machinery.

THE ROLE OF POSTHARVEST MACHINERIES AND PACKAGING IN MINIMIZING AFLATOXIN CONTAMINATION IN PEANUT

Directory of Open Access Journals (Sweden)

Raffi Paramawati

2016-10-01

Full Text Available As a tropical country with relatively high humidity and temperature, Indonesia is struggling with aflatoxin which frequently contaminates peanut. Aflatoxin is a carcinogenic toxic substance that could cause liver cancer. Due to the increasing concern on food safety, the Indonesian Drugs and Foods Agency specifies the maximum aflatoxin allowed in peanut as much as 20 ppb. However, researches showed that aflatoxin contamination in peanut in Indonesia is much higher than the threshold. The study was carried out to observe the effect of using postharvest machineries and packaging  treatments on aflatoxin contamination in peanut. Reduction of postharvest processes was conducted by using series of machineries, e.g. thresher, dryer, and sheller. Packaging treatments, e.g. vacuum plastic pack, hermetic glass chamber, and polyethylene (PE plastic wrap were carried out during storage at ambient temperature (25-27°C. The results showed that using machineries in postharvest handling produced peanut free from aflatoxin contamination. However, without effective packaging, the aflatoxin level would increase during storage. Hermetic packaging could protect peanut from the mold as indicated by low level of aflatoxin contamination.

Large-Scale Analysis Exploring Evolution of Catalytic Machineries and Mechanisms in Enzyme Superfamilies.

Science.gov (United States)

Furnham, Nicholas; Dawson, Natalie L; Rahman, Syed A; Thornton, Janet M; Orengo, Christine A

2016-01-29

Enzymes, as biological catalysts, form the basis of all forms of life. How these proteins have evolved their functions remains a fundamental question in biology. Over 100 years of detailed biochemistry studies, combined with the large volumes of sequence and protein structural data now available, means that we are able to perform large-scale analyses to address this question. Using a range of computational tools and resources, we have compiled information on all experimentally annotated changes in enzyme function within 379 structurally defined protein domain superfamilies, linking the changes observed in functions during evolution to changes in reaction chemistry. Many superfamilies show changes in function at some level, although one function often dominates one superfamily. We use quantitative measures of changes in reaction chemistry to reveal the various types of chemical changes occurring during evolution and to exemplify these by detailed examples. Additionally, we use structural information of the enzymes active site to examine how different superfamilies have changed their catalytic machinery during evolution. Some superfamilies have changed the reactions they perform without changing catalytic machinery. In others, large changes of enzyme function, in terms of both overall chemistry and substrate specificity, have been brought about by significant changes in catalytic machinery. Interestingly, in some superfamilies, relatives perform similar functions but with different catalytic machineries. This analysis highlights characteristics of functional evolution across a wide range of superfamilies, providing insights that will be useful in predicting the function of uncharacterised sequences and the design of new synthetic enzymes. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Electric machinery and drives in thermal power stations

International Nuclear Information System (INIS)

1974-01-01

The following subjects were dealt with during the VDE meeting: 1) Requirements made by the electric network on the generators and their excitation equipment, and the influence thereof on their design; 2) requirements made by the power station process on the electric drives and the influence thereof on type and design; 3) requirements made on protective measures from the point of the electric power station machinery. (TK) [de

Targeting Transcription Elongation Machinery for Breast Cancer Therapy

Science.gov (United States)

2017-05-01

ABSTRACT: This project focuses on the important but under-studied role of the P-TEFb- dependent transcription elongation machinery in human breast...molecule CDK9 inhibitors can be used to halt breast cancer metastasis. 8 experimental groups to test various drug dosage and frequency regimes will...tumor cells, which are said to be ’ addicted ’ to this protein. Consistently, pharmacological inhibition of Hsp90 has demonstrated great promise in

Progress in control equipment for fuel-handling machinery

International Nuclear Information System (INIS)

Nutting, B.A.

1986-01-01

The paper outlines the development of the equipment used to control the fuel-handling machinery associated with nuclear reactors, from the early electromechanical equipment, through solid-state switching logic to programmable controllers and microprocessors. The control techniques have developed along with the technology, and modern systems offer versatility, reliability and ease of design, operation and maintenance. Future trends and developments are discussed together with possible limiting factors. (author)

Protein phosphorylation and its role in archaeal signal transduction

Science.gov (United States)

Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C.; Albers, Sonja-Verena; Siebers, Bettina

2016-01-01

Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

Characterisation and properties of homo- and heterogenously phosphorylated nanocellulose.

Science.gov (United States)

Kokol, Vanja; Božič, Mojca; Vogrinčič, Robert; Mathew, Aji P

2015-07-10

Nano-sized cellulose ester derivatives having phosphoryl side groups were synthesised by phosphorylation of nanofibrilated cellulose (NFC) and nanocrystaline cellulose (NCC), using different heterogeneous (in water) and homogeneous (in molten urea) processes with phosphoric acid as phosphoryl donor. The phosphorylation mechanism, efficacy, stability, as well as its influence on the NC crystallinity and thermal properties, were evaluated using ATR-FTIR and (13)C NMR spectroscopies, potentiometric titration, capillary electrophoresis, X-ray diffraction, colorimetry, thermogravimmetry and SEM. Phosphorylation under both processes created dibasic phosphate and monobasic tautomeric phosphite groups at C6 and C3 positioned hydroxyls of cellulose, yielded 60-fold (∼1,173 mmol/kg) and 2-fold (∼1.038 mmol/kg) higher surface charge density for p-NFC and p-NCC, respectively, under homogenous conditions. None of the phosphorylations affected neither the NC crystallinity degree nor the structure, and noticeably preventing the derivatives from weight loss during the pyrolysis process. The p-NC showed high hydrolytic stability to water at all pH mediums. Reusing of the treatment bath was examined after the heterogeneous process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean; Rayapuram, Naganand; Pflieger, Delphine; Hirt, Heribert

2014-01-01

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean

2014-07-10

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Application of logical analysis of data to machinery-related accident prevention based on scarce data

International Nuclear Information System (INIS)

Jocelyn, Sabrina; Chinniah, Yuvin; Ouali, Mohamed-Salah; Yacout, Soumaya

2017-01-01

This paper deals with the application of Logical Analysis of Data (LAD) to machinery-related occupational accidents, using belt-conveyor-related accidents as an example. LAD is a pattern recognition and classification approach. It exploits the advancement in information technology and computational power in order to characterize the phenomenon under study. The application of LAD to machinery-related accident prevention is innovative. Ideally, accidents do not occur regularly, and as a result, companies have little data about them. The first objective of this paper is to demonstrate the feasibility of using LAD as an algorithm to characterize a small sample of machinery-related accidents with an adequate average classification accuracy. The second is to show that LAD can be used for prevention of machinery-related accidents. The results indicate that LAD is able to characterize different types of accidents with an average classification accuracy of 72–74%, which is satisfactory when compared with other studies dealing with large amounts of data where such a level of accuracy is considered adequate. The paper shows that the quantitative information provided by LAD about the patterns generated can be used as a logical way to prioritize risk factors. This prioritization helps safety practitioners make decisions regarding safety measures for machines. - Highlights: • LAD is presented as an innovative approach to prevent machinery-related accidents. • LAD is applied to a very small database of belt-conveyor-related accidents. • Despite scarce data, LAD generates patterns with adequate classification accuracy. • The patterns characterize different types of belt-conveyor-related accidents. • The patterns are useful to belt conveyor risk identification and risk estimation.

EXPLORING LINKS AMONG INVENTORY AND FINANCIAL PERFORMANCE IN THE AGRICULTURAL MACHINERY INDUSTRY

Directory of Open Access Journals (Sweden)

Dimitris Folinas

2014-10-01

Full Text Available The paper aims to investigate the effects of Inventory Turnover and Inventory Days on firm performance in the United Kingdom agricultural machinery industry by examining past literature reviews and empirical evidence of a primary research. Specific performance measures such as Earnings before Interest and Tax to Sales Ratio, Gross Profit to Sales Ratio, and Return on Assets are examined by conducting statistical analyses to determine the correlations between inventory and financial performance in agricultural machinery industry. The analysis of Inventory Turnover with financial performance measures doesn’t indicate any links between these variables. Furthermore, based on the results, Inventory Days plays a role in the financial performance of organisations however to varying degrees.

Phosphorylation sites of Arabidopsis MAP Kinase Substrate 1 (MKS1)

DEFF Research Database (Denmark)

Caspersen, M.B.; Qiu, J.-L.; Zhang, X.

2007-01-01

The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophore......The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified...... phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal...... at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine...

3-phosphorylated and -thiophosphorylated 2-thiazolidine- and 2-oxazolidine-thiones

International Nuclear Information System (INIS)

Vorob'eva, N.N.; Razvodovskaya, L.V.; Negrebetskii, V.V.; Grapov, A.F.; Mel'nikov, N.N.

1987-01-01

We investigated the phosphorylation and thiophosphorylation of 2-thiazolidine- and 2-oxazolidine-thiones. The presence in the heterocycle of the ambident triad HN-C=S can also lead to two series of phosphorylation products formed at the nitrogen and at the sulfur atom. It was therefore of interest to determine the dependence of the site of the phosphorylation on the structures of the heterocycle and off the phosphorylating agent. The formation of the N-phosphorylation products is confirmed by the 1 H NMR spectra, in which the signals of protons of the methylene group of the heteroring (C 4 H 2 ) are split on account of interaction with the phosphorus atom ( 3 JPH 0.5-2.3 Hz). We observed analogous values of 3 JPH constants for 2-aminothiazolines phosphorylated on the endocyclic nitrogen atom. In the 13 C NMR spectra of these compounds there are also coupling constants for the interaction of the carbon atoms C 4 and C 5 of the heterocycle with the phosphorus atom. The existence of the compounds as N-phosphorylated heterocycles is evidenced also by the 31 P chemical shifts

Phosphorylation of p37 is important for Golgi disassembly at mitosis

International Nuclear Information System (INIS)

Kaneko, Yayoi; Tamura, Kaori; Totsukawa, Go; Kondo, Hisao

2010-01-01

Research highlights: → p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis. → Phosphorylated p37 does not bind to Golgi membranes. → p37 phosphorylation inhibits p97/p37-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled at early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 by Cdc2 results in mitotic inhibition of the p97/p47 pathway . In this study, we demonstrate that p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis, and this phosphorylated p37 does not bind to Golgi membranes. Using an in vitro Golgi reassembly assay, we show that mutated p37(S56D, T59D), which mimics mitotic phosphorylation, does not cause any cisternal regrowth, indicating that p37 phosphorylation inhibits the p97/p37 pathway. Our results demonstrate that p37 phosphorylation on Serine-56 and Threonine-59 is important for Golgi disassembly at mitosis.

FOREWORD: The XXV IAHR Symposium on Hydraulic Machinery and Systems marks half a century tradition

Science.gov (United States)

Susan-Resiga, Romeo

2010-05-01

IAHR75_logoUPT90_logoARFT_logo International Association of Hydro-Environment Engineering and Research'Politehnica' University of TimisoaraRomanian Academy - Timisoara Branch The 25th edition of the IAHR Symposium on Hydraulic Machinery and Systems, held in Timisoara, Romania, 20-24 September 2010, jointly organized by the 'Politehnica' University of Timisoara and the Romanian Academy - Timisoara Branch, marks a half century tradition of these prestigious symposia. However, it is the first time that Romania hosts such a symposium, and for good reasons. The Romanian electrical power system has a total of 20,630 MW installed power, out of which 6,422 MW in hydropower plants. The energy produced in hydropower facilities was in 2008 of 17,105 GWh from a total of 64,772 GWh electrical energy production. Moreover, for the period 2009-2015, new hydropower capacities are going to be developed, with a total of 2,157 MW installed power and an estimated 5,770 GWh/year energy production. Within the same period of time, the refurbishment, modernization and repair programs will increase the actual hydropower production with an estimated 349 GWh/year. The 'Politehnica' University of Timisoara is proud to host the 25th IAHR Symposium on Hydraulic Machinery and Systems, in the year of its 90th anniversary. The 'Politehnica' University of Timisoara is one of the largest and most well-known technical universities from Central and Eastern Europe. It was founded in 1920, a short time after the union into one state of all the Romanian territories, following the end of the First World War, in order to respond to the need engineers felt by the Romanian society at that time, within the economical development framework. During its 90 years of existence, 'Politehnica' University of Timisoara educated over 100,000 engineers, greatly appreciated both in Romania and abroad, for their competence and seriousness. King Ferdinand I of Romania said while visiting the recently established

Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides

Science.gov (United States)

Bertran-Vicente, Jordi; Penkert, Martin; Nieto-Garcia, Olaia; Jeckelmann, Jean-Marc; Schmieder, Peter; Krause, Eberhard; Hackenberger, Christian P. R.

2016-09-01

In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICBGlc, which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes.

Effects of protein phosphorylation on color stability of ground meat.

Science.gov (United States)

Li, Meng; Li, Xin; Xin, Jianzeng; Li, Zheng; Li, Guixia; Zhang, Yan; Du, Manting; Shen, Qingwu W; Zhang, Dequan

2017-03-15

The influence of protein phosphorylation on meat color stability was investigated in this study. Phosphatase and protein kinase inhibitors were added to minced ovine Longissimus thoracis et lumborum (LTL) muscle to manipulate the global phosphorylation of sarcoplasmic proteins. The data obtained show that the rate and extent of pH decline, along with lactate accumulation in postmortem muscle, were related to protein phosphorylation. Analysis of meat color and the relative content of myoglobin redox forms revealed that meat color stability was inversely related to the phosphorylation of sarcoplasmic proteins. Thus, this study suggests that protein phosphorylation may be involved in meat color development by regulating glycolysis and the redox stability of myoglobin. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

Directory of Open Access Journals (Sweden)

Thomas H Sanderson

Full Text Available Recent advancements in isolation techniques for cytochrome c (Cytc have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Designing a machinery control system (MCS) security testbed

OpenAIRE

Desso, Nathan H.

2014-01-01

Approved for public release; distribution is unlimited Industrial control systems (ICS) face daily cyber security threats, can have a significant impact to the security of our nation, and present a difficult challenge to defend. Critical infrastructures, including military systems like the machinery control systems (MCS) found onboard modern U.S. warships, are affected because of their use of commercial automation solutions. The increase of automated control systems within the U.S. Navy sa...

Fault diagnosis in rotating machinery by vibration analysis

International Nuclear Information System (INIS)

Behzad, M.; Asayesh, M.

2002-01-01

Dynamic behavior of unbalanced bent shaft has been investigated in this research. Finite element method is used for unbalance response calculation of a bent shaft. The result shows the effect of bent on the unbalance response. The angle between bent vector and unbalance force, position and type of supports, shaft diameter and disk position can affect the outcome. The results of this research can significantly help in fault diagnosis in rotating machinery

PPARγ1 phosphorylation enhances proliferation and drug resistance in human fibrosarcoma cells

Energy Technology Data Exchange (ETDEWEB)

Pang, Xiaojuan; Shu, Yuxin; Niu, Zhiyuan; Zheng, Wei; Wu, Haochen [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Lu, Yan, E-mail: luyan@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Shen, Pingping, E-mail: ppshen@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Model Animal Research Center (MARC), Nanjing University, Nanjing (China)

2014-03-10

Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) is the most important post-translational modification. The function of PPARγ phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPARγ1 and tumors remains unclear. Here we investigated the role of PPARγ1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPARγ1, the results suggested that phosphorylation attenuated PPARγ1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPARγ1 promoted HT1080 cell proliferation and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21{sup Waf1/Cip1} and p27{sup Kip1} descended in PPARγ1{sup S84D} stable HT1080 cell, whereas the expression of p18{sup INK4C} was not changed. Moreover, compared to the PPARγ1{sup S84A}, PPARγ1{sup S84D} up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPARγ1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPARγ1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPARγ1 could act as anticancer drugs. - Highlights: • Phosphorylation attenuates PPARγ1 transcriptional activity. • Phosphorylated PPARγ1 promotes HT1080 cells proliferation. • PPARγ1 phosphorylation regulates cell cycle by mediating expression of cell cycle regulators. • PPARγ1 phosphorylation reduces sensitivity to agonist and anticancer drug. • Our findings establish PPARγ1 phosphorylation as a critical event in HT1080

Analysis of electric machinery and drive systems

CERN Document Server

Krause, Paul C; Sudhoff, Scott D; Pekarek, Steven

2013-01-01

Introducing a new edition of the popular reference on machine analysis Now in a fully revised and expanded edition, this widely used reference on machine analysis boasts many changes designed to address the varied needs of engineers in the electric machinery, electric drives, and electric power industries. The authors draw on their own extensive research efforts, bringing all topics up to date and outlining a variety of new approaches they have developed over the past decade. Focusing on reference frame theory that has been at the core of this work since the first edition, th

Electrification of agricultural machinery; Elektrifizierung von Landmaschinen

Energy Technology Data Exchange (ETDEWEB)

Goetz, Manuel; Grad, Karl; Weinmann, Olrik [ZF Friedrichshafen AG, Friedrichshafen (Germany)

2012-10-15

As early as 2009, ZF exhibited a generator system for agricultural machinery at Agritechnica under the name Terra+ which consisted of an electric motor in the transmission housing. As part of the ElecTra project, the company is now presenting its first tractor prototype with this generator system at Agritechnica 2011. The project involves combining the electrification of the tractor's auxiliary systems with electric drives for an attachment. The electrification of the implement was carried out in cooperation with Amazone, manufacturer of implements for the agricultural industry. (orig.)

The Machinery for Enforcement of Domestic Arbitral Awards in Nigeria

African Journals Online (AJOL)

Nnamdi Azikiwe University Journal of International Law and Jurisprudence ... Arbitration is a private means of resolving dispute which is resorted to, chiefly because the parties choose to avoid as much as possible employing the state machinery for dispute resolution, namely the court and its dreaded time consuming ...

Guidelines for meeting emission regulations for rotating machinery with mechanical seals

Science.gov (United States)

Mechanical seals have played a major role during many years in minimizing emissions to atmosphere in rotating machinery. A review is presented of the continuing innovations and new technologies to improve mechanical seal performance in terms of emissions.

The in vivo phosphorylation sites of rat brain dynamin I

DEFF Research Database (Denmark)

Graham, Mark E; Anggono, Victor; Bache, Nicolai

2007-01-01

-824). To resolve the discrepancy and to better understand the biological roles of dynI phosphorylation, we undertook a systematic identification of all phosphorylation sites in rat brain nerve terminal dynI. Using phosphoamino acid analysis, exclusively phospho-serine residues were found. Thr(780) phosphorylation...... of their relative abundance and relative responses to depolarization. The multiple phospho-sites suggest subtle regulation of synaptic vesicle endocytosis by new protein kinases and new protein-protein interactions. The homologous dynI and dynIII phosphorylation indicates a high mechanistic similarity. The results...

Industry sector analysis, Mexico: Oil and gas field machinery and equipment. Export Trade Information

International Nuclear Information System (INIS)

1990-04-01

The Industry Sector Analyses (I.S.A.) for oil and gas field machinery and equipment contains statistical and narrative information on projected market demand, end-users, receptivity of Mexican consumers to U.S. products, the competitive situation - Mexican production, total import market, U.S. market position, foreign competition, and competitive factors, and market access - Mexican tariffs, non-tariff barriers, standards, taxes and distribution channels. The I.S.A. provides the United States industry with meaningful information regarding the Mexican market for oil and gas field machinery and equipment

Phosphorylation of mouse serine racemase regulates D-serine synthesis

DEFF Research Database (Denmark)

Foltyn, Veronika N; Zehl, Martin; Dikopoltsev, Elena

2010-01-01

Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator D-serine, which plays a major role in synaptic plasticity and N-methyl D-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis and in v...... with a phosphorylation-deficient SR mutant indicate that Thr71 phosphorylation increases SR activity, suggesting a novel mechanism for regulating D-serine production....

Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

Directory of Open Access Journals (Sweden)

Neil Arvin Bretaña

Full Text Available Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase

Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

Science.gov (United States)

Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

2012-01-01

Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

The floating signifier “talent” in talent management - and why talent management has become a religion

DEFF Research Database (Denmark)

Adamsen, Billy

Within business life the term “talent” has been used by business leaders and consultants for decades to describe upcoming business managers with certain gifts and skills. Within science, especially within talent management, “talent” has been the fulcrum of most research and most research questions...... “talent” has changed semantically, and transformed itself into a floating signifier, and through its multiplicity of meaning, has turned the discipline of talent management into a religion, where believing seems to be more prevalent than knowing. The consequences of talent management being a religion...... have had - for years - focused on how the environment, culture, psychology and competencies influence the “talent”. To a certain extent science today can explain how certain internal and external variables can influence the development and the performance of a “talent”, but they are still unable...

The iron-sulfur cluster assembly machineries in plants: current knowledge and open questions

Science.gov (United States)

Couturier, Jérémy; Touraine, Brigitte; Briat, Jean-François; Gaymard, Frédéric; Rouhier, Nicolas

2013-01-01

Many metabolic pathways and cellular processes occurring in most sub-cellular compartments depend on the functioning of iron-sulfur (Fe-S) proteins, whose cofactors are assembled through dedicated protein machineries. Recent advances have been made in the knowledge of the functions of individual components through a combination of genetic, biochemical and structural approaches, primarily in prokaryotes and non-plant eukaryotes. Whereas most of the components of these machineries are conserved between kingdoms, their complexity is likely increased in plants owing to the presence of additional assembly proteins and to the existence of expanded families for several assembly proteins. This review focuses on the new actors discovered in the past few years, such as glutaredoxin, BOLA and NEET proteins as well as MIP18, MMS19, TAH18, DRE2 for the cytosolic machinery, which are integrated into a model for the plant Fe-S cluster biogenesis systems. It also discusses a few issues currently subjected to an intense debate such as the role of the mitochondrial frataxin and of glutaredoxins, the functional separation between scaffold, carrier and iron-delivery proteins and the crosstalk existing between different organelles. PMID:23898337

Application of Functional Link Artificial Neural Network for Prediction of Machinery Noise in Opencast Mines

Directory of Open Access Journals (Sweden)

Santosh Kumar Nanda

2011-01-01

Full Text Available Functional link-based neural network models were applied to predict opencast mining machineries noise. The paper analyzes the prediction capabilities of functional link neural network based noise prediction models vis-à-vis existing statistical models. In order to find the actual noise status in opencast mines, some of the popular noise prediction models, for example, ISO-9613-2, CONCAWE, VDI, and ENM, have been applied in mining and allied industries to predict the machineries noise by considering various attenuation factors. Functional link artificial neural network (FLANN, polynomial perceptron network (PPN, and Legendre neural network (LeNN were used to predict the machinery noise in opencast mines. The case study is based on data collected from an opencast coal mine of Orissa, India. From the present investigations, it could be concluded that the FLANN model give better noise prediction than the PPN and LeNN model.

Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.

Science.gov (United States)

Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide

2010-07-30

Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway. Copyright 2010 Elsevier Inc. All rights reserved.

Contraction regulates site-specific phosphorylation of TBC1D1 in skeletal muscle.

Science.gov (United States)

Vichaiwong, Kanokwan; Purohit, Suneet; An, Ding; Toyoda, Taro; Jessen, Niels; Hirshman, Michael F; Goodyear, Laurie J

2010-10-15

TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab-GAP (GTPase-activating protein) that is highly expressed in skeletal muscle, but little is known about TBC1D1 regulation and function. We studied TBC1D1 phosphorylation on three predicted AMPK (AMP-activated protein kinase) phosphorylation sites (Ser231, Ser660 and Ser700) and one predicted Akt phosphorylation site (Thr590) in control mice, AMPKα2 inactive transgenic mice (AMPKα2i TG) and Akt2-knockout mice (Akt2 KO). Muscle contraction significantly increased TBC1D1 phosphorylation on Ser231 and Ser660, tended to increase Ser700 phosphorylation, but had no effect on Thr590. AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) also increased phosphorylation on Ser231, Ser660 and Ser700, but not Thr590, whereas insulin only increased Thr590 phosphorylation. Basal and contraction-stimulated TBC1D1 Ser231, Ser660 and Ser700 phosphorylation were greatly reduced in AMPKα2i TG mice, although contraction still elicited a small increase in phosphorylation. Akt2 KO mice had blunted insulin-stimulated TBC1D1 Thr590 phosphorylation. Contraction-stimulated TBC1D1 Ser231 and Ser660 phosphorylation were normal in high-fat-fed mice. Glucose uptake in vivo was significantly decreased in tibialis anterior muscles overexpressing TBC1D1 mutated on four predicted AMPK phosphorylation sites. In conclusion, contraction causes site-specific phosphorylation of TBC1D1 in skeletal muscle, and TBC1D1 phosphorylation on AMPK sites regulates contraction-stimulated glucose uptake. AMPK and Akt regulate TBC1D1 phosphorylation, but there must be additional upstream kinases that mediate TBC1D1 phosphorylation in skeletal muscle.

Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

Energy Technology Data Exchange (ETDEWEB)

Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly, E-mail: anatoly_zhitkovich@brown.edu

2015-07-15

Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in

Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A)

International Nuclear Information System (INIS)

Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

2017-01-01

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. - Highlights: • We investigated the mechanism regulating subcellular localization of CDKL5. • DYRK1A was identified as an enzyme that bound to and phosphorylated CDKL5. • The phosphorylation site of CDKL5 was Ser-308, in the vicinity of the NLS. • When DYRK1A was co-expressed, the cytosolic CDKL5 was significantly increased. • In conclusion, DYRK1A regulates CDKL5 localization via phosphorylation on Ser-308.

DIESEL ENGINE SYSTEMS. AGRICULTURAL MACHINERY--SERVICE OCCUPATIONS, MODULE NUMBER 15.

Science.gov (United States)

Ohio State Univ., Columbus. Center for Vocational and Technical Education.

ONE OF A SERIES DESIGNED TO HELP TEACHERS PREPARE POSTSECONDARY STUDENTS FOR AGRICULTURAL MACHINERY SERVICE OCCUPATIONS AS PARTS MEN, MECHANICS, MECHANIC'S HELPERS, AND SERVICE SUPERVISORS, THIS GUIDE AIMS TO DEVELOP STUDENT UNDERSTANDING OF THE CONSTRUCTION AND OPERATING PRINCIPLES OF DIESEL ENGINES. IT WAS DEVELOPED BY A NATIONAL TASK FORCE ON…

Raptor is phosphorylated by cdc2 during mitosis.

Directory of Open Access Journals (Sweden)

Dana M Gwinn

2010-02-01

Full Text Available The appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1. As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process.We have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells.This study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct

Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

Science.gov (United States)

Meiler, Eugenia; Nieto-Pelegrín, Elvira; Martinez-Quiles, Narcisa

2012-01-01

Background Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes. Methodology/Principal Findings In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation. Conclusions/Significance Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results from the FIT system by examining endogenous cortactin in different cell types. Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading. PMID:22479425

Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants

Directory of Open Access Journals (Sweden)

Slavka eBekesova

2015-05-01

Full Text Available The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn2+ or Zn2+ used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™ offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies.

PKCδ-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function

International Nuclear Information System (INIS)

Greene, Michael W.; Ruhoff, Mary S.; Roth, Richard A.; Kim, Jeong-a; Quon, Michael J.; Krause, Jean A.

2006-01-01

The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKCδ on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKCδ-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKCδ catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1

Updating the model TREMOD - Mobile Machinery (TREMOD-MM); Aktualisierung des Modells TREMOD - Mobile Machinery (TREMOD-MM)

Energy Technology Data Exchange (ETDEWEB)

Helms, Hinrich; Lambrecht, Udo; Knoerr, Wolfram [ifeu - Institut fuer Energie- und Umweltforschung Heidelberg gGmbH, Heidelberg (Germany)

2010-05-15

In the context of the project ''Development of a model for the computation of the air pollutant emissions and the fuel consumption of combustion engines in mobile devices and machines'', the Institute for Energy and Environmental Research GmbH (Heidelberg, Federal Republic of Germany) has created the model TREMOD-MM (TREMOD Mobile Machinery). Thus a detailed computation of the emissions from mobile devices and machines in the agriculture, construction industry, forestry and gardening as well as the sport shipping and passenger shipping can be accomplished. Strongly differentiated data are considered to the age structure, engine performance, use and emission behaviour. Thus it is possible to compute the emissions for different scenarios in high degree of detail.

Axin-mediated CKI phosphorylation of beta-catenin at Ser 45

DEFF Research Database (Denmark)

Amit, Sharon; Hatzubai, Ada; Birman, Yaara

2002-01-01

The Wnt pathway controls numerous developmental processes via the beta-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of beta-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic beta-catenin associates with APC and axin...... and is continuously phosphorylated by GSK-3beta, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3beta from phosphorylating beta-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of beta......-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces beta-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate beta...

Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

DEFF Research Database (Denmark)

Mijakovic, Ivan; Petranovic, Dina; Macek, B

2006-01-01

for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (Sc......SSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically...... by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle.

Science.gov (United States)

Copeland, O'Neal; Sadayappan, Sakthivel; Messer, Andrew E; Steinen, Ger J M; van der Velden, Jolanda; Marston, Steven B

2010-12-01

A unique feature of MyBP-C in cardiac muscle is that it has multiple phosphorylation sites. MyBP-C phosphorylation, predominantly by PKA, plays an essential role in modulating contractility as part of the cellular response to β-adrenergic stimulation. In vitro studies indicate MyBP-C can be phosphorylated at Serine 273, 282, 302 and 307 (mouse sequence) but little is known about the level of MyBP-C phosphorylation or the sites phosphorylated in heart muscle. Since current methodologies are limited in specificity and are not quantitative we have investigated the use of phosphate affinity SDS-PAGE together with a total anti MyBP-C antibody and a range of phosphorylation site-specific antibodies for the main sites (Ser-273, -282 and -302). With these newly developed methods we have been able to make a detailed quantitative analysis of MyBP-C phosphorylation in heart tissue in situ. We have found that MyBP-C is highly phosphorylated in non-failing human (donor) heart or mouse heart; tris and tetra-phosphorylated species predominate and less than 10% of MyBP-C is unphosphorylated (0, 9.3 ± 1%: 1P, 13.4 ± 2.7%: 2P, 10.5 ± 3.3%: 3P, 28.7 ± 3.7%: 4P, 36.4 ± 2.7%, n=21). Total phosphorylation was 2.7 ± 0.07 mol Pi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C was unphosphorylated. Total phosphorylation levels were 23% of normal in failing heart myofibrils (0, 60.1 ± 2.8%: 1P, 27.8 ± 2.8%: 2P, 4.8 ± 2.0%: 3P, 3.7 ± 1.2%: 4P, 2.8 ± 1.3%, n=19) and 39% of normal in myectomy samples. The site-specific antibodies showed a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser-273, Ser-282 and Ser-302 were all present in the 4P band of MyBP-C but none of them were significant in the 1P band, indicating that there must be at least one other site of MyBP-C phosphorylation in human heart. The pattern of phosphorylation at the

Phosphorylation of αB-crystallin: Role in stress, aging and patho-physiological conditions.

Science.gov (United States)

Bakthisaran, Raman; Akula, Kranthi Kiran; Tangirala, Ramakrishna; Rao, Ch Mohan

2016-01-01

αB-crystallin, once thought to be a lenticular protein, is ubiquitous and has critical roles in several cellular processes that are modulated by phosphorylation. Serine residues 19, 45 and 59 of αB-crystallin undergo phosphorylation. Phosphorylation of S45 is mediated by p44/42 MAP kinase, whereas S59 phosphorylation is mediated by MAPKAP kinase-2. Pathway involved in S19 phosphorylation is not known. The review highlights the role of phosphorylation in (i) oligomeric structure, stability and chaperone activity, (ii) cellular processes such as apoptosis, myogenic differentiation, cell cycle regulation and angiogenesis, and (iii) aging, stress, cardiomyopathy-causing αB-crystallin mutants, and in other diseases. Depending on the context and extent of phosphorylation, αB-crystallin seems to confer beneficial or deleterious effects. Phosphorylation alters structure, stability, size distribution and dynamics of the oligomeric assembly, thus modulating chaperone activity and various cellular processes. Phosphorylated αB-crystallin has a tendency to partition to the cytoskeleton and hence to the insoluble fraction. Low levels of phosphorylation appear to be protective, while hyperphosphorylation has negative implications. Mutations in αB-crystallin, such as R120G, Q151X and 464delCT, associated with inherited myofibrillar myopathy lead to hyperphosphorylation and intracellular inclusions. An ongoing study in our laboratory with phosphorylation-mimicking mutants indicates that phosphorylation of R120GαB-crystallin increases its propensity to aggregate. Phosphorylation of αB-crystallin has dual role that manifests either beneficial or deleterious consequences depending on the extent of phosphorylation and interaction with cytoskeleton. Considering that disease-causing mutants of αB-crystallin are hyperphosphorylated, moderation of phosphorylation may be a useful strategy in disease management. This article is part of a Special Issue entitled Crystallin

PhosphoRice: a meta-predictor of rice-specific phosphorylation sites

Directory of Open Access Journals (Sweden)

Que Shufu

2012-02-01

Full Text Available Abstract Background As a result of the growing body of protein phosphorylation sites data, the number of phosphoprotein databases is constantly increasing, and dozens of tools are available for predicting protein phosphorylation sites to achieve fast automatic results. However, none of the existing tools has been developed to predict protein phosphorylation sites in rice. Results In this paper, the phosphorylation site predictors, NetPhos 2.0, NetPhosK, Kinasephos, Scansite, Disphos and Predphosphos, were integrated to construct meta-predictors of rice-specific phosphorylation sites using several methods, including unweighted voting, unreduced weighted voting, reduced unweighted voting and weighted voting strategies. PhosphoRice, the meta-predictor produced by using weighted voting strategy with parameters selected by restricted grid search and conditional random search, performed the best at predicting phosphorylation sites in rice. Its Matthew's Correlation Coefficient (MCC and Accuracy (ACC reached to 0.474 and 73.8%, respectively. Compared to the best individual element predictor (Disphos_default, PhosphoRice archieved a significant increase in MCC of 0.071 (P Conclusions PhosphoRice is a powerful tool for predicting unidentified phosphorylation sites in rice. Compared to the existing methods, we found that our tool showed greater robustness in ACC and MCC. PhosphoRice is available to the public at http://bioinformatics.fafu.edu.cn/PhosphoRice.

Hybridisation of powertrains for construction machinery; Hybridisierung von Antriebsstraengen fuer Baumaschinen

Energy Technology Data Exchange (ETDEWEB)

Mohr, Mark; Goetz, Manuel; Fellmann, Martin [ZF Friedrichshafen AG (Germany). Vorentwicklung Arbeitsmaschinen-Antriebe; Brehmer, Udo [ZF Passau GmbH (Germany). Entwicklung Baumaschinengetriebe

2010-04-15

Increasing fuel prices and increased environmental requirements lead, for construction machinery, to an increased importance of measures to reduce fuel consumption. The hybrid drive in this sector could become an economical option by using synergies for example with the commercial vehicle section. (orig.)

Energy-autonomous wireless vibration sensor for condition-based maintenance of machinery

NARCIS (Netherlands)

Wang, Z.; Bouwens, F.; Vullers, R.; Petré, F.; Devos, S.

2011-01-01

This paper addresses the development of an energy-autonomous wireless vibration sensor for condition-based monitoring of machinery. Such technology plays an increasingly important role in modern manufacturing industry. In this work, energy harvesting is realized by resorting to a custom designed

Cell secretion machinery: Studies using the AFM

International Nuclear Information System (INIS)

Jena, Bhanu P.

2006-01-01

A new field in biology, 'nano-cell biology', has emerged from the successful use of force microscopy in understanding the structure and dynamics of cells and biomolecules, at nm resolution and in real time. Atomic force microscopy, in combination with conventional tools and approaches (electron microscopy, electrophysiology, X-ray diffraction, photon correlation spectroscopy, mass spectroscopy, biochemistry, and molecular biology), has revealed for the first time, the universal molecular machinery and mechanism of secretion in cells. Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are packaged and stored in membranous sacs or vesicles within the cell. When the cell needs to secrete these products, the secretory vesicles containing them, dock and fuse at plasma membrane-associated supramolecular structures called Porosome, to release their contents. Specialized cells for neurotransmission, enzyme secretion, or hormone release utilize a highly regulated secretory process. During secretion, swelling of secretory vesicles results in a build-up of intravesicular pressure, allowing expulsion of vesicular contents. The extent of vesicle swelling dictates the amount of vesicular contents expelled. The discovery of the porosome as the universal secretory machinery, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution into artificial lipid membrane, have been determined. The molecular mechanism of secretory vesicle swelling, and the fusion of opposing bilayers, i.e., the fusion of secretory vesicle membrane at the base of the porosome membrane, has also been resolved

Motion control of multi-actuator hydraulic systems for mobile machineries: Recent advancements and future trends

Science.gov (United States)

Xu, Bing; Cheng, Min

2018-06-01

This paper presents a survey of recent advancements and upcoming trends in motion control technologies employed in designing multi-actuator hydraulic systems for mobile machineries. Hydraulic systems have been extensively used in mobile machineries due to their superior power density and robustness. However, motion control technologies of multi-actuator hydraulic systems have faced increasing challenges due to stringent emission regulations. In this study, an overview of the evolution of existing throttling control technologies is presented, including open-center and load sensing controls. Recent advancements in energy-saving hydraulic technologies, such as individual metering, displacement, and hybrid controls, are briefly summarized. The impact of energy-saving hydraulic technologies on dynamic performance and control solutions are also discussed. Then, the advanced operation methods of multi-actuator mobile machineries are reviewed, including coordinated and haptic controls. Finally, challenges and opportunities of advanced motion control technologies are presented by providing an overall consideration of energy efficiency, controllability, cost, reliability, and other aspects.

Robust IFE Based Order Analysis of Rotating Machinery in Virtual Instrument

International Nuclear Information System (INIS)

Guo, Y; Chi, Y L; Huang, Y Y; Qin, S R

2006-01-01

Character analysis plays an important role in fault-find and diagnosis of rotating machinery. Order analysis is one of the major methods in character analysis for the analysis of non-stationary vibration signals in run-up or coast down of rotating machinery. An order analysis method, which employs instantaneous frequency estimation based on time-frequency analysis, is introduced. In contrast with traditional order analysis methods, this method avoids the use of tachometer and other special hardware; hence it makes the application of order analysis simplified. The order analysis introduced in the paper with the character that only software is depended for order tracking makes it specially satisfy the requirement of Virtual Instruments. Corresponding order analysis items, such as order spectrum, order spectrum matrix and tracking order spectrum etc., which are applied in Virtual Instruments, are also introduced. A test example is provided to demonstrate the validity of the method presented

Calcium-regulated in vivo protein phosphorylation in Zea mays L. root tips

Science.gov (United States)

Raghothama, K. G.; Reddy, A. S.; Friedmann, M.; Poovaiah, B. W.

1987-01-01

Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(beta-aminoethyl ether)-N-N' -tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with 32P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.

Mediator phosphorylation prevents stress response transcription during non-stress conditions.

Science.gov (United States)

Miller, Christian; Matic, Ivan; Maier, Kerstin C; Schwalb, Björn; Roether, Susanne; Strässer, Katja; Tresch, Achim; Mann, Matthias; Cramer, Patrick

2012-12-28

The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.

Cereal bioengineering: Amylopectin-free and hyper-phosphorylated barley starch

DEFF Research Database (Denmark)

Carciofi, Massimiliano; Shaik, Shahnoor Sultana; Jensen, Susanne Langgård

Barley lines producing grains with either amylopectin-free or hyper-phosphorylated starches were made by transgenic methods. Cereals producing these kind of starches have not been reported before. Amylopectin-free barley was generated by simultaneously silencing the three genes encoding the starch...... and T1) of transgenic grains was tenfold higher than from vector control and wild type grains. Amylose content was not affected in hyper-phosphorylated grains. Hyper-phosphorylated starch granules had several pores on the surfaces, similar to pores seen on enzymatically semi-degraded granules...

Cereal bioengineering: Amylopectin-free and hyper-phosphorylated barley starch

DEFF Research Database (Denmark)

Carciofi, Massimiliano; Shaik, Shahnoor Sultana; Jensen, Susanne Langgård

2011-01-01

Barley lines producing grains with either amylopectin-free or hyper-phosphorylated starches were made by transgenic methods. Cereals producing these kind of starches have not been reported before. Amylopectin-free barley was generated by simultaneously silencing the three genes encoding the starch...... and T1) of transgenic grains was tenfold higher than from vector control and wild type grains. Amylose content was not affected in hyper-phosphorylated grains. Hyper-phosphorylated starch granules had several pores on the surfaces, similar to pores seen on enzymatically semi-degraded granules...

Infancia, significante en falta de significación Infância, significante em falta de significação Childhood, a signifier lacking significations

Directory of Open Access Journals (Sweden)

Mercedes Minnicelli

2009-04-01

Full Text Available Considerar o termo "infância" como um "significante sempre em falta de significação" permite-nos uma rede argumentativa sustentada no discurso psicanalítico, segundo a perspectiva de Giorgio Agamben, que coloca a infância em relação à linguagem. Ponto de partida para fazer a seguinte tese: A infância - como significante - é, para a linguagem, o que o desejo é para a lei. Infância foi, é e será um significante sempre em falta de significação, instituindo-se pela linguagem e no campo da linguagem. O estatuto da legalidade subjetiva deve distinguir-se do código jurídico para poder colocar a tensão possível entre um e outro. É preciso esclarecer o sentido que damos ao termo instituição, distinguindo-o do sentido jurídico que ele recebeu e ainda conserva, propondo recuperar seu valor em termos de escritura da lei na constituição subjetiva, marco epistêmico que nos brinda a psicanálise.Considering the term "childhood" as a "signifier always lacking signification" enables us to establish an argumentative network supported by the psychoanalytical discourse. According to Giorgio Agamben, childhood is related to language, and this is the starting point for the following thesis: childhood - as a signifier - stands for language as desire stands for law. It was, is and will be a signifier always in lack of signification, establishing itself by language and in the field of language. The statute of the subjective legality must be distinguished from the judicial code enabling the enactment of a possible tension between them. The meaning given to the term institution must be cleared up: it should be distinguished from the legal sense it has been given and still retains, proposing a retrieval of its value in terms of the writing of law as subjective constitution, an epistemic landmark raised by psychoanalysis.

Signifying quantum benchmarks for qubit teleportation and secure quantum communication using Einstein-Podolsky-Rosen steering inequalities

Science.gov (United States)

Reid, M. D.

2013-12-01

The demonstration of quantum teleportation of a photonic qubit from Alice to Bob usually relies on data conditioned on detection at Bob's location. I show that Bohm's Einstein-Podolsky-Rosen (EPR) paradox can be used to verify that the quantum benchmark for qubit teleportation has been reached, without postselection. This is possible for scenarios insensitive to losses at the generation station, and with efficiencies of ηB>1/3 for the teleportation process. The benchmark is obtained if it is shown that Bob can “steer” Alice's record of the qubit as stored by Charlie. EPR steering inequalities involving m measurement settings can also be used to confirm quantum teleportation, for efficiencies ηB>1/m, if one assumes trusted detectors for Charlie and Alice. Using proofs of monogamy, I show that two-setting EPR steering inequalities can signify secure teleportation of the qubit state.

Research on monitoring technology of axial gap change about high-speed rotating machinery

International Nuclear Information System (INIS)

Zhang Xiaochan; Liu Fanglei; Hu Shihua; Xie Qing; Li Zhen

2014-01-01

This paper describes that the only measuring point of high-speed rotating machinery (speed monitoring transducer) measuring the operation of the axial gap change and application. According to mechanism analysis the speed monitoring transducer's signal, prove its amplitude changes including the axial gap change information. To carry out the speed monitoring transducer qualitative and quantitative axial gap change research, Find the output signal amplitude and clearance change corresponding relationship formula of speed monitoring transducer, define the measurement method. Based on the above analsis, manufacture the single channel measurement devices and multiple unit measurement system, provide an important fault decision of high-speed rotating machinery, it can be applied to new equipment development and production. (authors)

Fault Severity Estimation of Rotating Machinery Based on Residual Signals

Directory of Open Access Journals (Sweden)

Fan Jiang

2012-01-01

Full Text Available Fault severity estimation is an important part of a condition-based maintenance system, which can monitor the performance of an operation machine and enhance its level of safety. In this paper, a novel method based on statistical property and residual signals is developed for estimating the fault severity of rotating machinery. The fast Fourier transformation (FFT is applied to extract the so-called multifrequency-band energy (MFBE from the vibration signals of rotating machinery with different fault severity levels in the first stage. Usually these features of the working conditions with different fault sensitivities are different. Therefore a sensitive features-selecting algorithm is defined to construct the feature matrix and calculate the statistic parameter (mean in the second stage. In the last stage, the residual signals computed by the zero space vector are used to estimate the fault severity. Simulation and experimental results reveal that the proposed method based on statistics and residual signals is effective and feasible for estimating the severity of a rotating machine fault.

Effects of high-gradient magnetic fields on living cell machinery

International Nuclear Information System (INIS)

Zablotskii, V; Lunov, O; Kubinova, S; Polyakova, T; Dejneka, A; Sykova, E

2016-01-01

A general interest in biomagnetic effects is related to fundamental studies of the influence of magnetic fields on living objects on the cellular and whole organism levels. Emerging technologies offer new directions for the use of high-gradient magnetic fields to control cell machinery and to understand the intracellular biological processes of the emerging field of nanomedicine. In this review we aim at highlighting recent advances made in identifying fundamental mechanisms by which magnetic gradient forces act on cell fate specification and cell differentiation. The review also provides an analysis of the currently available magnetic systems capable of generating magnetic fields with spatial gradients of up to 10 MT m −1 , with the focus on their suitability for use in cell therapy. Relationships between experimental factors and underlying biophysical mechanisms and assumptions that would ultimately lead to a deeper understanding of cell machinery and the development of more predictive models for the evaluation of the effects of magnetic fields on cells, tissue and organisms are comprehensively discussed. (topical review)

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

Science.gov (United States)

Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

2005-01-01

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

Inhibition of peptide aggregation by means of enzymatic phosphorylation

Directory of Open Access Journals (Sweden)

Kristin Folmert

2016-11-01

Full Text Available As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP-dependent protein kinase that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β-sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated.

Vibration sensor data denoising using a time-frequency manifold for machinery fault diagnosis.

Science.gov (United States)

He, Qingbo; Wang, Xiangxiang; Zhou, Qiang

2013-12-27

Vibration sensor data from a mechanical system are often associated with important measurement information useful for machinery fault diagnosis. However, in practice the existence of background noise makes it difficult to identify the fault signature from the sensing data. This paper introduces the time-frequency manifold (TFM) concept into sensor data denoising and proposes a novel denoising method for reliable machinery fault diagnosis. The TFM signature reflects the intrinsic time-frequency structure of a non-stationary signal. The proposed method intends to realize data denoising by synthesizing the TFM using time-frequency synthesis and phase space reconstruction (PSR) synthesis. Due to the merits of the TFM in noise suppression and resolution enhancement, the denoised signal would have satisfactory denoising effects, as well as inherent time-frequency structure keeping. Moreover, this paper presents a clustering-based statistical parameter to evaluate the proposed method, and also presents a new diagnostic approach, called frequency probability time series (FPTS) spectral analysis, to show its effectiveness in fault diagnosis. The proposed TFM-based data denoising method has been employed to deal with a set of vibration sensor data from defective bearings, and the results verify that for machinery fault diagnosis the method is superior to two traditional denoising methods.

DETERMINATION OF AGRICULTURAL MACHINERY OPERATORS’ OPINIONS ABOUT THE CABIN COMFORT IN ESKİŞEHİR

Directory of Open Access Journals (Sweden)

Özge ACARBAŞ BALTACI

2015-08-01

Full Text Available Comfort has a great importance in the interior design of tractor and agricultural machinery cabins. Operators are exposed to muscoskeletal system disorders since they spend long time periods during the day in these vehicles. There is a few work in the literature reporting operators’ opinions about cabin comfort of these machineries. In this study, a questionnaire was conducted in order to get information about agricultural machinery operators’ opinions about the comfort of their vehicles. Tractor cabins and combine harvester machine cabins were selected as machineries. The study was conducted in Eskişehir in Turkey. Questionnaire was composed of four groups of questions and five ordered response levels were used in the Likert's scale. Demographic questions, general questions about the machine, personal evaluation questions and open ended questions were asked to the operators. After the questionnaire completed, collected data were classified according to the machine type. Frequency tables were used to present the results. Visibility and the accessibility were the most satisfied issues for the tractor operators with 55.9% and 55.4% percentages, respectively. Seat comfort has the highest satisfaction degree with 43.7% for the combine harvester operators. Cronbach Alpha reliability coefficient was used for the satisfaction questions in the applied questionnaire. The reliability of the study was high with coefficients of 0.878 and 0.940 for the tractor and combine harvester questionnaires, respectively. This study will support design and development process of new products by considering operator opinions.

Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB.

Directory of Open Access Journals (Sweden)

Andaleeb Sajid

2011-03-01

Full Text Available The integrated functions of 11 Ser/Thr protein kinases (STPKs and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP is regulated through phosphorylation by STPKs.PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn(2+-ions and inorganic phosphate (Pi. PstP is differentially phosphorylated on the cytosolic domain with Thr(137, Thr(141, Thr(174 and Thr(290 being the target residues of PknB while Thr(137 and Thr(174 are phosphorylated by PknA. The Mn(2+-ion binding residues Asp(38 and Asp(229 are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP(c (D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn(2+/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.

Conflicting views on the membrane fusion machinery and the fusion pore

DEFF Research Database (Denmark)

Sørensen, Jakob B

2009-01-01

of the assembly of the fusogenic SNARE-complex. Here, I review conflicting views on the function of the core fusion machinery consisting of the SNAREs, Munc18, complexin, and synaptotagmin. Munc18 controls docking of vesicles to the plasma membrane and initial SNARE-complex assembly, whereas complexin...

Tampering with springs: phosphorylation of titin affecting the mechanical function of cardiomyocytes.

Science.gov (United States)

Hamdani, Nazha; Herwig, Melissa; Linke, Wolfgang A

2017-06-01

Reversible post-translational modifications of various cardiac proteins regulate the mechanical properties of the cardiomyocytes and thus modulate the contractile performance of the heart. The giant protein titin forms a continuous filament network in the sarcomeres of striated muscle cells, where it determines passive tension development and modulates active contraction. These mechanical properties of titin are altered through post-translational modifications, particularly phosphorylation. Titin contains hundreds of potential phosphorylation sites, the functional relevance of which is only beginning to emerge. Here, we provide a state-of-the-art summary of the phosphorylation sites in titin, with a particular focus on the elastic titin spring segment. We discuss how phosphorylation at specific amino acids can reduce or increase the stretch-induced spring force of titin, depending on where the spring region is phosphorylated. We also review which protein kinases phosphorylate titin and how this phosphorylation affects titin-based passive tension in cardiomyocytes. A comprehensive overview is provided of studies that have measured altered titin phosphorylation and titin-based passive tension in myocardial samples from human heart failure patients and animal models of heart disease. As our understanding of the broader implications of phosphorylation in titin progresses, this knowledge could be used to design targeted interventions aimed at reducing pathologically increased titin stiffness in patients with stiff hearts.

Technical development of fluid machinery area

International Nuclear Information System (INIS)

Chung, Kyung Nam; Kim, Jin Young; Kim, Yang Ik

2008-01-01

In this paper, recent research activity of Hyundai Heavy Industries in the fluid machinery area is introduced. Technical development has been carried out in pumps, turbines, construction equipment, side thrusters, engine lubrication flow, etc. Here the technology of pumps and cooling of construction equipment will be dealt with. We have actively used computational fluid dynamics in the performance analysis of pump models and the design of new models of various industrial pumps or marine pumps. And a cooling analysis system composed of 3D flow analysis and 1D cooling analysis has been established, and applied to the design of cooling systems of new models of excavators and wheel loaders. The above mentioned technology is presented in details, and some future works are mentioned

The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation.

Science.gov (United States)

Beavis, A D; Lehninger, A L

1986-07-15

Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.

Haloperidol Regulates the State of Phosphorylation of Ribosomal Protein S6 via Activation of PKA and Phosphorylation of DARPP-32

Science.gov (United States)

Valjent, Emmanuel; Bertran-Gonzalez, Jesus; Bowling, Heather; Lopez, Sébastien; Santini, Emanuela; Matamales, Miriam; Bonito-Oliva, Alessandra; Hervé, Denis; Hoeffer, Charles; Klann, Eric; Girault, Jean-Antoine; Fisone, Gilberto

2011-01-01

Administration of typical antipsychotic drugs, such as haloperidol, promotes cAMP-dependent signaling in the medium spiny neurons (MSNs) of the striatum. In this study, we have examined the effect of haloperidol on the state of phosphorylation of the ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit. We found that haloperidol increases the phosphorylation of rpS6 at the dual site Ser235/236, which is involved in the regulation of mRNA translation. This effect was exerted in the MSNs of the indirect pathway, which express specifically dopamine D2 receptors (D2Rs) and adenosine A2 receptors (A2ARs). The effect of haloperidol was decreased by blockade of A2ARs or by genetic attenuation of the Gαolf protein, which couples A2ARs to activation of adenylyl cyclase. Moreover, stimulation of cAMP-dependent protein kinase A (PKA) increased Ser235/236 phosphorylation in cultured striatal neurons. The ability of haloperidol to promote rpS6 phosphorylation was abolished in knock-in mice deficient for PKA activation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa. In contrast, pharmacological or genetic inactivation of p70 rpS6 kinase 1, or extracellular signal-regulated kinases did not affect haloperidol-induced rpS6 phosphorylation. These results identify PKA as a major rpS6 kinase in neuronal cells and suggest that regulation of protein synthesis through rpS6 may be a potential target of antipsychotic drugs. PMID:21814187

Recent Advances in Precision Machinery and Manufacturing Technology

DEFF Research Database (Denmark)

Liu, Chien-Hung; Hsieh, Wen-Hsiang; Chang, Zong-Yu

2014-01-01

Precision machinery and manufacturing technology are be- coming more important in current and future technologies. New knowledge in this field will aid in the advancement of various technologies that are needed to gain industrial competitiveness. To this end, the special issue aims to disseminate...... the latest advancements of relevant fundamental and applied research works of high quality to the inter- national community. The topics of the accepted articles in the special issue include precision manufacturing pro- cesses, measurements and control, robotics and automation, machine tools, advanced...

Construction and Deciphering of Human Phosphorylation-Mediated Signaling Transduction Networks.

Science.gov (United States)

Zhang, Menghuan; Li, Hong; He, Ying; Sun, Han; Xia, Li; Wang, Lishun; Sun, Bo; Ma, Liangxiao; Zhang, Guoqing; Li, Jing; Li, Yixue; Xie, Lu

2015-07-02

Protein phosphorylation is the most abundant reversible covalent modification. Human protein kinases participate in almost all biological pathways, and approximately half of the kinases are associated with disease. PhoSigNet was designed to store and display human phosphorylation-mediated signal transduction networks, with additional information related to cancer. It contains 11 976 experimentally validated directed edges and 216 871 phosphorylation sites. Moreover, 3491 differentially expressed proteins in human cancer from dbDEPC, 18 907 human cancer variation sites from CanProVar, and 388 hyperphosphorylation sites from PhosphoSitePlus were collected as annotation information. Compared with other phosphorylation-related databases, PhoSigNet not only takes the kinase-substrate regulatory relationship pairs into account, but also extends regulatory relationships up- and downstream (e.g., from ligand to receptor, from G protein to kinase, and from transcription factor to targets). Furthermore, PhoSigNet allows the user to investigate the impact of phosphorylation modifications on cancer. By using one set of in-house time series phosphoproteomics data, the reconstruction of a conditional and dynamic phosphorylation-mediated signaling network was exemplified. We expect PhoSigNet to be a useful database and analysis platform benefiting both proteomics and cancer studies.

Cholinergic regulation of protein phosphorylation in bovine adrenal chromaffin cells

International Nuclear Information System (INIS)

Haycock, J.W.; Browning, M.D.; Greengard, P.

1988-01-01

Chromaffin cells were isolated from bovine adrenal medullae and maintained in primary culture. After prelabeling with 32 PO 4 , exposure of the chromaffin cells to acetylcholine increased the phosphorylation of a M/sub r/ ≅ 100,000 protein and a M/sub r/ ≅ 60,000 protein (tyrosine hydroxylase), visualized after separation of total cellular proteins in NaDodSO 4 /polyacrylamide gels. Immunoprecipitation with antibodies to three known phosphoproteins (100-kDa, 87-kDa, and protein III) revealed an acetylcholine-dependent phosphorylation of these proteins. These three proteins were also shown to be present in bovine adrenal chromaffin cells by immunolabeling techniques. 100-kDa is a M/sub r/ ≅ 100,000 protein selectively phosphorylated by calcium/calmodulin-dependent protein kinase III, 87-kDa is a M/sub r/ ≅ 87,000 protein selectively phosphorylated by protein kinase C, and protein III is a phosphoprotein doublet of M/sub r/ ≅ 74,000 (IIIa) and M/sub r/ ≅ 55,000 (IIIb) phosphorylated by cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase I. The data demonstrate that cholinergic activation of chromaffin cells increases the phosphorylation of several proteins and that several protein kinase systems may be involved in these effects

A Novel Fault Diagnosis Method for Rotating Machinery Based on a Convolutional Neural Network.

Science.gov (United States)

Guo, Sheng; Yang, Tao; Gao, Wei; Zhang, Chen

2018-05-04

Fault diagnosis is critical to ensure the safety and reliable operation of rotating machinery. Most methods used in fault diagnosis of rotating machinery extract a few feature values from vibration signals for fault diagnosis, which is a dimensionality reduction from the original signal and may omit some important fault messages in the original signal. Thus, a novel diagnosis method is proposed involving the use of a convolutional neural network (CNN) to directly classify the continuous wavelet transform scalogram (CWTS), which is a time-frequency domain transform of the original signal and can contain most of the information of the vibration signals. In this method, CWTS is formed by discomposing vibration signals of rotating machinery in different scales using wavelet transform. Then the CNN is trained to diagnose faults, with CWTS as the input. A series of experiments is conducted on the rotor experiment platform using this method. The results indicate that the proposed method can diagnose the faults accurately. To verify the universality of this method, the trained CNN was also used to perform fault diagnosis for another piece of rotor equipment, and a good result was achieved.

An unusual protein kinase phosphorylates the chemotactic receptor of Dictystelium discoideum

International Nuclear Information System (INIS)

Meier, K.; Klein, C.

1988-01-01

The authors report the cAMP-dependent phosphorylation of the chemotactic receptor of Dictyostelium discoideum in partially purified plasma membranes. The protein kinase responsible for receptor phosphorylation is associated with this fraction and preferentially phosphorylates the ligand-occupied form of the receptor. 8-Azido[ 32 P]cAMP labeling of the cell surface has shown that the cAMP receptor exists in two forms. A 45-kDa protein is predominant on unstimulated cells. cAMP stimulation results in an increased receptor phosphorylation such that the receptor migrates on NaDodSO 4 /PAGE as a 47-kDa protein. Phosphorylation of the chemotactic receptor is not detected in membrane preparations unless cAMP is added to the incubation mixture. Only under those conditions is the phosphorylated 47-kDa form observed. The requirement for cAMP reflects the fact that the kinase involved preferentially uses the ligand-occupied receptor as a substrate. In vitro phosphorylation of the receptor does not involve tyrosine residues. The enzyme does not appear to be a cAMP- or cGMP-dependent protein kinase nor is it sensitive to guanine nucleotides, Ca 2+ /calmodulin, Ca 2+ /phospholipid, or EGTA. Similarities with the β-adrenergic receptor protein kinase are discussed

Bad phosphorylation as a target of inhibition in oncology.

Science.gov (United States)

Bui, Ngoc-Linh-Chi; Pandey, Vijay; Zhu, Tao; Ma, Lan; Basappa; Lobie, Peter E

2018-02-28

Bcl-2 agonist of cell death (BAD) is a BH3-only member of the Bcl-2 family which possesses important regulatory function in apoptosis. BAD has also been shown to possess many non-apoptotic functions closely linked to cancer including regulation of glycolysis, autophagy, cell cycle progression and immune system development. Interestingly, BAD can be either pro-apoptotic or pro-survival depending on the phosphorylation state of three specific serine residues (human S75, S99 and S118). Expression of BAD and BAD phosphorylation patterns have been shown to influence tumor initiation and progression and play a predictive role in disease prognosis, drug response and chemosensitivity in various cancers. This review aims to summarize the current evidence on the functional role of BAD phosphorylation in human cancer and evaluate the potential utility of modulating BAD phosphorylation in cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Outer membrane targeting of Pseudomonas aeruginosa proteins shows variable dependence on the components of Bam and Lol machineries.

Science.gov (United States)

Hoang, Hanh H; Nickerson, Nicholas N; Lee, Vincent T; Kazimirova, Anastasia; Chami, Mohamed; Pugsley, Anthony P; Lory, Stephen

2011-01-01

In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively. Targeting of lipidated and nonlipidated proteins to the outer membrane (OM) compartment in Gram-negative bacteria involves the transfer across the periplasm utilizing the Lol and Bam machineries, respectively. We show that depletion of Bam and Lol components in Pseudomonas aeruginosa does not lead to a general OM protein translocation defect

The physiological link between metabolic rate depression and tau phosphorylation in mammalian hibernation.

Directory of Open Access Journals (Sweden)

Jens T Stieler

Full Text Available Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimer's disease (AD. Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation in vivo and the causes for its increased stage of phosphorylation in AD are still not well understood, a fact that is primarily based on the lack of adequate animal models. Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with

29 CFR 1918.98 - Qualifications of machinery operators and supervisory training.

Science.gov (United States)

2010-07-01

...) Qualification of machinery operators. (1) Only an employee determined by the employer to be competent by reason...). Therefore, employers who act in accordance with the employment provisions (Title I) of the ADA (42 U.S.C... authority; elements of accidents prevention; attitudes, leadership and motivation; hazards of longshoring...

Dedicated monitoring and machinery protection systems on reciprocating compressors

Energy Technology Data Exchange (ETDEWEB)

Grande, Alvaro; Wenisch, Markus [Hoerbiger Ventilwerke GmbH and Co KG, Wien (Austria); Jacobs, Denis [HOERBIGER do Brasil Industria de Equipamentos, Cajamar, SP (Brazil)

2012-07-01

Growing demands on reciprocating compressors (recips) in the process gas industry require particular solutions for machinery protection and performance monitoring systems. Compared to rotating equipment, monitoring systems for recips have to consider the special mechanical and physical characteristics, such as oscillating masses, variable vibration behaviour and varying operating conditions. Furthermore, they provide valuable information about the performance of cylinder related components allowing the operator the optimization of efficiency and availability, and therefore increase production. (author)

Fault diagnosis of rotating machinery using an improved HHT based on EEMD and sensitive IMFs

International Nuclear Information System (INIS)

Lei, Yaguo; Zuo, Ming J

2009-01-01

A Hilbert–Huang transform (HHT) is a time–frequency technique and has been widely applied to analyzing vibration signals in the field of fault diagnosis of rotating machinery. It analyzes the vibration signals using intrinsic mode functions (IMFs) extracted using empirical mode decomposition (EMD). However, EMD sometimes cannot reveal the signal characteristics accurately because of the problem of mode mixing. Ensemble empirical mode decomposition (EEMD) was developed recently to alleviate this problem. The IMFs generated by EEMD have different sensitivity to faults. Some IMFs are sensitive and closely related to the faults but others are irrelevant. To enhance the accuracy of the HHT in fault diagnosis of rotating machinery, an improved HHT based on EEMD and sensitive IMFs is proposed in this paper. Simulated signals demonstrate the effectiveness of the improved HHT in diagnosing the faults of rotating machinery. Finally, the improved HHT is applied to diagnosing an early rub-impact fault of a heavy oil catalytic cracking machine set, and the application results prove that the improved HHT is superior to the HHT based on all IMFs of EMD

Solid polymer electrolyte from phosphorylated chitosan

Energy Technology Data Exchange (ETDEWEB)

Fauzi, Iqbal, E-mail: arcana@chem.itb.ac.id; Arcana, I Made, E-mail: arcana@chem.itb.ac.id [Inorganic and Physical Chemistry Research Groups, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132 (Indonesia)

2014-03-24

Recently, the need of secondary battery application continues to increase. The secondary battery which using a liquid electrolyte was indicated had some weakness. A solid polymer electrolyte is an alternative electrolytes membrane which developed in order to replace the liquid electrolyte type. In the present study, the effect of phosphorylation on to polymer electrolyte membrane which synthesized from chitosan and lithium perchlorate salts was investigated. The effect of the component’s composition respectively on the properties of polymer electrolyte, was carried out by analyzed of it’s characterization such as functional groups, ion conductivity, and thermal properties. The mechanical properties i.e tensile resistance and the morphology structure of membrane surface were determined. The phosphorylation processing of polymer electrolyte membrane of chitosan and lithium perchlorate was conducted by immersing with phosphoric acid for 2 hours, and then irradiated on a microwave for 60 seconds. The degree of deacetylation of chitosan derived from shrimp shells was obtained around 75.4%. Relative molecular mass of chitosan was obtained by viscometry method is 796,792 g/mol. The ionic conductivity of chitosan membrane was increase from 6.33 × 10{sup −6} S/cm up to 6.01 × 10{sup −4} S/cm after adding by 15 % solution of lithium perchlorate. After phosphorylation, the ionic conductivity of phosphorylated lithium chitosan membrane was observed 1.37 × 10{sup −3} S/cm, while the tensile resistance of 40.2 MPa with a better thermal resistance. On the strength of electrolyte membrane properties, this polymer electrolyte membrane was suggested had one potential used for polymer electrolyte in field of lithium battery applications.

Abrasive Wear of Alloyed Cast Steels Applied for Heavy Machinery

Directory of Open Access Journals (Sweden)

Studnicki A.

2015-03-01

Full Text Available In the paper the results and analysis of abrasive wear studies were shown for two grades of cast steels: low-alloyed cast steel applied for heavy machinery parts such as housing, covers etc. and chromium cast steels applied for kinetic nodes of pin-sleeve type. Studies were performed using the modified in Department of Foundry pin-on-disc method.

Phosphorylation of proteins in Clostridium thermohydrosulfuricum

International Nuclear Information System (INIS)

Londesborough, J.

1986-01-01

Cell extracts of the thermophile Clostridium thermohydrosulfuricum catalyzed the phosphorylation by (γ- 32 P)ATP of several endogenous proteins with M/sub r/s between 13,000 and 100,000. Serine and tyrosine were the main acceptors. Distinct substrate proteins were found in the soluble (e.g., proteins p66, p63, and p53 of M/sub r/s 66,000, 63,000, and 53,000, respectively) and particulate (p76 and p30) fractions, both of which contained protein kinase and phosphatase activity. The soluble fraction suppressed the phosphorylation of particulate proteins and contained a protein kinase inhibitor. Phosphorylation of p53 was promoted by 10μM fructose 1,6-bisphosphate or glucose 1,6-bisphosphate and suppressed by hexose monophosphates, whereas p30 and p13 were suppressed by 5 μM brain (but not spinach) calmodulin. Polyamines, including the odd polyamines characteristic of thermophiles, modulated the labeling of most of the phosphoproteins. Apart from p66, all the proteins labeled in vitro were also rapidly labeled in intact cells by 32 P/sub i/. Several proteins strongly labeled in vivo were labeled slowly or not at all in vitro

Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A).

Science.gov (United States)

Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

2017-01-08

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. Copyright © 2016 Elsevier Inc. All rights reserved.

Musite, a tool for global prediction of general and kinase-specific phosphorylation sites.

Science.gov (United States)

Gao, Jianjiong; Thelen, Jay J; Dunker, A Keith; Xu, Dong

2010-12-01

Reversible protein phosphorylation is one of the most pervasive post-translational modifications, regulating diverse cellular processes in various organisms. High throughput experimental studies using mass spectrometry have identified many phosphorylation sites, primarily from eukaryotes. However, the vast majority of phosphorylation sites remain undiscovered, even in well studied systems. Because mass spectrometry-based experimental approaches for identifying phosphorylation events are costly, time-consuming, and biased toward abundant proteins and proteotypic peptides, in silico prediction of phosphorylation sites is potentially a useful alternative strategy for whole proteome annotation. Because of various limitations, current phosphorylation site prediction tools were not well designed for comprehensive assessment of proteomes. Here, we present a novel software tool, Musite, specifically designed for large scale predictions of both general and kinase-specific phosphorylation sites. We collected phosphoproteomics data in multiple organisms from several reliable sources and used them to train prediction models by a comprehensive machine-learning approach that integrates local sequence similarities to known phosphorylation sites, protein disorder scores, and amino acid frequencies. Application of Musite on several proteomes yielded tens of thousands of phosphorylation site predictions at a high stringency level. Cross-validation tests show that Musite achieves some improvement over existing tools in predicting general phosphorylation sites, and it is at least comparable with those for predicting kinase-specific phosphorylation sites. In Musite V1.0, we have trained general prediction models for six organisms and kinase-specific prediction models for 13 kinases or kinase families. Although the current pretrained models were not correlated with any particular cellular conditions, Musite provides a unique functionality for training customized prediction models

Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

International Nuclear Information System (INIS)

Deaciuc, I.V.; Spitzer, J.A.

1989-01-01

The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with [32P]H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis

The selective phosphorylation of a guanine nucleotide-binding regulatory protein

International Nuclear Information System (INIS)

Carlson, K.E.

1989-01-01

Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the α subunit of G i and other G proteins in solution. However, the occurrence of the phosphorylation of G 1 within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which the α subunits of G i undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [γ 32 P]ATP and [ 32 P]H 3 PO 4 , respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G iα -despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G zα , or antibodies for both G zα and G iα , precipitated a 40-kDa phosphoprotein

ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation

Science.gov (United States)

Lievens, Patricia Marie-Jeanne; Kuznetsova, Tatiana; Kochlamazashvili, Gaga; Cesca, Fabrizia; Gorinski, Natalya; Galil, Dalia Abdel; Cherkas, Volodimir; Ronkina, Natalia; Lafera, Juri; Gaestel, Matthias

2016-01-01

The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis. PMID:27247265

Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

Science.gov (United States)

Waraky, Ahmed; Lin, Yingbo; Warsito, Dudi; Haglund, Felix; Aleem, Eiman; Larsson, Olle

2017-11-03

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases ( e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G 1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

Energy Technology Data Exchange (ETDEWEB)

Marasini, Carlotta, E-mail: marasini@ge.ibf.cnr.it [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy); Galeno, Lauretta; Moran, Oscar [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy)

2012-07-06

Highlights: Black-Right-Pointing-Pointer CFTR mutations produce cystic fibrosis. Black-Right-Pointing-Pointer Chloride transport depends on the regulatory domain phosphorylation. Black-Right-Pointing-Pointer Regulatory domain is intrinsically disordered. Black-Right-Pointing-Pointer Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and {beta}-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of {alpha}-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two

Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

International Nuclear Information System (INIS)

Marasini, Carlotta; Galeno, Lauretta; Moran, Oscar

2012-01-01

Highlights: ► CFTR mutations produce cystic fibrosis. ► Chloride transport depends on the regulatory domain phosphorylation. ► Regulatory domain is intrinsically disordered. ► Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and β-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of α-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two conditions, monitoring the changes of the mean residue ellipticity measured at 222 nm as a function of temperature

Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins

International Nuclear Information System (INIS)

Watkins, D.T.

1991-01-01

The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing [32P]ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules

LRRK2 mediated Rab8a phosphorylation promotes lipid storage.

Science.gov (United States)

Yu, Miao; Arshad, Muhammad; Wang, Wenmin; Zhao, Dongyu; Xu, Li; Zhou, Linkang

2018-02-27

Several mutations in leucine rich repeat kinase 2 (LRRK2) gene have been associated with pathogenesis of Parkinson's disease (PD), a neurodegenerative disorder marked by resting tremors, and rigidity, leading to Postural instability. It has been revealed that mutations that lead to an increase of kinase activity of LRRK2 protein are significantly associated with PD pathogenesis. Recent studies have shown that some Rab GTPases, especially Rab8, serve as substrates of LRRK2 and undergo phosphorylation in its switch II domain upon interaction. Current study was performed in order to find out the effects of the phosphorylation of Rab8 and its mutants on lipid metabolism and lipid droplets growth. The phosphorylation status of Rab8a was checked by phos-tag gel. Point mutant construct were generated to investigate the function of Rab8a. 3T3L1 cells were transfected with indicated plasmids and the lipid droplets were stained with Bodipy. Fluorescent microscopy experiments were performed to examine the sizes of lipid droplets. The interactions between Rab8a and Optineurin were determined by immunoprecipitation and western blot. Our assays demonstrated that Rab8a was phosphorylated by mutated LRRK2 that exhibits high kinase activity. Phosphorylation of Rab8a on amino acid residue T72 promoted the formation of large lipid droplets. T72D mutant of Rab8a had higher activity to promote the formation of large lipid droplets compared with wild type Rab8a, with increase in average diameter of lipid droplets from 2.10 μm to 2.46 μm. Moreover, phosphorylation of Rab8a weakened the interaction with its effector Optineurin. Y1699C mutated LRRK2 was able to phosphorylate Rab8a and phosphorylation of Rab8a on site 72 plays important role in the fusion and enlargement of lipid droplets. Taken together, our study suggests an indirect relationship between enhanced lipid storage capacity and PD pathogenesis.

Impaired degradation of WNK by Akt and PKA phosphorylation of KLHL3.

Science.gov (United States)

Yoshizaki, Yuki; Mori, Yutaro; Tsuzaki, Yoshihito; Mori, Takayasu; Nomura, Naohiro; Wakabayashi, Mai; Takahashi, Daiei; Zeniya, Moko; Kikuchi, Eriko; Araki, Yuya; Ando, Fumiaki; Isobe, Kiyoshi; Nishida, Hidenori; Ohta, Akihito; Susa, Koichiro; Inoue, Yuichi; Chiga, Motoko; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi; Sohara, Eisei

2015-11-13

Mutations in with-no-lysine kinase (WNK) 1, WNK4, Kelch-like 3 (KLHL3), and Cullin3 result in an inherited hypertensive disease, pseudohypoaldosteronism type II. WNK activates the Na-Cl cotransporter (NCC), increasing sodium reabsorption in the kidney. Further, KLHL3, an adapter protein of Cullin3-based E3 ubiquitin ligase, has been recently found to bind to WNK, thereby degrading them. Insulin and vasopressin have been identified as powerful activators of WNK signaling. In this study, we investigated effects of Akt and PKA, key downstream substrates of insulin and vasopressin signaling, respectively, on KLHL3. Mass spectrometry analysis revealed that KLHL3 phosphorylation at S433. Phospho-specific antibody demonstrated defective binding between phosphorylated KLHL3 and WNK4. Consistent with the fact that S433 is a component of Akt and PKA phosphorylation motifs, in vitro kinase assay demonstrated that Akt and PKA can phosphorylate KLHL3 at S433, that was previously reported to be phosphorylated by PKC. Further, forskolin, a representative PKA stimulator, increased phosphorylation of KLHL3 at S433 and WNK4 protein expression in HEK293 cells by inhibiting the KLHL3 effect that leads to WNK4 degradation. Insulin also increased phosphorylation of KLHL3 at S433 in cultured cells. In conclusion, we found that Akt and PKA phosphorylated KLHL3 at S433, and phosphorylation of KLHL3 by PKA inhibited WNK4 degradation. This could be a novel mechanism on how insulin and vasopressin physiologically activate the WNK signal. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of phosphorylation on antioxidant activities of pumpkin (Cucurbita pepo, Lady godiva) polysaccharide.

Science.gov (United States)

Song, Yi; Ni, Yuanying; Hu, Xiaosong; Li, Quanhong

2015-11-01

Phosphorylated derivatives of pumpkin polysaccharide with different degree of substitution were synthesized using POCl3 and pyridine. Antioxidant activities and cytoprotective effects of unmodified polysaccharide and phosphorylated derivatives were investigated employing various in vitro systems. Results showed that high ratio of POCl3/pyridine could increase the degree of substitution and no remarkable degradation occurred in the phosphorylation process. Characteristic absorption of phosphorylation appeared both in the IR and (31)P NMR spectrum. The df values between 2.27 and 2.55 indicated the relatively expanded conformation of the phosphorylated derivatives. All the phosphorylated polysaccharides exhibited higher antioxidant activities. H2O2-induced oxidative damages on rat thymic lymphocyte were also prevented by the derivatives. In general, phosphorylation could improve the antioxidant activities of pumpkin polysaccharide both in vitro and in a cell system. Copyright © 2015 Elsevier B.V. All rights reserved.

MODELING ENVIRONMENTAL IMPACT OF MACHINERY SECTORS TO PROMOTE SUSTAINABLE DEVELOPMENT OF THAILAND

Directory of Open Access Journals (Sweden)

Pruethsan Sutthichaimethee

2016-01-01

Full Text Available The objective of this research is to propose an indicator to evaluate environmental impacts from the machinery sectors of Thailand, leading to more sustainable consumption and production in this sector of the economy. The factors used to calculate the forward linkage, backward linkage and real benefit included the total environmental costs. The highest total environmental cost was railway equipment need to be resolved immediately because it uses natural resources in carrying capacity, higher than standard environmental cost, and contribute to low real benefit. Electric accumulator & battery, secondary special industrial machinery, motorcycle, bicycle & other carriages, and engines and turbines need to monitor closely because they are able to link to other production sectors more than other production sector do and they have high environmental cost. In order to decide the sustainable development strategy of the country, there is a need to use this research to support decision-making.

Phosphorylation of acidic ribosomal proteins from rabbit reticulocytes by a ribosome-associated casein kinase

DEFF Research Database (Denmark)

Issinger, O G

1977-01-01

Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate polyacryl......Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate...

In vitro phosphorylation of insulin receptor substrate 1 by protein kinase C-zeta: functional analysis and identification of novel phosphorylation sites.

Science.gov (United States)

Sommerfeld, Mark R; Metzger, Sabine; Stosik, Magdalene; Tennagels, Norbert; Eckel, Jürgen

2004-05-18

Protein kinase C-zeta (PKC-zeta) participates both in downstream insulin signaling and in the negative feedback control of insulin action. Here we used an in vitro approach to identify PKC-zeta phosphorylation sites within insulin receptor substrate 1 (IRS-1) and to characterize the functional implications. A recombinant IRS-1 fragment (rIRS-1(449)(-)(664)) containing major tyrosine motifs for interaction with phosphatidylinositol (PI) 3-kinase strongly associated to the p85alpha subunit of PI 3-kinase after Tyr phosphorylation by the insulin receptor. Phosphorylation of rIRS-1(449)(-)(664) by PKC-zeta induced a prominent inhibition of this process with a mixture of classical PKC isoforms being less effective. Both PKC-zeta and the classical isoforms phosphorylated rIRS-1(449)(-)(664) on Ser(612). However, modification of this residue did not reduce the affinity of p85alpha binding to pTyr-containing peptides (amino acids 605-615 of rat IRS-1), as determined by surface plasmon resonance. rIRS-1(449)(-)(664) was then phosphorylated by PKC-zeta using [(32)P]ATP and subjected to tryptic phosphopeptide mapping based on two-dimensional HPLC coupled to mass spectrometry. Ser(498) and Ser(570) were identified as novel phosphoserine sites targeted by PKC-zeta. Both sites were additionally confirmed by phosphopeptide mapping of the corresponding Ser --> Ala mutants of rIRS-1(449)(-)(664). Ser(570) was specifically targeted by PKC-zeta, as shown by immunoblotting with a phosphospecific antiserum against Ser(570) of IRS-1. Binding of p85alpha to the S570A mutant was less susceptible to inhibition by PKC-zeta, when compared to the S612A mutant. In conclusion, our in vitro data demonstrate a strong inhibitory action of PKC-zeta at the level of IRS-1/PI 3-kinase interaction involving multiple serine phosphorylation sites. Whereas Ser(612) appears not to participate in the negative control of insulin signaling, Ser(570) may at least partly contribute to this process.

Tyrosine phosphorylation switching of a G protein.

Science.gov (United States)

Li, Bo; Tunc-Ozdemir, Meral; Urano, Daisuke; Jia, Haiyan; Werth, Emily G; Mowrey, David D; Hicks, Leslie M; Dokholyan, Nikolay V; Torres, Matthew P; Jones, Alan M

2018-03-30

Heterotrimeric G protein complexes are molecular switches relaying extracellular signals sensed by G protein-coupled receptors (GPCRs) to downstream targets in the cytoplasm, which effect cellular responses. In the plant heterotrimeric GTPase cycle, GTP hydrolysis, rather than nucleotide exchange, is the rate-limiting reaction and is accelerated by a receptor-like regulator of G signaling (RGS) protein. We hypothesized that posttranslational modification of the Gα subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr 166 in the Arabidopsis Gα subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occur. We also identified a receptor-like kinase that phosphorylates AtGPA1 in a Tyr 166 -dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr 166 forms a salt bridge in this interface and potentially affects the RGS protein-accelerated GTPase cycle. Using a Tyr 166 phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound Gα substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr 166 in AtGPA1 changes the binding pattern with AtRGS1 and thereby attenuates the steady-state rate of the GTPase cycle. We coin this newly identified mechanism "substrate phosphoswitching." © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Exposure to Tumescent Solution Significantly Increases Phosphorylation of Perilipin in Adipocytes.

Science.gov (United States)

Keskin, Ilknur; Sutcu, Mustafa; Eren, Hilal; Keskin, Mustafa

2017-02-01

Lidocaine and epinephrine could potentially decrease adipocyte viability, but these effects have not been substantiated. The phosphorylation status of perilipin in adipocytes may be predictive of cell viability. Perilipin coats lipid droplets and restricts access of lipases; phospho-perilipin lacks this protective function. The authors investigated the effects of tumescent solution containing lidocaine and epinephrine on the phosphorylation status of perilipin in adipocytes. In this in vitro study, lipoaspirates were collected before and after tumescence from 15 women who underwent abdominoplasty. Fat samples were fixed, sectioned, and stained for histologic and immunohistochemical analyses. Relative phosphorylation of perilipin was inferred from pixel intensities of immunostained adipocytes observed with confocal microscopy. For adipocytes collected before tumescent infiltration, 10.08% of total perilipin was phosphorylated. In contrast, 30.62% of total perilipin was phosphorylated for adipocytes collected from tumescent tissue (P < .01). The tumescent technique increases the relative phosphorylation of perilipin in adipocytes, making these cells more vulnerable to lipolysis. Tumescent solution applied for analgesia or hemostasis of the donor site should contain the lowest possible concentrations of lidocaine and epinephrine. LEVEL OF EVIDENCE 5. © 2016 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.

Identification and analysis of the RNA degrading complexes and machinery of Giardia lamblia using an in silico approach.

Science.gov (United States)

Williams, Christopher W; Elmendorf, Heidi G

2011-11-29

RNA degradation is critical to the survival of all cells. With increasing evidence for pervasive transcription in cells, RNA degradation has gained recognition as a means of regulating gene expression. Yet, RNA degradation machinery has been studied extensively in only a few eukaryotic organisms, including Saccharomyces cerevisiae and humans. Giardia lamblia is a parasitic protist with unusual genomic traits: it is binucleated and tetraploid, has a very compact genome, displays a theme of genomic minimalism with cellular machinery commonly comprised of a reduced number of protein components, and has a remarkably large population of long, stable, noncoding, antisense RNAs. Here we use in silico approaches to investigate the major RNA degradation machinery in Giardia lamblia and compare it to a broad array of other parasitic protists. We have found key constituents of the deadenylation and decapping machinery and of the 5'-3' RNA degradation pathway. We have similarly found that all of the major 3'-5' RNA degradation pathways are present in Giardia, including both exosome-dependent and exosome-independent machinery. However, we observe significant loss of RNA degradation machinery genes that will result in important differences in the protein composition, and potentially functionality, of the various RNA degradation pathways. This is most apparent in the exosome, the central mediator of 3'-5' degradation, which apparently contains an altered core configuration in both Giardia and Plasmodium, with only four, instead of the canonical six, distinct subunits. Additionally the exosome in Giardia is missing both the Rrp6, Nab3, and Nrd1 proteins, known to be key regulators of noncoding transcript stability in other cells. These findings suggest that although the full complement of the major RNA degradation mechanisms were present - and likely functional - early in eukaryotic evolution, the composition and function of the complexes is more variable than previously

Assessment of exposure to manganese in welding operations during the assembly of heavy excavation machinery accessories.

Science.gov (United States)

Smargiassi, A; Baldwin, M; Savard, S; Kennedy, G; Mergler, D; Zayed, J

2000-10-01

Welder exposure to metals in various industrial sectors is poorly characterized. We had the opportunity to carry out an exploratory study to characterize manganese exposure in welding operations in a recently established Quebec factory that assembled accessories for heavy excavation machinery. Ten workers were sampled for total manganese for at least two consecutive days out of three followed by two consecutive days for respirable manganese (with a size selective sampler with a median cut-off of 4 microns), during a typical week in the summer of 1998. Parts being welded were characterized as large or small. Small parts were those being welded on tables during subassembly. Workers were divided into two groups according to the parts they were welding. Seventy-eight percent of the total manganese exposure levels of welding operations during the assembly of large accessories of heavy excavation machinery exceeded the manganese American Conference of Governmental Industrial Hygienists (ACGIH) threshold limit value (TLV) of 0.20 mg/m3 (GM 0.24 mg/m3, n = 14) while none exceeded the TLV during the assembly of small pieces (GM 0.06 mg/m3, n = 8). Welding operations during the assembly of large heavy excavation machinery accessories may pose a significant health hazard. Considering the importance of task-related variables affecting exposure among workers, further studies are needed to better characterize exposure determinants of welding operations during the assembly of heavy excavation machinery accessories.

Solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters

International Nuclear Information System (INIS)

Valtorta, F.; Schiebler, W.; Jahn, R.; Ceccarelli, B.; Greengard, P.

1986-01-01

A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phyosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides

Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies.

Science.gov (United States)

Lier, Clemens; Becker, Stephan; Biedenkopf, Nadine

2017-12-01

Zaire Ebolavirus (EBOV) causes a severe feverish disease with high case fatality rates. Transcription of EBOV is dependent on the activity of the nucleocapsid protein VP30 which represents an essential viral transcription factor. Activity of VP30 is regulated via phosphorylation at six N-terminal serine residues. Recent data demonstrated that dynamic phosphorylation and dephosphorylation of serine residue 29 is essential for transcriptional support activity of VP30. To analyze the spatio/temporal dynamics of VP30 phosphorylation, we generated a peptide antibody recognizing specifically VP30 phosphorylated at serine 29. Using this antibody we could demonstrate that (i) the majority of VP30 molecules in EBOV-infected cells is dephosphorylated at the crucial position serine 29, (ii) both, VP30 phosphorylation and dephosphorylation take place in viral inclusion bodies that are induced by the nucleoprotein NP and (iii) NP influences the phosphorylation state of VP30. Copyright © 2017 Elsevier Inc. All rights reserved.

Tyrosine phosphorylation in human lymphomas

NARCIS (Netherlands)

Haralambieva, E; Jones, M.; Roncador, GM; Cerroni, L; Lamant, L; Ott, G; Rosenwald, A; Sherman, C; Thorner, P; Kusec, R; Wood, KM; Campo, E; Falini, B; Ramsay, A; Marafioti, T; Stein, H; Kluin, PM; Pulford, K; Mason, DY

2002-01-01

In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated

ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

Energy Technology Data Exchange (ETDEWEB)

Inesta-Vaquera, Francisco A. [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain); Campbell, David G.; Arthur, J. Simon C. [MRC Protein Phosphorylation Unit, Sir James Black Building, School of Life Sciences, University of Dundee, Dundee DD1 5EH (United Kingdom); Cuenda, Ana, E-mail: acuenda@cnb.csic.es [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain)

2010-08-13

Research highlights: {yields} hDlg is phosphorylated during mitosis in multiple residues. {yields} Prospho-hDlg is excluded from the midbody during mitosis. {yields} hDlg is not phosphorylated by p38{gamma} or JNK1/2 during mitosis. {yields} ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

International Nuclear Information System (INIS)

Inesta-Vaquera, Francisco A.; Campbell, David G.; Arthur, J. Simon C.; Cuenda, Ana

2010-01-01

Research highlights: → hDlg is phosphorylated during mitosis in multiple residues. → Prospho-hDlg is excluded from the midbody during mitosis. → hDlg is not phosphorylated by p38γ or JNK1/2 during mitosis. → ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

Model and simulation of Na+/K+ pump phosphorylation in the presence of palytoxin.

Science.gov (United States)

Rodrigues, Antônio M; Almeida, Antônio-Carlos G; Infantosi, Antonio F C; Teixeira, Hewerson Z; Duarte, Mario A

2008-02-01

The ATP hydrolysis reactions responsible for the Na(+)/K(+)-ATPase phosphorylation, according to recent experimental evidences, also occur for the PTX-Na(+)/K(+) pump complex. Moreover, it has been demonstrated that PTX interferes with the enzymes phosphorylation status. However, the reactions involved in the PTX-Na(+)/K(+) pump complex phosphorylation are not very well established yet. This work aims at proposing a reaction model for PTX-Na(+)/K(+) pump complex, with similar structure to the Albers-Post model, to contribute to elucidate the PTX effect over Na(+)/K(+)-ATPase phosphorylation and dephosphorylation. Computational simulations with the proposed model support several hypotheses and also suggest: (i) phosphorylation promotes an increase of the open probability of induced channels; (ii) PTX reduces the Na(+)/K(+) pump phosphorylation rate; (iii) PTX may cause conformational changes to substates where the Na(+)/K(+)-ATPase may not be phosphorylated; (iv) PTX can bind to substates of the two principal states E1 and E2, with highest affinity to phosphorylated enzymes and with ATP bound to its low-affinity sites. The proposed model also allows previewing the behavior of the PTX-pump complex substates for different levels of intracellular ATP concentrations.

Pro-Tumorigenic Phosphorylation of p120 Catenin in Renal and Breast Cancer.

Directory of Open Access Journals (Sweden)

Antonis Kourtidis

Full Text Available Altered protein expression and phosphorylation are common events during malignant transformation. These perturbations have been widely explored in the context of E-cadherin cell-cell adhesion complexes, which are central in the maintenance of the normal epithelial phenotype. A major component of these complexes is p120 catenin (p120, which binds and stabilizes E-cadherin to promote its adhesive and tumor suppressing function. However, p120 is also an essential mediator of pro-tumorigenic signals driven by oncogenes, such as Src, and can be phosphorylated at multiple sites. Although alterations in p120 expression have been extensively studied by immunohistochemistry (IHC in the context of tumor progression, little is known about the status and role of p120 phosphorylation in cancer. Here we show that tyrosine and threonine phosphorylation of p120 in two sites, Y228 and T916, is elevated in renal and breast tumor tissue samples. We also show that tyrosine phosphorylation of p120 at its N-terminus, including at the Y228 site is required for its pro-tumorigenic potential. In contrast, phosphorylation of p120 at T916 does not affect this p120 function. However, phosphorylation of p120 at T916 interferes with epitope recognition of the most commonly used p120 antibody, namely pp120. As a result, this antibody selectively underrepresents p120 levels in tumor tissues, where p120 is phosphorylated. Overall, our data support a role of p120 phosphorylation as a marker and mediator of tumor transformation. Importantly, they also argue that the level and localization of p120 in human cancer tissues immunostained with pp120 needs to be re-evaluated.

Age-related changes in the synthesis and phosphorylation of proteins

International Nuclear Information System (INIS)

Butler, J.A.; Heydari, A.; Richardson, A.

1986-01-01

It is well documented that the protein synthetic activity of liver tissue decreases significantly with age. However, very little information is available on the effect of age on the synthesis or phosphorylation of individual proteins. Hepatocytes were isolated from 5- to 30-month-old male Fischer F344 rats, and proteins were labeled with either [ 3 H]-valine or [ 32 P]-phosphate. Two-dimensional polyacrylamide gel electrophoresis was used to monitor the synthesis and phosphorylation of a wide variety of proteins. A dramatic increase or decrease in the synthesis of approximately 2 to 3% of the proteins was observed. Most of the proteins whose synthesis increased with age were found to be plasma proteins, e.g., acute phase proteins, synthesized by the liver. In general, the synthesis of most proteins decreased 20 to 40% with age. The phosphorylation of most proteins (over 200) did not appear to change with age. However the phosphorylation of two acidic proteins (molecular weights of 148 Kd and 130 Kd and pIs of 5.4 and 5.36, respectively) decreased with age while the phosphorylation of a basic protein (molecular weight of 57 Kd and pI of 8.09) increased with age

In vivo phosphorylation of a peptide tag for protein purification.

Science.gov (United States)

Goux, Marine; Fateh, Amina; Defontaine, Alain; Cinier, Mathieu; Tellier, Charles

2016-05-01

To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag. The level of the co-expressed CKIIα was controlled by the arabinose promoter and optimal phosphorylation was obtained with 2 % (w/v) arabinose as inductor. The effectiveness of the phosphorylation system was demonstrated by electrophoretic mobility shift assay (NUT-PAGE) and staining with a specific phosphoprotein-staining gel. The resulting phosphorylated tag was also used to purify the phosphoprotein by immobilized metal affinity chromatography, which relies on the specific interaction of phosphate moieties with Fe(III). The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.

A Mass Spectrometry-Based Predictive Strategy Reveals ADAP1 is Phosphorylated at Tyrosine 364

Energy Technology Data Exchange (ETDEWEB)

Littrell, BobbiJo R [National Renewable Energy Laboratory (NREL), Golden, CO (United States)

2018-04-16

The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry-based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition. ADAP1 was immunoprecipitated from extracts of HEK293 cells stably-transfected with ADAP1 cDNA. Immunoprecipitated ADAP1 was digested with proteolytic enzymes and analyzed by LC-MS in MS1 mode by high-resolution quadrupole time-of-flight mass spectrometry (QTOF-MS). Peptide molecular features were extracted using an untargeted data mining algorithm. Extracted peptide neutral masses were matched against the ADAP1 amino acid sequence with phosphorylation included as a predicted modification. Peptides with predicted phosphorylation sites were analyzed by targeted LC-MS2. Acquired MS2 spectra were then analyzed using database search engines to confirm phosphorylation. Spectra of phosphorylated peptides were validated by manual interpretation. Further confirmation was performed by manipulating phospho-peptide abundance using calf intestinal phosphatase (CIP) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Of five predicted phosphopeptides, one, comprised of the sequence AVDRPMLPQEYAVEAHFK, was confirmed to be phosphorylated on a Tyrosine at position 364. Pre-treatment of cells with PMA prior to immunoprecipitation increased the ratio of phosphorylated to unphosphorylated peptide as determined by area counts of extracted ion chromatograms (EIC). Addition of CIP to immunoprecipitation reactions eliminated the phosphorylated form. A novel phosphorylation site was identified at Tyrosine 364. Phosphorylation at this site is increased by treatment with PMA. PMA promotes membrane translocation and activation of protein kinase C (PKC), indicating that Tyrosine 364

Monitoring of vibrating machinery using artificial neural networks

International Nuclear Information System (INIS)

Alguindigue, I.E.; Loskiewicz-Buczak, A.

1991-01-01

The primary source of vibration in complex engineering systems is rotating machinery. Vibration signatures collected from these components render valuable information about the operational state of the system and may be used to perform diagnostics. For example, the low frequency domain contains information about unbalance, misalignment, instability in journal bearing and mechanical looseness; analysis of the medium frequency range can render information about faults in meshing gear teeth; while the high frequency domain will contain information about incipient faults in rolling-element bearings. Trend analysis may be performed by comparing the vibration spectrum for each machine with a reference spectrum and evaluating the vibration magnitude changes at different frequencies. This form of analysis for diagnostics is often performed by maintenance personnel monitoring and recording transducer signals and analyzing the signals to identify the operating condition of the machine. With the advent of portable fast Fourier transform (FFT) analyzers and ''laptop'' computers, it is possible to collect and analyze vibration data an site and detect incipient failures several weeks or months before repair is necessary. It is often possible to estimate the remaining life of certain systems once a fault has been detected. RMS velocity, acceleration, displacements, peak value, and crest factor readings can be collected from vibration sensors. To exploit all the information embedded in these signals, a robust and advanced analysis technique is required. Our goal is to design a diagnostic system using neural network technology, a system such as this would automate the interpretation of vibration data coming from plant-wide machinery and permit efficient on-line monitoring of these components

Mechanism of APC/CCDC20 activation by mitotic phosphorylation.

Science.gov (United States)

Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A; Brunner, Michael R; Davidson, Iain F; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A; Peters, Jan-Michael

2016-05-10

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/C(CDC20) activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/C(CDC20) activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/C(CDC20) activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.

Phosphorylation variation during the cell cycle scales with structural propensities of proteins.

Directory of Open Access Journals (Sweden)

Stefka Tyanova

Full Text Available Phosphorylation at specific residues can activate a protein, lead to its localization to particular compartments, be a trigger for protein degradation and fulfill many other biological functions. Protein phosphorylation is increasingly being studied at a large scale and in a quantitative manner that includes a temporal dimension. By contrast, structural properties of identified phosphorylation sites have so far been investigated in a static, non-quantitative way. Here we combine for the first time dynamic properties of the phosphoproteome with protein structural features. At six time points of the cell division cycle we investigate how the variation of the amount of phosphorylation correlates with the protein structure in the vicinity of the modified site. We find two distinct phosphorylation site groups: intrinsically disordered regions tend to contain sites with dynamically varying levels, whereas regions with predominantly regular secondary structures retain more constant phosphorylation levels. The two groups show preferences for different amino acids in their kinase recognition motifs - proline and other disorder-associated residues are enriched in the former group and charged residues in the latter. Furthermore, these preferences scale with the degree of disorderedness, from regular to irregular and to disordered structures. Our results suggest that the structural organization of the region in which a phosphorylation site resides may serve as an additional control mechanism. They also imply that phosphorylation sites are associated with different time scales that serve different functional needs.

Rat1p maintains RNA polymerase II CTD phosphorylation balance

DEFF Research Database (Denmark)

Jimeno-González, Silvia; Schmid, Manfred; Malagon, Francisco

2014-01-01

. Here we describe a function of Rat1p in regulating phosphorylation levels of the C-terminal domain (CTD) of the largest RNAPII subunit, Rpb1p, during transcription elongation. The rat1-1 mutant exhibits highly elevated levels of CTD phosphorylation as well as RNAPII distribution and transcription...... termination defects. These phenotypes are all rescued by overexpression of the CTD phosphatase Fcp1p, suggesting a functional relationship between the absence of Rat1p activity, elevated CTD phosphorylation, and transcription defects. We also demonstrate that rat1-1 cells display increased RNAPII...

Exercise increases TBC1D1 phosphorylation in human skeletal muscle

Science.gov (United States)

Jessen, Niels; An, Ding; Lihn, Aina S.; Nygren, Jonas; Hirshman, Michael F.; Thorell, Anders

2011-01-01

Exercise and weight loss are cornerstones in the treatment and prevention of type 2 diabetes, and both interventions function to increase insulin sensitivity and glucose uptake into skeletal muscle. Studies in rodents demonstrate that the underlying mechanism for glucose uptake in muscle involves site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 (TBC1D4) and TBC1D1. Multiple kinases, including Akt and AMPK, phosphorylate TBC1D1 and AS160 on distinct residues, regulating their activity and allowing for GLUT4 translocation. In contrast to extensive rodent-based studies, the regulation of AS160 and TBC1D1 in human skeletal muscle is not well understood. In this study, we determined the effects of dietary intervention and a single bout of exercise on TBC1D1 and AS160 site-specific phosphorylation in human skeletal muscle. Ten obese (BMI 33.4 ± 2.4, M-value 4.3 ± 0.5) subjects were studied at baseline and after a 2-wk dietary intervention. Muscle biopsies were obtained from the subjects in the resting (basal) state and immediately following a 30-min exercise bout (70% V̇o2 max). Muscle lysates were analyzed for AMPK activity and Akt phosphorylation and for TBC1D1 and AS160 phosphorylation on known or putative AMPK and Akt sites as follows: AS160 Ser711 (AMPK), TBC1D1 Ser231 (AMPK), TBC1D1 Ser660 (AMPK), TBC1D1 Ser700 (AMPK), and TBC1D1 Thr590 (Akt). The diet intervention that consisted of a major shift in the macronutrient composition resulted in a 4.2 ± 0.4 kg weight loss (P < 0.001) and a significant increase in insulin sensitivity (M value 5.6 ± 0.6), but surprisingly, there was no effect on expression or phosphorylation of any of the muscle-signaling proteins. Exercise increased muscle AMPKα2 activity but did not increase Akt phosphorylation. Exercise increased phosphorylation on AS160 Ser711, TBC1D1 Ser231, and TBC1D1 Ser660 but had no effect on TBC1D1 Ser700. Exercise did not increase TBC1D1 Thr590 phosphorylation or TBC1D1/AS160 PAS

Activation of purified calcium channels by stoichiometric protein phosphorylation

Energy Technology Data Exchange (ETDEWEB)

Nunoki, K.; Florio, V.; Catterall, W.A. (Univ. of Washington, Seattle (USA))

1989-09-01

Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of {sup 45}Ca{sup 2+} uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of {sup 45}Ca{sup 2+} uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd{sup 2+}, Ni{sup 2+}, and Mg{sup 2+}. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.

Activation of purified calcium channels by stoichiometric protein phosphorylation

International Nuclear Information System (INIS)

Nunoki, K.; Florio, V.; Catterall, W.A.

1989-01-01

Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of 45 Ca 2+ uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of 45 Ca 2+ uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd 2+ , Ni 2+ , and Mg 2+ . The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels

Conceptual design and analysis of roads and road construction machinery for initial lunar base operations

Science.gov (United States)

Sines, Jeffrey L.; Banks, Joel; Efatpenah, Keyanoush

1990-01-01

Recent developments have made it possible for scientists and engineers to consider returning to the Moon to build a manned lunar base. The base can be used to conduct scientific research, develop new space technology, and utilize the natural resources of the Moon. Areas of the base will be separated, connected by a system of roads that reduce the power requirements of vehicles traveling on them. Feasible road types for the lunar surface were analyzed and a road construction system was designed for initial lunar base operations. A model was also constructed to show the system configuration and key operating features. The alternate designs for the lunar road construction system were developed in four stages: analyze and select a road type; determine operations and machinery needed to produce the road; develop machinery configurations; and develop alternates for several machine components. A compacted lunar soil road was selected for initial lunar base operations. The only machinery required to produce this road were a grader and a compactor. The road construction system consists of a main drive unit which is used for propulsion, a detachable grader assembly, and a towed compactor.

The Antioxidant Machinery of Young and Senescent Human Umbilical Vein Endothelial Cells and Their Microvesicles

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Guillermo Bodega

2017-01-01

Full Text Available We examine the antioxidant role of young and senescent human umbilical vein endothelial cells (HUVECs and their microvesicles (MVs. Proteomic and Western blot studies have shown young HUVECs to have a complete and well-developed antioxidant system. Their MVs also contain antioxidant molecules, though of a smaller and more specific range, specialized in the degradation of hydrogen peroxide and the superoxide anion via the thioredoxin-peroxiredoxin system. Senescence was shown to be associated with a large increase in the size of the antioxidant machinery in both HUVECs and their MVs. These responses might help HUVECs and their MVs deal with the more oxidising conditions found in older cells. Functional analysis confirmed the antioxidant machinery of the MVs to be active and to increase in size with senescence. No glutathione or nonpeptide antioxidant (ascorbic acid and vitamin E activity was detected in the MVs. Endothelial cells and MVs seem to adapt to higher ROS concentrations in senescence by increasing their antioxidant machinery, although this is not enough to recover completely from the senescence-induced ROS increase. Moreover, MVs could be involved in the regulation of the blood plasma redox status by functioning as ROS scavengers.

A sequential fuzzy diagnosis method for rotating machinery using ant colony optimization and possibility theory

Energy Technology Data Exchange (ETDEWEB)

Sun, Hao; Ping, Xueliang; Cao, Yi; Lie, Ke [Jiangnan University, Wuxi (China); Chen, Peng [Mie University, Mie (Japan); Wang, Huaqing [Beijing University, Beijing (China)

2014-04-15

This study proposes a novel intelligent fault diagnosis method for rotating machinery using ant colony optimization (ACO) and possibility theory. The non-dimensional symptom parameters (NSPs) in the frequency domain are defined to reflect the features of the vibration signals measured in each state. A sensitive evaluation method for selecting good symptom parameters using principal component analysis (PCA) is proposed for detecting and distinguishing faults in rotating machinery. By using ACO clustering algorithm, the synthesizing symptom parameters (SSP) for condition diagnosis are obtained. A fuzzy diagnosis method using sequential inference and possibility theory is also proposed, by which the conditions of the machinery can be identified sequentially. Lastly, the proposed method is compared with a conventional neural networks (NN) method. Practical examples of diagnosis for a V-belt driving equipment used in a centrifugal fan are provided to verify the effectiveness of the proposed method. The results verify that the faults that often occur in V-belt driving equipment, such as a pulley defect state, a belt defect state and a belt looseness state, are effectively identified by the proposed method, while these faults are difficult to detect using conventional NN.

Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

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Lei eShi

2014-09-01

Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

Phosphorylation regulates SIRT1 function.

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Tsutomu Sasaki

Full Text Available BACKGROUND: SIR2 is an NAD(+-dependent deacetylase [1]-[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo[9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported. METHODOLOGY/PRINCIPAL FINDINGS: We identified 13 residues in SIRT1 that are phosphorylated in vivo using mass spectrometry. Dephosphorylation by phosphatases in vitro resulted in decreased NAD(+-dependent deacetylase activity. We identified cyclinB/Cdk1 as a cell cycle-dependent kinase that forms a complex with and phosphorylates SIRT1. Mutation of two residues phosphorylated by Cyclin B/Cdk1 (threonine 530 and serine 540 disturbs normal cell cycle progression and fails to rescue proliferation defects in SIRT1-deficient cells [12], [13]. CONCLUSIONS/SIGNIFICANCE: Pharmacological manipulation of SIRT1 activity is currently being tested as a means of extending lifespan in mammals. Treatment of obese mice with resveratrol, a pharmacological activator of SIRT1, modestly but significantly improved longevity and, perhaps more importantly, offered some protection against the development of type 2 diabetes mellitus and metabolic syndrome [14]-[16]. Understanding the endogenous mechanisms that regulate the level and activity of SIRT1, therefore, has obvious relevance to human health and disease. Our results identify

Histones and their phosphorylation during germination of rice seeds

International Nuclear Information System (INIS)

Iqbal Ahmed, C.M.; Padayatti, J.D.

1980-01-01

Histones from nuclei of rice embryos were identified by their mobilities on 15% acid-urea polyacrylamide gel electrophoreogram, incorporation of ( 3 H)lysine and ( 14 C) arginine and lack of incorporation of ( 3 H)tryptophan. The ratio of histone to DNA in ungerminated embryos was 2.7 which decreased during germination reaching unity by 48 hr. There was preferential phosphorylation of lysine-rich histones, which paralleled the synthesis of DNA. In the presence of cytosine arabinoside and mitomycin-C, which inhibited the synthesis of DNA to the extend of 75-80% the phosphorylation of lysine-rich histone was reduced by 50-60% suggesting the dependence of phosphorylation on the ongoing synthesis of DNA. (auth.)

Single-well monitoring of protein-protein interaction and phosphorylation-dephosphorylation events.

Science.gov (United States)

Arcand, Mathieu; Roby, Philippe; Bossé, Roger; Lipari, Francesco; Padrós, Jaime; Beaudet, Lucille; Marcil, Alexandre; Dahan, Sophie

2010-04-20

We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.

Industrial policy and technology diffusion : evidence from paper making machinery in Indonesia

NARCIS (Netherlands)

Dijk, van M.; Szirmai, A.

2006-01-01

In this paper, we analyze the diffusion and adoption of paper making machinery in the Indonesian pulp and paper industry, from 1923 till 2000. We develop a machine level index of technological sophistication (mach), which measures the technological distance of each paper machine to the world

dbPAF: an integrative database of protein phosphorylation in animals and fungi.

Science.gov (United States)

Ullah, Shahid; Lin, Shaofeng; Xu, Yang; Deng, Wankun; Ma, Lili; Zhang, Ying; Liu, Zexian; Xue, Yu

2016-03-24

Protein phosphorylation is one of the most important post-translational modifications (PTMs) and regulates a broad spectrum of biological processes. Recent progresses in phosphoproteomic identifications have generated a flood of phosphorylation sites, while the integration of these sites is an urgent need. In this work, we developed a curated database of dbPAF, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. From the scientific literature and public databases, we totally collected and integrated 54,148 phosphoproteins with 483,001 phosphorylation sites. Multiple options were provided for accessing the data, while original references and other annotations were also present for each phosphoprotein. Based on the new data set, we computationally detected significantly over-represented sequence motifs around phosphorylation sites, predicted potential kinases that are responsible for the modification of collected phospho-sites, and evolutionarily analyzed phosphorylation conservation states across different species. Besides to be largely consistent with previous reports, our results also proposed new features of phospho-regulation. Taken together, our database can be useful for further analyses of protein phosphorylation in human and other model organisms. The dbPAF database was implemented in PHP + MySQL and freely available at http://dbpaf.biocuckoo.org.

Phosphorylated benzimedazoles. 8. Synthesis of phosphorylated with /sup 32/P benzimidazoles

Energy Technology Data Exchange (ETDEWEB)

Makarov, A M; Matevosyan, G L; Zavlin, P M [Leningradskij Sel' skokhozyajstvennyj Inst. (USSR)

1983-03-01

Accessible methods of synthesis and identification of phosphorylated benzimidazoles with specific activity close to the maximum permissible with labelled /sup 32/P are developed. These methods permit to determine the permissible residual amounts of the above preparations in nutrition products and the maximum permissible amounts of growth regulators in different objects of the environment, because it is impossible to detect, for example, tri(1-benzimidazolido)phosphate with other physico-chemical methods with the existing concentration of 10/sup -9/%.

Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III Complexes

Directory of Open Access Journals (Sweden)

Jun Sumaoka

2016-01-01

Full Text Available Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear TbIII complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr, have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer and phosphothreonine (pThr, pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.

New Fault Recognition Method for Rotary Machinery Based on Information Entropy and a Probabilistic Neural Network.

Science.gov (United States)

Jiang, Quansheng; Shen, Yehu; Li, Hua; Xu, Fengyu

2018-01-24

Feature recognition and fault diagnosis plays an important role in equipment safety and stable operation of rotating machinery. In order to cope with the complexity problem of the vibration signal of rotating machinery, a feature fusion model based on information entropy and probabilistic neural network is proposed in this paper. The new method first uses information entropy theory to extract three kinds of characteristics entropy in vibration signals, namely, singular spectrum entropy, power spectrum entropy, and approximate entropy. Then the feature fusion model is constructed to classify and diagnose the fault signals. The proposed approach can combine comprehensive information from different aspects and is more sensitive to the fault features. The experimental results on simulated fault signals verified better performances of our proposed approach. In real two-span rotor data, the fault detection accuracy of the new method is more than 10% higher compared with the methods using three kinds of information entropy separately. The new approach is proved to be an effective fault recognition method for rotating machinery.

Phosphorylation of Nanog is Essential to Regulate Bmi1 and Promote Tumorigenesis

Science.gov (United States)

Xie, Xiujie; Piao, Longzhu; Cavey, Greg S.; Old, Matthew; Teknos, Theodoros N.; Mapp, Anna K; Pan, Quintin

2014-01-01

Emerging evidence indicates that Nanog is intimately involved in tumorigenesis in part through regulation of the cancer initiating cell population. However, the regulation and role of Nanog in tumorigenesis are still poorly understood. In this study, human Nanog was identified to be phosphorylated by human PKCε at multiple residues including T200 and T280. Our work indicated that phosphorylation at T200 and T280 modulates Nanog function through several regulatory mechanisms. Results with phosphorylation-insensitive and phosphorylation-mimetic mutant Nanog revealed that phosphorylation at T200 and T280 enhance Nanog protein stability. Moreover, phosphorylation-insensitive T200A and T280A mutant Nanog had a dominant-negative function to inhibit endogenous Nanog transcriptional activity. Inactivation of Nanog was due to impaired homodimerization, DNA binding, promoter occupancy, and p300, a transcriptional co-activator, recruitment resulting in a defect in target gene promoter activation. Ectopic expression of phosphorylation-insensitive T200A or T280A mutant Nanog reduced cell proliferation, colony formation, invasion, migration, and the cancer initiating cell population in head and neck squamous cell carcinoma (HNSCC) cells. The in vivo cancer initiating ability was severely compromised in HNSCC cells expressing phosphorylation-insensitive T200A or T280A mutant Nanog; 87.5% (14/16), 12.5% (1/8), and 0% (0/8) for control, T200A, and T280A, respectively. Nanog occupied the Bmi1 promoter to directly transactivate and regulate Bmi1. Genetic ablation and rescue experiments demonstrated that Bmi1 is a critical downstream signaling node for the pleiotropic, pro-oncogenic effects of Nanog. Taken together, our study revealed, for the first time, that post-translational phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis. PMID:23708658

Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

Energy Technology Data Exchange (ETDEWEB)

Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao, E-mail: hk228@med.kyushu-u.ac.jp

2013-04-05

Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

Effective reaction rates in diffusion-limited phosphorylation-dephosphorylation cycles

Science.gov (United States)

Szymańska, Paulina; Kochańczyk, Marek; Miekisz, Jacek; Lipniacki, Tomasz

2015-02-01

We investigate the kinetics of the ubiquitous phosphorylation-dephosphorylation cycle on biological membranes by means of kinetic Monte Carlo simulations on the triangular lattice. We establish the dependence of effective macroscopic reaction rate coefficients as well as the steady-state phosphorylated substrate fraction on the diffusion coefficient and concentrations of opposing enzymes: kinases and phosphatases. In the limits of zero and infinite diffusion, the numerical results agree with analytical predictions; these two limits give the lower and the upper bound for the macroscopic rate coefficients, respectively. In the zero-diffusion limit, which is important in the analysis of dense systems, phosphorylation and dephosphorylation reactions can convert only these substrates which remain in contact with opposing enzymes. In the most studied regime of nonzero but small diffusion, a contribution linearly proportional to the diffusion coefficient appears in the reaction rate. In this regime, the presence of opposing enzymes creates inhomogeneities in the (de)phosphorylated substrate distributions: The spatial correlation function shows that enzymes are surrounded by clouds of converted substrates. This effect becomes important at low enzyme concentrations, substantially lowering effective reaction rates. Effective reaction rates decrease with decreasing diffusion and this dependence is more pronounced for the less-abundant enzyme. Consequently, the steady-state fraction of phosphorylated substrates can increase or decrease with diffusion, depending on relative concentrations of both enzymes. Additionally, steady states are controlled by molecular crowders which, mostly by lowering the effective diffusion of reactants, favor the more abundant enzyme.

A Diagnosis Method for Rotation Machinery Faults Based on Dimensionless Indexes Combined with K-Nearest Neighbor Algorithm

Directory of Open Access Journals (Sweden)

Jianbin Xiong

2015-01-01

Full Text Available It is difficult to well distinguish the dimensionless indexes between normal petrochemical rotating machinery equipment and those with complex faults. When the conflict of evidence is too big, it will result in uncertainty of diagnosis. This paper presents a diagnosis method for rotation machinery fault based on dimensionless indexes combined with K-nearest neighbor (KNN algorithm. This method uses a KNN algorithm and an evidence fusion theoretical formula to process fuzzy data, incomplete data, and accurate data. This method can transfer the signals from the petrochemical rotating machinery sensors to the reliability manners using dimensionless indexes and KNN algorithm. The input information is further integrated by an evidence synthesis formula to get the final data. The type of fault will be decided based on these data. The experimental results show that the proposed method can integrate data to provide a more reliable and reasonable result, thereby reducing the decision risk.

Rapid changes in protein phosphorylation associated with gravity perception in corn roots

International Nuclear Information System (INIS)

McFadden, J.J.; Poovaiah, B.W.

1987-01-01

A previous paper from this laboratory showed calcium- and calmodulin-dependent in vivo protein phosphorylation in corn root tips. The authors show that rapid changes in calcium-dependent protein phosphorylation are involved in light-dependent graviperception in corn root tips. Corn seedlings (Zea mays L, cv Merit) were grown in the dark for 3 d, then apical root segments were harvested in dim green light to measure in vivo protein phosphorylation. Segments were incubated with 0.5 mCi 32 P for 1 h, then immediately frozen in liquid N 2 or first treated with either 7 min light, or 7 min light plus 1 mM EGTA and 10 μM A23187. Labeled proteins were separated by 2D gel electrophoresis and detected by autoradiography. Light caused rapid and specific promotion of phosphorylation of 5 polypeptides. The increases in protein phosphorylation were reversed by treating with EGTA and A23187. The authors postulate that these changes in protein phosphorylation are an essential part of the light-dependent gravity response in Merit roots

Industrial policy and technology diffusion : evidence from paper making machinery in Indonesia

NARCIS (Netherlands)

Dijk, van M.; Szirmai, A.

2005-01-01

In this paper, we analyse the diffusion and adoption of paper making machinery in the Indonesian pulp and paper industry, from 1923 till 2000. The two main questions in this paper are: (1) What patterns of technology diffusion can be discerned in the Indonesian paper manufacturing sector? (2) What

46 CFR 167.15-25 - Inspection standards for hulls, boilers and machinery.

Science.gov (United States)

2010-10-01

... SCHOOLS PUBLIC NAUTICAL SCHOOL SHIPS Inspections § 167.15-25 Inspection standards for hulls, boilers and... 46 Shipping 7 2010-10-01 2010-10-01 false Inspection standards for hulls, boilers and machinery... inspection. These rules may be purchased from the American Bureau of Shipping (ABS), ABS Plaza, 16855...

Stabilization of Microtubule-Unbound Tau via Tau Phosphorylation at Ser262/356 by Par-1/MARK Contributes to Augmentation of AD-Related Phosphorylation and Aβ42-Induced Tau Toxicity.

Directory of Open Access Journals (Sweden)

Kanae Ando

2016-03-01

Full Text Available Abnormal accumulation of the microtubule-interacting protein tau is associated with neurodegenerative diseases including Alzheimer's disease (AD. β-amyloid (Aβ lies upstream of abnormal tau behavior, including detachment from microtubules, phosphorylation at several disease-specific sites, and self-aggregation into toxic tau species in AD brains. To prevent the cascade of events leading to neurodegeneration in AD, it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. Currently, however, these mechanisms remain unclear. In this study, using transgenic Drosophila co-expressing human tau and Aβ, we found that tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau in the early phase of tau mismetabolism, leading to neurodegeneration. Aβ increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aβ42, and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aβ-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD.

Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis

DEFF Research Database (Denmark)

Miller, Martin Lee; Brunak, Søren; Olsen, JV

2010-01-01

and phosphorylation sites were grouped according to their cell cycle kinetics and compared to publicly available messenger RNA microarray data. Most detected phosphorylation sites and more than 20% of all quantified proteins showed substantial regulation, mainly in mitotic cells. Kinase-motif analysis revealed global...

Transportation of part supply improvement in agricultural machinery assembly plant

Science.gov (United States)

Saysaman, Anusit; Chutima, Parames

2018-02-01

This research focused on the problem caused by the transportation of part supply in agricultural machinery assembly plant in Thailand, which is one of the processes that are critical to the whole production process. If poorly managed, it will affect transportation of part supply, the emergence of sink cost, quality problems, and the ability to respond to the needs of the customers in time. Since the competition in the agricultural machinery market is more intense, the efficiency of part transportation process has to be improved. In this study, the process of transporting parts of the plant was studied and it was found that the efficiency of the process of transporting parts from the sub assembly line to its main assembly line was 83%. The approach to the performance improvement is done by using the Lean tool to limit wastes based on the ECRS principle and applying pull production system by changing the transportation method to operate as milkrun for transportation of parts to synchronize with the part demands of the main assembly line. After the transportation of parts from sub-assembly line to the main assembly line was improved, the efficiency raised to 98% and transportation process cost was saved to 540,000 Baht per year.

Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

International Nuclear Information System (INIS)

Diaz, Jason; Wang, Xin; Tsang, Sabrina H.; Jiao, Jing; You, Jianxin

2014-01-01

Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells

Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

Energy Technology Data Exchange (ETDEWEB)

Diaz, Jason; Wang, Xin; Tsang, Sabrina H. [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States); Jiao, Jing [Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 (United States); You, Jianxin, E-mail: jianyou@mail.med.upenn.edu [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States)

2014-07-08

Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

Assessment of the most significant causes of transportation and machinery accidents on collieries

CSIR Research Space (South Africa)

Oberholzer, JW

1995-08-01

Full Text Available The purpose of this study is to identify those areas, classified according to the SAMRASS data base system under the codes relating to underground transport and machinery type accidents that give cause to the greatest amount of accidents...

Identification and quantitation of signal molecule-dependent protein phosphorylation

KAUST Repository

Groen, Arnoud J.

2013-09-03

Phosphoproteomics is a fast-growing field that aims at characterizing phosphorylated proteins in a cell or a tissue at a given time. Phosphorylation of proteins is an important regulatory mechanism in many cellular processes. Gel-free phosphoproteome technique involving enrichment of phosphopeptide coupled with mass spectrometry has proven to be invaluable to detect and characterize phosphorylated proteins. In this chapter, a gel-free quantitative approach involving 15N metabolic labelling in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through the identification and quantification of responsive phospho(proteins). © Springer Science+Business Media New York 2013.

Phosphorylation site dynamics of early T-cell receptor signaling

DEFF Research Database (Denmark)

Chylek, Lily A; Akimov, Vyacheslav; Dengjel, Jörn

2014-01-01

In adaptive immune responses, T-cell receptor (TCR) signaling impacts multiple cellular processes and results in T-cell differentiation, proliferation, and cytokine production. Although individual protein-protein interactions and phosphorylation events have been studied extensively, we lack...... that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites...... a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found...

Characterization of a novel phosphorylation site in the sodium-chloride cotransporter, NCC

DEFF Research Database (Denmark)

Rosenbaek, L L; Assentoft, M; Pedersen, N B

2012-01-01

The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho-specific antib......The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho......-related proline-alanine-rich kinase and oxidative stress-response kinases (SPAK and OSR1) were not able to phosphorylate NCC at S124. Protein kinase arrays identified multiple kinases that were able to bind to the region surrounding S124. Four of these kinases (IRAK2, CDK6/Cyclin D1, NLK and m...

Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation

Directory of Open Access Journals (Sweden)

Raj Kumar

2008-08-01

Full Text Available Raj Kumar1, William J Calhoun21Division of Gastroenterology; 2Division of Allergy, Pulmonary, Immunology, Critical Care, and Sleep (APICS, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USAAbstract: Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade

Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation

Energy Technology Data Exchange (ETDEWEB)

Freire, Paula Paccielli, E-mail: freirepp@hotmail.com; Alves, Carlos Augusto Barnabe; Deus, Adriana Fernandes de [Departamento de Clínica Médica - Faculdade de Medicina de Botucatu - Universidade Estadual Paulista, Botucatu, SP (Brazil); Leopoldo, Ana Paula Lima; Leopoldo, André Soares [Centro de Educação Física e Desportos - Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Silva, Danielle Cristina Tomaz da; Tomasi, Loreta Casquel de; Campos, Dijon Henrique Salomé; Cicogna, Antonio Carlos [Departamento de Clínica Médica - Faculdade de Medicina de Botucatu - Universidade Estadual Paulista, Botucatu, SP (Brazil)

2014-07-15

The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP), alters the structure of protein kinase A (PKA) and leads to phospholamban (PLB) phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA. To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation. Male Wistar rats were randomly distributed into two groups: control (n = 14), fed with normocaloric diet; and obese (n = 13), fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index, and protein expressions of phosphatase 1 (PP-1), PKA, PLB, phosphorylated phospholamban at serine16 (PPLB-Ser16) were assessed by Western blot. Obesity caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hyperleptinemia and did not alter the protein expression of PKA, PP-1, PLB, PPLB-Ser16. Obesity does not promote an imbalance between myocardial PLB phosphorylation and dephosphorylation via beta-adrenergic system.

Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation

International Nuclear Information System (INIS)

Freire, Paula Paccielli; Alves, Carlos Augusto Barnabe; Deus, Adriana Fernandes de; Leopoldo, Ana Paula Lima; Leopoldo, André Soares; Silva, Danielle Cristina Tomaz da; Tomasi, Loreta Casquel de; Campos, Dijon Henrique Salomé; Cicogna, Antonio Carlos

2014-01-01

The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP), alters the structure of protein kinase A (PKA) and leads to phospholamban (PLB) phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA. To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation. Male Wistar rats were randomly distributed into two groups: control (n = 14), fed with normocaloric diet; and obese (n = 13), fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index, and protein expressions of phosphatase 1 (PP-1), PKA, PLB, phosphorylated phospholamban at serine16 (PPLB-Ser16) were assessed by Western blot. Obesity caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hyperleptinemia and did not alter the protein expression of PKA, PP-1, PLB, PPLB-Ser16. Obesity does not promote an imbalance between myocardial PLB phosphorylation and dephosphorylation via beta-adrenergic system

Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation

Directory of Open Access Journals (Sweden)

Paula Paccielli Freire

2014-07-01

Full Text Available Background: The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP, alters the structure of protein kinase A (PKA and leads to phospholamban (PLB phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA. Objective: To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation. Methods: Male Wistar rats were randomly distributed into two groups: control (n = 14, fed with normocaloric diet; and obese (n = 13, fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index, and protein expressions of phosphatase 1 (PP-1, PKA, PLB, phosphorylated phospholamban at serine16 (PPLB-Ser16 were assessed by Western blot. Results: Obesity caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hyperleptinemia and did not alter the protein expression of PKA, PP-1, PLB, PPLB-Ser16. Conclusion: Obesity does not promote an imbalance between myocardial PLB phosphorylation and dephosphorylation via beta-adrenergic system.

Synthesis of Isomeric Phosphoubiquitin Chains Reveals that Phosphorylation Controls Deubiquitinase Activity and Specificity

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Nicolas Huguenin-Dezot

2016-07-01

Full Text Available Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages.

The adaptive response of lichens to mercury exposure involves changes in the photosynthetic machinery

International Nuclear Information System (INIS)

Nicolardi, Valentina; Cai, Giampiero; Parrotta, Luigi; Puglia, Michele; Bianchi, Laura; Bini, Luca; Gaggi, Carlo

2012-01-01

Lichens are an excellent model to study the bioaccumulation of heavy metals but limited information is available on the molecular mechanisms occurring during bioaccumulation. We investigated the changes of the lichen proteome during exposure to constant concentrations of mercury. We found that most of changes involves proteins of the photosynthetic pathway, such as the chloroplastic photosystem I reaction center subunit II, the oxygen-evolving protein and the chloroplastic ATP synthase β-subunit. This suggests that photosynthesis is a target of the toxic effects of mercury. These findings are also supported by changes in the content of photosynthetic pigments (chlorophyll a and b, and β-carotene). Alterations to the photosynthetic machinery also reflect on the structure of thylakoid membranes of algal cells. Response of lichens to mercury also involves stress-related proteins (such as Hsp70) but not cytoskeletal proteins. Results suggest that lichens adapt to mercury exposure by changing the metabolic production of energy. - Highlights: ► Lichens exposed to Hg° vapors accumulate this metal irreversibly. ► Hg° interferes with physiological processes of the epiphytic lichen Evernia prunastri. ► Hg° promotes changes in the concentration of photosynthetic pigments. ► Hg° treatment causes changes in the ultrastructure of the photobiont plastids. ► Hg° induces changes in the protein machinery involved in the photosynthesis pathway. - Mercury affects the photosynthetic protein machinery of lichens.

Distribution pattern of histone H3 phosphorylation at serine 10

Indian Academy of Sciences (India)

We evaluated the pattern of H3 phosphorylation using immunodetection during mitosis and meiosis in both diploid and tetraploid genotypes of Brachiaria species. Results revealed differences in chromosome distribution of H3S10ph when mitosis and meiosis were compared. Whole chromosomes were phosphorylated ...

46 CFR 62.50-30 - Additional requirements for periodically unattended machinery plants.

Science.gov (United States)

2010-10-01

... include machinery plants and spaces that are automated to the degree that they are self-regulating and... engineers' accommodations. Other than fire or flooding alarms, this may be accomplished by summarized visual... in § 111.103 of this chapter; and (7) Voice communications with the bridge. (i) Oil leakage. Leakages...

Palytoxin and the sodium/potassium pump—phosphorylation and potassium interaction

International Nuclear Information System (INIS)

Rodrigues, Antônio M; De Almeida, Antônio-Carlos G; Infantosi, Antonio F C

2009-01-01

We proposed a reaction model for investigating interactions between K + and the palytoxin–sodium–potassium (PTX–Na + /K + ) pump complex under conditions where enzyme phosphorylation may occur. The model is composed of (i) the Albers–Post model for Na + /K + –ATPase, describing Na + and K + pumping; (ii) the reaction model proposed for Na + /K + –ATPase interactions with its ligands (Na + , K + , ATP, ADP and P) and with PTX. A mathematical model derived for representing the reactions was used to simulate experimental studies of the PTX-induced current, in different concentrations for the pump ligands. The simulations allow interpretation of the simultaneous action of Na + /K + –ATPase phosphorylation and K + on the PTX-induced channels. The results suggest that (i) phosphorylation increases the PTX toxic effect, increasing its affinity and reducing the K + occlusion rate, and (ii) K + causes channel blockage, increases the toxin dissociation rate and impedes the induced channel phosphorylation, implying reduction of the PTX toxic effect

Stress induces pain transition by potentiation of AMPA receptor phosphorylation.

Science.gov (United States)

Li, Changsheng; Yang, Ya; Liu, Sufang; Fang, Huaqiang; Zhang, Yong; Furmanski, Orion; Skinner, John; Xing, Ying; Johns, Roger A; Huganir, Richard L; Tao, Feng

2014-10-08

Chronic postsurgical pain is a serious issue in clinical practice. After surgery, patients experience ongoing pain or become sensitive to incident, normally nonpainful stimulation. The intensity and duration of postsurgical pain vary. However, it is unclear how the transition from acute to chronic pain occurs. Here we showed that social defeat stress enhanced plantar incision-induced AMPA receptor GluA1 phosphorylation at the Ser831 site in the spinal cord and greatly prolonged plantar incision-induced pain. Interestingly, targeted mutation of the GluA1 phosphorylation site Ser831 significantly inhibited stress-induced prolongation of incisional pain. In addition, stress hormones enhanced GluA1 phosphorylation and AMPA receptor-mediated electrical activity in the spinal cord. Subthreshold stimulation induced spinal long-term potentiation in GluA1 phosphomimetic mutant mice, but not in wild-type mice. Therefore, spinal AMPA receptor phosphorylation contributes to the mechanisms underlying stress-induced pain transition. Copyright © 2014 the authors 0270-6474/14/3413737-10$15.00/0.

Tau Phosphorylation by GSK3 in Different Conditions

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Jesús Avila

2012-01-01

Full Text Available Almost a 20% of the residues of tau protein are phosphorylatable amino acids: serine, threonine, and tyrosine. In this paper we comment on the consequences for tau of being a phosphoprotein. We will focus on serine/threonine phosphorylation. It will be discussed that, depending on the modified residue in tau molecule, phosphorylation could be protective, in processes like hibernation, or toxic like in development of those diseases known as tauopathies, which are characterized by an hyperphosphorylation and aggregation of tau.

Phosphorylation and disassembly of intermediate filaments in mitotic cells

International Nuclear Information System (INIS)

Chou, Yinghao; Rosevear, E.; Goldman, R.D.

1989-01-01

As baby hamster kidney (BHK-21) cells enter mitosis, networks of intermediate filaments (IFs) are transformed into cytoplasmic aggregates of protofilaments. Coincident with this morphological change, the phosphate content of vimentin increases from 0.3 mol of P i per mol of protein in interphase to 1.9 mol of P i per mol of protein in mitosis. A similar increase in phosphate content is observed with desmin, from 0.5 mol of P i per mol of protein to 1.5 mol of P i per mol of protein. Fractionation of mitotic cell lysates by hydroxylapatite column chromatography reveals the presence of two IF protein kinase activities, designated as IF protein kinase I and IF protein kinase II. Comparison of two-dimensional 32 P-labeled phosphopeptide maps of vimentin and desmin phosphorylated in vivo in mitosis, and in vitro using partially purified kinase fractions, reveals extensive similarity in the two sets of phosphorylation sites. Phosphorylation of in vitro polymerized IFs by IF protein kinase II induces complete disassembly as determined by negative-stain electron microscopy. The results support the idea that the disassembly of IFs in mitosis is regulated by the phosphorylation of its subunit proteins

Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

OpenAIRE

Vassin, Vitaly M.; Wold, Marc S.; Borowiec, James A.

2004-01-01

Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with re...

Phosphorylation regulates human T-cell leukemia virus type 1 Rex function

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Ward Michael

2009-11-01

Full Text Available Abstract Background Human T-cell leukemia virus type 1 (HTLV-1 is a pathogenic complex deltaretrovirus, which is the causative agent of adult T-cell leukemia/lymphoma (ATL and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic viral gene products, HTLV-1 encodes the positive regulatory proteins Tax and Rex along with viral accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes important in viral replication and cellular transformation. Rex is a nucleolar-localizing shuttling protein that acts post-transcriptionally by binding and facilitating the export of the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV-1 Rex (Rex-1 is a phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore, it has been proposed that Rex-1 function may be regulated through site-specific phosphorylation. Results We conducted a phosphoryl mapping of Rex-1 over-expressed in transfected 293 T cells using a combination of affinity purification and liquid chromatography tandem mass spectrometry. We achieved 100% physical coverage of the Rex-1 polypeptide and identified five novel phosphorylation sites at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed evidence of two previously identified residues, Ser-70 and Thr-174, but found no evidence of phosphorylation at Ser-177. The functional significance of these phosphorylation events was evaluated using a Rex reporter assay and site-directed mutational analysis. Our results indicate that phosphorylation at Ser-97 and Thr-174 is critical for Rex-1 function. Conclusion We have mapped completely the site-specific phosphorylation of Rex-1 identifying a total of seven residues; Thr-22, Ser-36, Thr-37, Ser-70, Ser-97, Ser-106, and Thr-174. Overall, this work is the first to completely map the phosphorylation sites in Rex-1 and provides important insight into

Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation

Science.gov (United States)

Chen, Long; Li, Zhiguo; Ahmad, Nihal; Liu, Xiaoqi

2016-01-01

Insulin receptor substrate (IRS) proteins play important roles by acting as a platform in transducing signals from transmembrane receptors upon growth factor stimulation. Although tyrosine phosphorylation on IRS proteins plays critical roles in signal transduction, phosphorylation of IRS proteins on serine/threonine residues are believed to play various regulatory roles on IRS protein function. However, studies on serine/threonine phosphorylation of IRS proteins are very limited, especially for insulin receptor substrate 2 (IRS2), one member of the IRS protein family. In this study, we identify Polo-like kinase 1 (Plk1) as the responsible kinase for phosphorylation of IRS2 on two serine residues, Ser 556 and Ser 1098. Phosphorylation of IRS2 on these two serine residues by Plk1 prevents the activation of the PI3K pathway upon growth factor stimulation by inhibiting the binding between IRS2 and the PI3K pathway components and increasing IRS2 protein degradation. Of significance, we show that IRS2 phosphorylation is cell cycle regulated and that Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation. PMID:25830382

Rapid changes in plasma membrane protein phosphorylation during initiation of cell wall digestion

International Nuclear Information System (INIS)

Blowers, D.P.; Boss, W.F.; Trewavas, A.J.

1988-01-01

Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [γ- 32 P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M r 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M r 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M r 22,000. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic 32 P also showed a response to the Driselase treatment. An enzymically active driselas preparation was required for the observed responses

Synthesis of rigid polyurethane foams from phosphorylated biopolyols.

Science.gov (United States)

de Haro, Juan Carlos; López-Pedrajas, Daniel; Pérez, Ángel; Rodríguez, Juan Francisco; Carmona, Manuel

2017-08-18

Renewable resources are playing a key role on the synthesis of biodegradable polyols. Moreover, the incorporation of covalently linked additives is increasing in importance in the polyurethane (PU) market. In this work, previously epoxidized grape seed oil and methyl oleate were transformed into phosphorylated biopolyols through an acid-catalyzed ring-opening hydrolysis in the presence of H 3 PO 4 . The formation of phosphate polyesters was confirmed by FT-IR and 31 P-NMR. However, the synthesis of a high-quality PU rigid foam was not possible using exclusively these polyols attending to their low hydroxyl value. In that way, different rigid PU foams were prepared from the phosphorylated biopolyols and the commercial polyol Alcupol R4520. It was observed that phosphorylated biopolyols can be incorporated up to a 57 wt.% in the PU synthesis without significant structural changes with respect to the commercial foam. Finally, thermogravimetric and EDAX analyses revealed an improvement of thermal stability by the formation of a protective phosphorocarbonaceous char layer.

NetPhosYeast: prediction of protein phosphorylation sites in yeast

DEFF Research Database (Denmark)

Ingrell, C.R.; Miller, Martin Lee; Jensen, O.N.

2007-01-01

sites compared to those in humans, suggesting the need for an yeast-specific phosphorylation site predictor. NetPhosYeast achieves a correlation coefficient close to 0.75 with a sensitivity of 0.84 and specificity of 0.90 and outperforms existing predictors in the identification of phosphorylation sites...

Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues

International Nuclear Information System (INIS)

Perez, L.; Aguilar, R.; Mendez, A.P.; de Jimenez, E.S.

1990-01-01

The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of [ 32 P] ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of 32 P incorporation and the electrophoretic patterns were dependent on 32 P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K m values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins

Methods for the Analysis of Protein Phosphorylation-Mediated Cellular Signaling Networks

Science.gov (United States)

White, Forest M.; Wolf-Yadlin, Alejandro

2016-06-01

Protein phosphorylation-mediated cellular signaling networks regulate almost all aspects of cell biology, including the responses to cellular stimulation and environmental alterations. These networks are highly complex and comprise hundreds of proteins and potentially thousands of phosphorylation sites. Multiple analytical methods have been developed over the past several decades to identify proteins and protein phosphorylation sites regulating cellular signaling, and to quantify the dynamic response of these sites to different cellular stimulation. Here we provide an overview of these methods, including the fundamental principles governing each method, their relative strengths and weaknesses, and some examples of how each method has been applied to the analysis of complex signaling networks. When applied correctly, each of these techniques can provide insight into the topology, dynamics, and regulation of protein phosphorylation signaling networks.

Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II

Science.gov (United States)

Zheng, Yupeng; John, Sam; Pesavento, James J.; Schultz-Norton, Jennifer R.; Schiltz, R. Louis; Baek, Sonjoon; Nardulli, Ann M.; Hager, Gordon L.; Kelleher, Neil L.

2010-01-01

Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. In this study, we show that specific sites in H1.2 and H1.4 of human cells are phosphorylated only during mitosis or during both mitosis and interphase. Antisera generated to individual H1.2/H1.4 interphase phosphorylations reveal that they are distributed throughout nuclei and enriched in nucleoli. Moreover, interphase phosphorylated H1.4 is enriched at active 45S preribosomal RNA gene promoters and is rapidly induced at steroid hormone response elements by hormone treatment. Our results imply that site-specific interphase H1 phosphorylation facilitates transcription by RNA polymerases I and II and has an unanticipated function in ribosome biogenesis and control of cell growth. Differences in the numbers, structure, and locations of interphase phosphorylation sites may contribute to the functional diversity of H1 variants. PMID:20439994

ATM phosphorylation in HepG2 cells following continuous low dose-rate irradiation

International Nuclear Information System (INIS)

Mei Quelin; Du Duanming; Chen Zaizhong; Liu Pengcheng; Yang Jianyong; Li Yanhao

2008-01-01

Objective: To investigate the change of ATM phosphorylation in HepG2 cells following a continuous low dose-rate irradiation. Methods: Cells were persistently exposed to low dose-rate (8.28 cGy/h) irradiation. Indirect immunofluorescence and Western blot were used to detect the expression of ATM phosphorylated proteins. Colony forming assay was used to observe the effect of a low dose-rate irradiation on HepG2 cell survival. Results: After 30 min of low dose-rate irradiation, the phosphorylation of ATM occurred. After 6 h persistent irradiation, the expression of ATM phosphorylated protein reached the peak value, then gradually decreased. After ATM phosphorylation was inhibited with Wortmannin, the surviving fraction of HepG2 cells was lower than that of the irradiation alone group at each time point (P<0.05). Conclusions: Continuous low dose-rate irradiation attenuated ATM phosphorylation, suggesting that continuous low dose-rate irradiation has a potential effect for increasing the radiosensitivity of HepG2 cells. (authors)

Targeting apoptotic machinery as approach for anticancer therapy: Smac mimetics as anticancer agents

Directory of Open Access Journals (Sweden)

Nevine M.Y. Elsayed

2015-06-01

Full Text Available Apoptosis is a chief regulator of cellular homeostasis. Impairment of apoptotic machinery is a main characteristic of several diseases such as cancer, where the evasion of apoptosis is a cardinal hallmark of cancer. Apoptosis is regulated by contribution of pro- and anti- apoptotic proteins, where caspases are the main executioners of the apoptotic machinery. IAP (inhibitors of apoptosis proteins is a family of endogenous inhibitors of apoptosis, which perform their function through interference with the function of caspases. Smac (second mitochondria-derived activator of caspases is endogenous inhibitor of IAPs, thus it is one of the major proapoptotic endogenous proteins. Thus, the development of Smac mimetics has evolved as an approach for anticancer therapy. Several Smac mimetic agents have been introduced to clinical trial such as birinapanet 12. Herein, the history of development of Smac mimetics along with the recent development in this field is briefly discussed.

4th International Conference on Condition Monitoring of Machinery in Non-Stationary Operations

CERN Document Server

Zimroz, Radoslaw; Bartelmus, Walter; Haddar, Mohamed

2016-01-01

The book provides readers with a snapshot of recent research and technological trends in the field of condition monitoring of machinery working under a broad range of operating conditions. Each chapter, accepted after a rigorous peer-review process, reports on an original piece of work presented and discussed at the 4th International Conference on Condition Monitoring of Machinery in Non-stationary Operations, CMMNO 2014, held on December 15-16, 2014, in Lyon, France. The contributions have been grouped into three different sections according to the main subfield (signal processing, data mining, or condition monitoring techniques) they are related to. The book includes both theoretical developments as well as a number of industrial case studies, in different areas including, but not limited to: noise and vibration; vibro-acoustic diagnosis; signal processing techniques; diagnostic data analysis; instantaneous speed identification; monitoring and diagnostic systems; and dynamic and fault modeling. This book no...

Research on a Rotating Machinery Fault Prognosis Method Using Three-Dimensional Spatial Representations

Directory of Open Access Journals (Sweden)

Xiaoni Dong

2016-01-01

Full Text Available Process models and parameters are two critical steps for fault prognosis in the operation of rotating machinery. Due to the requirement for a short and rapid response, it is important to study robust sensor data representation schemes. However, the conventional holospectrum defined by one-dimensional or two-dimensional methods does not sufficiently present this information in both the frequency and time domains. To supply a complete holospectrum model, a new three-dimensional spatial representation method is proposed. This method integrates improved three-dimensional (3D holospectra and 3D filtered orbits, leading to the integration of radial and axial vibration features in one bearing section. The results from simulation and experimental analysis on a complex compressor show that the proposed method can present the real operational status and clearly reveal early faults, thus demonstrating great potential for condition-based maintenance prediction in industrial machinery.

Developing an Online Learning Media Using Smartphone for the Electrical Machinery Course

Science.gov (United States)

Muchlas

2018-01-01

This research is aimed to prepare a desktop-based learning media that can be used to support an online lab activities using android smartphones in Electrical Machinery Course at the Department of Electrical Engineering for the undergraduate level. This work uses a conceptual development model which integrates some sub systems of internet…

Characterization and validation of new tools for measuring site-specific cardiac troponin I phosphorylation.

Science.gov (United States)

Thoemmes, Stephen F; Stutzke, Crystal A; Du, Yanmei; Browning, Michael D; Buttrick, Peter M; Walker, Lori A

2014-01-31

Phosphorylation of cardiac troponin I is a well established mechanism by which cardiac contractility is modulated. However, there are a number of phosphorylation sites on TnI which contribute singly or in combination to influence cardiac function. Accordingly, methods for accurately measuring site-specific TnI phosphorylation are needed. Currently, two strategies are employed: mass spectrometry, which is costly, difficult and has a low throughput; and Western blotting using phospho-specific antibodies, which is limited by the availability of reagents. In this report, we describe a cohort of new site-specific TnI phosphoantibodies, generated against physiologically relevant phosphorylation sites, that are superior to the current commercially available antibodies: to phospho-serine 22/23 which shows a >5-fold phospho-specificity for phosphorylated TnI; to phospho-serine 43, which has >3-fold phospho-specificity for phosphorylated TnI; and phospho-serine 150 which has >2-fold phospho-specificity for phosphorylated TnI. These new antibodies demonstrated greater sensitivity and specificity for the phosphorylated TnI than the most widely used commercially available reagents. For example, at a protein load of 20 μg of total cardiac extract, a commercially available antibody recognized both phosphorylated and dephosphorylated TnI to the same degree. At the same protein load our phospho-serine 22/23 antibody exhibited no cross-reactivity with dephosphorylated TnI. These new tools should allow a more accurate assessment and a better understanding of the role of TnI phosphorylation in the response of the heart to pathologic stress. Copyright © 2013 Elsevier B.V. All rights reserved.

A novel post-translational modification in nerve terminals: O-linked N-acetylglucosamine phosphorylation

DEFF Research Database (Denmark)

Graham, Mark E; Thaysen-Andersen, Morten; Bache, Nicolai

2011-01-01

Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved......NAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking...... phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification....

Pinpointing Phosphorylation Sites: Quantitative Filtering and a Novel Site-specific x-Ion Fragment

DEFF Research Database (Denmark)

Kelstrup, Christian D; Hekmat, Omid; Francavilla, Chiara

2011-01-01

Phosphoproteomics deals with the identification and quantification of thousands of phosphopeptides. Localizing the phosphorylation site is however much more difficult than establishing the identity of a phosphorylated peptide. Further, recent findings have raised doubts of the validity of the site......-phase phosphate rearrangement reactions during collision-induced dissociation (CID) and used these spectra to devise a quantitative filter that by comparing signal intensities of putative phosphorylated fragment ions with their nonphosphorylated counterparts allowed us to accurately pinpoint which fragment ions...... contain a phosphorylated residue and which ones do not. We also evaluated higher-energy collisional dissociation (HCD) and found this to be an accurate method for correct phosphorylation site localization with no gas-phase rearrangements observed above noise level. Analyzing a large set of HCD spectra...

Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

International Nuclear Information System (INIS)

Yoshida, Ikuma; Ibuki, Yuko

2014-01-01

Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications

Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

Energy Technology Data Exchange (ETDEWEB)

Yoshida, Ikuma; Ibuki, Yuko, E-mail: ibuki@u-shizuoka-ken.ac.jp

2014-12-15

Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.

Impact of SNPs on Protein Phosphorylation Status in Rice (Oryza sativa L.

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Shoukai Lin

2016-11-01

Full Text Available Single nucleotide polymorphisms (SNPs are widely used in functional genomics and genetics research work. The high-quality sequence of rice genome has provided a genome-wide SNP and proteome resource. However, the impact of SNPs on protein phosphorylation status in rice is not fully understood. In this paper, we firstly updated rice SNP resource based on the new rice genome Ver. 7.0, then systematically analyzed the potential impact of Non-synonymous SNPs (nsSNPs on the protein phosphorylation status. There were 3,897,312 SNPs in Ver. 7.0 rice genome, among which 9.9% was nsSNPs. Whilst, a total 2,508,261 phosphorylated sites were predicted in rice proteome. Interestingly, we observed that 150,197 (39.1% nsSNPs could influence protein phosphorylation status, among which 52.2% might induce changes of protein kinase (PK types for adjacent phosphorylation sites. We constructed a database, SNP_rice, to deposit the updated rice SNP resource and phosSNPs information. It was freely available to academic researchers at http://bioinformatics.fafu.edu.cn. As a case study, we detected five nsSNPs that potentially influenced heterotrimeric G proteins phosphorylation status in rice, indicating that genetic polymorphisms showed impact on the signal transduction by influencing the phosphorylation status of heterotrimeric G proteins. The results in this work could be a useful resource for future experimental identification and provide interesting information for better rice breeding.

In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage

International Nuclear Information System (INIS)

Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Samarth, Ravindra Mahadeo; Tomita, Masanori; Matsumoto, Yoshihisa

2016-01-01

XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells

Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury.

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Godar, Rebecca J; Ma, Xiucui; Liu, Haiyan; Murphy, John T; Weinheimer, Carla J; Kovacs, Attila; Crosby, Seth D; Saftig, Paul; Diwan, Abhinav

2015-01-01

Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEB-mediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fasting-induced autophagy in myocardial ischemia-reperfusion injury.

Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

Science.gov (United States)

Godar, Rebecca J; Ma, Xiucui; Liu, Haiyan; Murphy, John T; Weinheimer, Carla J; Kovacs, Attila; Crosby, Seth D; Saftig, Paul; Diwan, Abhinav

2015-01-01

Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEB-mediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fasting-induced autophagy in myocardial ischemia-reperfusion injury. PMID:26103523

Changes in phosphorylation of myofibrillar proteins during postmortem development of porcine muscle

DEFF Research Database (Denmark)

Huang, Honggang; Larsen, Martin Røssel; Lametsch, Rene

2012-01-01

A gel-based phosphoproteomic study was performed to investigate the postmortem (PM) changes in protein phosphorylation of the myofibrillar proteins in three groups of pigs with different pH decline rates, from PM 1 h to 24 h. The global phosphorylation level in the group with a fast pH decline rate...... was higher than that in the slow and intermediate groups at early PM time, but became the lowest at 24 h. The protein phosphorylation level of seven individual protein bands was only significantly (ppH...... phosphorylated protein bands with the highest scores. The results indicate that the phosphorylation pattern of myofibrillar proteins in PM muscle is mainly changed with PM time, but only to a minor extent influenced by the rate of pH decline, suggesting that the phosphorylation of myofibrillar proteins may...

Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis.

Science.gov (United States)

Cai, Hui; Zhang, Yu; Lu, Mijia; Liang, Xueya; Jennings, Ryan; Niewiesk, Stefan; Li, Jianrong

2016-08-15

Human metapneumovirus (hMPV) is a major causative agent of upper- and lower-respiratory-tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1, which plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated, but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization, whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using a reverse genetics system, recombinant hMPVs (rhMPVs) lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wild-type hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication, and pathogenesis in vivo The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that phosphorylation of M2-1 is essential for virus

Diagnostic suite neuro-fuzzy in an advanced alarm monitoring and predictive diagnostic system for rotating machinery

International Nuclear Information System (INIS)

Geruzzi, P.

1999-01-01

The 'Foxboro SCADA', former 'Automation Systems Division' of Nuovo Pignone, at the end of eighty years, has been involved in the development of a flexible and powerful Diagnostic System for Rotating Machinery designed and manufactured in other divisions of the Company. This system amalgamates, in a single computer, all the functionality nowadays necessary to correctly manage locally and remotely the Evolutionary Maintenance of rotating machines as well as the relevant plants. It's specially designed to plan preventive and emergency maintenance procedures and to help the maintenance staff/service in preventing the occurrence of failures or severe damage to complete turbo-machinery plant including turbine, compressor and other machines. The system is designed to supervise and to analyze the operating state of one or more turbo- machinery units such as turbo-compressors, turbo-generators and turbo-pumps giving an effective support to plan preventive and breakdown maintenance monitoring the performances of each turbo-group's element and analyzing a large number of thermodynamic and mechanical parameters related to high pressure turbines, low pressure turbines, combustion chambers, axial compressors and load (compressors, generators, and pumps). A brief presentation of the system is provided (author) (ml)

HSP20 phosphorylation and airway smooth muscle relaxation

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Mariam Ba

2009-06-01

Full Text Available Mariam Ba1, Cherie A Singer1, Manoj Tyagi2, Colleen Brophy3, Josh E Baker4, Christine Cremo4, Andrew Halayko5, William T Gerthoffer21Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA; 2Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA; 3Harrington Department of Biochemistry, Arizona State University, Tempe, AZ, USA; 4Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, USA; 5Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, MB, CanadaAbstract: HSP20 (HSPB6 is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbacholinduced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1, and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200–500 kDa to smaller complexes (<60 kDa. The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway

Kinome analysis of receptor-induced phosphorylation in human natural killer cells.

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Sebastian König

Full Text Available BACKGROUND: Natural killer (NK cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244 and DNAM-1 (CD226, act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome are involved in NK cell activation. RESULTS: A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2, FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. CONCLUSIONS: The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.

About phosphorylation of lappaconitine

International Nuclear Information System (INIS)

Burdelnaya, E.V.; Turmukhambetov, A.Zh.

2005-01-01

In the article chemical modifications of alkaloid lappaconitine are investigated. It was shown that synthesis of the phosphorylated derivatives are the ways to create new biologically active compounds. Interaction of lappaconitine with phosphorus pentachloride was used to obtain new phosphoric derivatives of alkaloid. The composition and structure of the new phosphorus-containing compounds were confirmed by elemental analysis: IR, UV and 13 C, 1 H, 31 P NMR -spectroscopy

Phosphorylation of p300 by ATM controls the stability of NBS1

International Nuclear Information System (INIS)

Jang, Eun Ryoung; Choi, Jae Duk; Jeong, Gajin; Lee, Jong-Soo

2010-01-01

Acetyltransferase, p300 is a transcriptional cofactor of signal-responsive transcriptional regulation. The surveillance kinase ataxia-telangiectasia mutated (ATM) plays a central role in regulation of a wide range of cellular DNA damage responses. Here, we investigated whether and how ATM mediates phosphorylation of p300 in response to DNA damage and how p300 phosphorylation is functionally linked to DNA damage. ATM-phosphorylated p300 in vitro and in vivo, in response to DNA damage. Phosphorylation of p300 proteins was observed upon γ-irradiation in ATM + cells but not ATM - cells. Importantly, expression of nonphosphorylatable serine to alanine form of p300 (S106A) destabilized both p300 and NBS1 proteins, after DNA damage. These data demonstrate that ATM transduces a DNA damage signal to p300, and that ATM-dependent phosphorylation of p300 is required for stabilization of NBS1 proteins in response to DNA damage.

Appetitive Pavlovian conditioned stimuli increase CREB phosphorylation in the nucleus accumbens.

Science.gov (United States)

Shiflett, Michael W; Mauna, Jocelyn C; Chipman, Amanda M; Peet, Eloise; Thiels, Edda

2009-10-01

The transcription factor cAMP response element-binding protein (CREB) in the nucleus accumbens (NAc) has been shown to regulate an animal's behavioral responsiveness to emotionally salient stimuli, and an increase in CREB phosphorylation in the NAc has been observed during exposure to rewarding stimuli, such as drugs of abuse. Here we show that CREB phosphorylation increases in the NAc also during exposure to cues that an animal has associated with delivery of natural rewards. Adult male Sprague-Dawley rats (rattus norvegicus) were trained to associate an auditory stimulus with delivery of food pellets, and CREB phosphorylation was examined in the striatum following training. We found that repeated tone-food pairings resulted in an increase in CREB phosphorylation in the NAc but not in the adjacent dorsal striatum or in the NAc 3h after the final training session. We further found that the cue itself, as opposed to the food pellets, the training context, or tone-food pairings, was sufficient to increase CREB phosphorylation in the NAc. These results suggest that the processing of primary rewarding stimuli and of environmental cues that predict them triggers similar accumbal signaling mechanisms.

Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

DEFF Research Database (Denmark)

Biondi, R M; Walz, K; Issinger, O G

1996-01-01

of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

Research Using Virtual Reality: Mobile Machinery Safety in the 21st Century

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Giselle P. Delgado

2013-04-01

Full Text Available Whole-body vibration is a significant health risk for between 4% and 7% of the work force in North America. In addition, many factors compound the health risks of heavy machinery operators. For example, twisted trunk and neck postures stiffen the spine and increase the transmission of vibration to the head. Similarly, workers adopt awkward postures in order to gain appropriate lines of sight for machine operations. Although the relative contribution of these various issues can be evaluated in field studies and models, we propose that virtual reality is a powerful medium for investigating issues related to health and safety in mining machine operators. We have collected field data of posture and vibration, as well as visual environment, for a forklift operating in a warehouse. This paper describes the process and outcome of this field data collection, and provides a discussion on the next steps to develop and test the virtual reality model to enable laboratory testing. Our ongoing studies will evaluate the interplay between posture and vibration under conditions replicating routine heavy machinery operations, such as underground mining.

49 CFR 1242.42 - Administration, repair and maintenance, machinery repair, equipment damaged, dismantling retired...

Science.gov (United States)

2010-10-01

... repair, equipment damaged, dismantling retired property, fringe benefits, other casualties and insurance, lease rentals, joint facility rents, other rents, depreciation, joint facility, repairs billed to others... maintenance, machinery repair, equipment damaged, dismantling retired property, fringe benefits, other...

Phosphorylation of rat thymus histones, its control and the effects thereon of γ-irradiation

International Nuclear Information System (INIS)

Fonagy, A.; Ord, M.G.; Stocken, L.A.

1977-01-01

The phosphate content of rat thymus histones was determined. As expected for a replicating tissue, histones 1 and 2B were more phosphorylated and had higher 32 P uptakes than did histones from resting liver nuclei; the other histones all showed 32 P uptake, but the phosphate content and uptake of histone 2A was about half that for liver histone 2A. When thymus nuclei were incubated in a slightly hypo-osmotic medium, non-histone proteins and phosphorylated histones were released into solution; this was enhanced if ATP was present in the medium. [γ- 32 P]ATP was incorporated into non-histone proteins, including Pl, and into the ADP-ribosylated form of histone 1; negligible 32 P was incorporated into the other, bound, histones. Histones 1 and 2B added to the incubation medium were extensively, and histones 2A and 4 slightly, phosphorylated. Histones released by increasing the ionic strength of the medium were phosphorylated. Added lysozyme and cytochrome c were neither bound nor phosphorylated, but added non-histone protein Pl was phosphorylated, causing other histones to be released from the nuclei, especially histones 2A and 3. The released histones were phosphorylated. γ-irradiation decreased 32 P uptake into the non-ADP-ribosylated histones 1 and 4; phosphorylation of histone 1 in vitro was unaffected. The importance of non-histone proteins, ATP availability and nuclear protein kinases to the control of histone phosphorylation in vivo is discussed. (author)

Postmortem Changes in Pork Muscle Protein Phosphorylation in Relation to the RN Genotype

DEFF Research Database (Denmark)

Lametsch, René; Larsen, Martin Røssel; Essén-Gustavsson, Birgitta

2011-01-01

Postmortem changes in pork muscle protein phosphorylation in relation to the RN(-) genotype were investigated using one-dimensional gel electrophoresis and a phosphor specific staining. The phosphorylation levels of several protein bands were found to be affected by the RN(-) genotype and to change...... of phosphorylation of these key enzymes during the postmortem metabolism. The results illustrate that the protein phosphorylation level of the muscle proteins could be interpreted as a global metabolic fingerprint containing information about the activity status of the enzymes in the postmortem metabolism....... during postmortem development. Glycogen phosphorylase, phosphofructokinase, and pyruvate kinase were found in protein bands affected by the RN(-) genotype, and the phosphorylation profile indicates that part of the increased rate and extended pH decline of the RN(-) genotype could be a consequence...

Characterization and Prediction of Protein Phosphorylation Hotspots in Arabidopsis thaliana.

Science.gov (United States)

Christian, Jan-Ole; Braginets, Rostyslav; Schulze, Waltraud X; Walther, Dirk

2012-01-01

The regulation of protein function by modulating the surface charge status via sequence-locally enriched phosphorylation sites (P-sites) in so called phosphorylation "hotspots" has gained increased attention in recent years. We set out to identify P-hotspots in the model plant Arabidopsis thaliana. We analyzed the spacing of experimentally detected P-sites within peptide-covered regions along Arabidopsis protein sequences as available from the PhosPhAt database. Confirming earlier reports (Schweiger and Linial, 2010), we found that, indeed, P-sites tend to cluster and that distributions between serine and threonine P-sites to their respected closest next P-site differ significantly from those for tyrosine P-sites. The ability to predict P-hotspots by applying available computational P-site prediction programs that focus on identifying single P-sites was observed to be severely compromised by the inevitable interference of nearby P-sites. We devised a new approach, named HotSPotter, for the prediction of phosphorylation hotspots. HotSPotter is based primarily on local amino acid compositional preferences rather than sequence position-specific motifs and uses support vector machines as the underlying classification engine. HotSPotter correctly identified experimentally determined phosphorylation hotspots in A. thaliana with high accuracy. Applied to the Arabidopsis proteome, HotSPotter-predicted 13,677 candidate P-hotspots in 9,599 proteins corresponding to 7,847 unique genes. Hotspot containing proteins are involved predominantly in signaling processes confirming the surmised modulating role of hotspots in signaling and interaction events. Our study provides new bioinformatics means to identify phosphorylation hotspots and lays the basis for further investigating novel candidate P-hotspots. All phosphorylation hotspot annotations and predictions have been made available as part of the PhosPhAt database at http://phosphat.mpimp-golm.mpg.de.

Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

DEFF Research Database (Denmark)

Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei

2015-01-01

Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial TRs on other types...

Methylmercury disrupts the balance between phosphorylated and non-phosphorylated cofilin in primary cultures of mice cerebellar granule cells A proteomic study

International Nuclear Information System (INIS)

Vendrell, Iolanda; Carrascal, Montserrat; Campos, Francisco; Abian, Joaquin; Sunol, Cristina

2010-01-01

Methylmercury is an environmental contaminant that is particularly toxic to the developing central nervous system; cerebellar granule neurons are especially vulnerable. Here, primary cultures of cerebellar granule cells (CGCs) were continuously exposed to methylmercury for up to 16 days in vitro (div). LC50 values were 508 ± 199, 345 ± 47, and 243 ± 45 nM after exposure for 6, 11, and 16 div, respectively. Proteins from cultured mouse CGCs were separated by 2DE. Seventy-one protein spots were identified by MALDI-TOF PMF and MALDI-TOF/TOF sequencing. Prolonged exposure to a subcytotoxic concentration of methylmercury significantly increased non-phosphorylated cofilin both in cell protein extracts (1.4-fold; p < 0.01) and in mitochondrial-enriched fractions (1.7-fold; p < 0.01). The decrease in P-cofilin induced by methylmercury was concentration-dependent and occurred after different exposure times. The percentage of P-cofilin relative to total cofilin significantly decreased to 49 ± 13% vs. control cells after exposure to 300 nM methylmercury for 5 div. The balance between the phosphorylated and non-phosphorylated form of cofilin regulates actin dynamics and facilitates actin filament turnover. Filamentous actin dynamics and reorganization are responsible of neuron shape change, migration, polarity formation, regulation of synaptic structures and function, and cell apoptosis. An alteration of the complex regulation of the cofilin phosphorylation/dephosphorylation pathway could be envisaged as an underlying mechanism compatible with reported signs of methylmercury-induced neurotoxicity.

Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

Science.gov (United States)

Morgan, Mark R.; Hamidi, Hellyeh; Bass, Mark D.; Warwood, Stacey; Ballestrem, Christoph; Humphries, Martin J.

2013-01-01

Summary Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration. PMID:23453597

Protein phosphorylation in bcterial signaling and regulation

KAUST Repository

Mijakovic, Ivan

2016-01-26

In 2003, it was demonstrated for the first time that bacteria possess protein-tyrosine kinases (BY-kinases), capable of phosphorylating other cellular proteins and regulating their activity. It soon became apparent that these kinases phosphorylate a number of protein substrates, involved in different cellular processes. More recently, we found out that BY-kinases can be activated by several distinct protein interactants, and are capable of engaging in cross-phosphorylation with other kinases. Evolutionary studies based on genome comparison indicate that BY-kinases exist only in bacteria. They are non-essential (present in about 40% bacterial genomes), and their knockouts lead to pleiotropic phenotypes, since they phosphorylate many substrates. Surprisingly, BY-kinase genes accumulate mutations at an increased rate (non-synonymous substitution rate significantly higher than other bacterial genes). One direct consequence of this phenomenon is no detectable co-evolution between kinases and their substrates. Their promiscuity towards substrates thus seems to be “hard-wired”, but why would bacteria maintain such promiscuous regulatory devices? One explanation is the maintenance of BY-kinases as rapidly evolving regulators, which can readily adopt new substrates when environmental changes impose selective pressure for quick evolution of new regulatory modules. Their role is clearly not to act as master regulators, dedicated to triggering a single response, but they might rather be employed to contribute to fine-tuning and improving robustness of various cellular responses. This unique feature makes BY-kinases a potentially useful tool in synthetic biology. While other bacterial kinases are very specific and their signaling pathways insulated, BY-kinase can relatively easily be engineered to adopt new substrates and control new biosynthetic processes. Since they are absent in humans, and regulate some key functions in pathogenic bacteria, they are also very promising

Folding machineries displayed on a cation-exchanger for the concerted refolding of cysteine- or proline-rich proteins

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Lee Dae-Hee

2009-03-01

Full Text Available Abstract Background Escherichia coli has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in E. coli frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant E. coli. Here, we describe the successful use of the immobilized folding machineries for in vitro refolding with the examples of high yield refolding of a ribonuclease A (RNase A and cyclohexanone monooxygenase (CHMO. Results We have generated refolding-facilitating media immobilized with three folding machineries, mini-chaperone (a monomeric apical domain consisting of residues 191–345 of GroEL and two foldases (DsbA and human peptidyl-prolyl cis-trans isomerase by mimicking oxidative refolding chromatography. For efficient and simple purification and immobilization simultaneously, folding machineries were fused with the positively-charged consecutive 10-arginine tag at their C-terminal. The immobilized folding machineries were fully functional when assayed in a batch mode. When the refolding-facilitating matrices were applied to the refolding of denatured and reduced RNase A and CHMO, both of which contain many cysteine and proline residues, RNase A and CHMO were recovered in 73% and 53% yield of soluble protein with full enzyme activity, respectively. Conclusion The refolding-facilitating media presented here could be a cost-efficient platform and should be applicable to refold a wide range of E. coli inclusion bodies in high yield with biological function.

Common extraction of Tc, Pd and Eu by phosphorylated calixarenes

International Nuclear Information System (INIS)

Babain, V.; Smirnov, I.; Kvasnitskiy, I.; Karavan, M.; Boiko, V.; Miroshnichenko, V.; Klimchuk, O.; Kalchenko, V.

2003-01-01

The present work is aimed at studying the extraction systems based on neutral organophosphorus extractants - phosphorylated calixarenes for recovery of Pd and Tc together with Am and Cm from high-level radioactive wastes. Extraction of Pd, Tc and Eu (Am) was studied for phosphorylated calixarenes in meta-nitrobenzotrifluoride (NBTF). Main results are presented in Table. On the basis of available data one can suggest that type and position of phosphor-organic substituents are not so important for extraction of Tc and Pd, as for Eu and Am extraction. The phosphorylated at upper rim calix[4]arenas with small alkyl substituents at phosphorus atom are of prime interest for joint recovery of europium, americium, technetium and palladium. (authors)

Necrosome core machinery: MLKL.

Science.gov (United States)

Zhang, Jing; Yang, Yu; He, Wenyan; Sun, Liming

2016-06-01

In the study of regulated cell death, the rapidly expanding field of regulated necrosis, in particular necroptosis, has been drawing much attention. The signaling of necroptosis represents a sophisticated form of a death pathway. Anti-caspase mechanisms (e.g., using inhibitors of caspases, or genetic ablation of caspase-8) switch cell fate from apoptosis to necroptosis. The initial extracellular death signals regulate RIP1 and RIP3 kinase activation. The RIP3-associated death complex assembly is necessary and sufficient to initiate necroptosis. MLKL was initially identified as an essential mediator of RIP1/RIP3 kinase-initiated necroptosis. Recent studies on the signal transduction using chemical tools and biomarkers support the idea that MLKL is able to make more functional sense for the core machinery of the necroptosis death complex, called the necrosome, to connect to the necroptosis execution. The experimental data available now have pointed that the activated MLKL forms membrane-disrupting pores causing membrane leakage, which extends the prototypical concept of morphological and biochemical events following necroptosis happening in vivo. The key role of MLKL in necroptosis signaling thus sheds light on the logic underlying this unique "membrane-explosive" cell death pathway. In this review, we provide the general concepts and strategies that underlie signal transduction of this form of cell death, and then focus specifically on the role of MLKL in necroptosis.

Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

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Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

2017-10-01

Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

A Simple Numerical Body Surface Mapping Parameter Signifies Successful Percutaneous Coronary Artery Intervention.

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Simonyi, Gábor; Kirschner, Róbert; Szűcs, Endre; Préda, István; Duray, Gábor; Medvegy, Nóra; Horvath, Bálint; Medvegy, Mihály

2016-03-01

In coronary artery disease (CAD), body surface potential mapping (BSPM) may reveal minor electrical potential changes appearing in the depolarization phase even if pathological changes are absent on the conventional 12-lead ECG. We hypothesized that a simple BSPM parameter, Max/Min signifies successful percutaneous coronary intervention (PCI). Ninety-two adult Caucasian patients with stable CAD and positive exercise test underwent coronary angiography. Seventy patients (age, 59 ± 8; 46 males) were revascularized by PCI (left anterior descending [LAD] in 38, right [RCA] in 17 and left circumflex [LCX] coronary artery in 15). Control groups contained 22 patients (age, 60 ± 8; 14 males) without intervention and 35 healthy subjects (age, 58 ± 2; 15 males). Left ventricular ejection fraction (LVEF, transthoracic echocardiography) and Max/Min BSPM parameter (63-lead Montreal system) were evaluated before and 4-40 days following coronary angiography. Max/Min was defined by the ratio of the highest maximum to the deepest minimum potential of all leads recorded by BSPM. Before PCI, Max/Min value of patients with LAD lesion (0.83 [0.74; 0.93]) was significantly lower while that with RCA lesion (1.63 [1.35; 1.99]) was significantly higher than that of healthy group (1.01 [0.970; 1.13]) (P intervention. LVEF significantly increased (from 46.50% [43.00; 51.00] to 49.00% [46.00; 51.00]) only after LAD PCI. Max/Min parameter is suitable to follow patients after LAD and RCA PCI. © 2015 Wiley Periodicals, Inc.

Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.

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Gilles Gouspillou

Full Text Available BACKGROUND: Mitochondrial dysfunctions appear strongly implicated in a wide range of pathologies. Therefore, there is a growing need in the determination of the normal and pathological integrated response of oxidative phosphorylation to cellular ATP demand. The present study intends to address this issue by providing a method to investigate mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria. METHODOLOGY/PRINCIPAL FINDINGS: The proposed method is based on the simultaneous monitoring of substrate oxidation (determined polarographically and phosphorylation (determined using the glucose-hexokinase glucose-6-phosphate dehydrogenase-NADP(+ enzymatic system rates, coupled to the determination of actual ADP and ATP concentrations by bioluminescent assay. This enzymatic system allows the study of oxidative phosphorylation during true steady states in a wide range of ADP concentrations. We demonstrate how the application of this method allows an accurate determination of mitochondrial affinity for ADP from both oxidation (K(mVox and phosphorylation (K(mVp rates. We also demonstrate that determination of K(mVox leads to an important overestimation of the mitochondrial affinity for ADP, indicating that mitochondrial affinity for ADP should be determined using phosphorylation rate. Finally, we show how this method allows the direct and precise determination of the mitochondrial coupling efficiency. Data obtained from rat skeletal muscle and liver mitochondria illustrate the discriminating capabilities of this method. CONCLUSIONS/SIGNIFICANCE: Because the proposed method allows the accurate determination of mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria, it also opens the route to a better understanding of functional consequences of mitochondrial adaptations/dysfunctions arising in various physiological/pathophysiological conditions.

A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-Crk signaling

Directory of Open Access Journals (Sweden)

Hanafusa Hidesaburo

2002-07-01

Full Text Available Abstract Background The adaptor protein p130Cas (Cas has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals. Results We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk. Conclusion Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.

Quantifying Kinase-Specific Phosphorylation Stoichiometry Using Stable Isotope Labeling In a Reverse In-Gel Kinase Assay

Energy Technology Data Exchange (ETDEWEB)

Li, Xiang; Cox, Jonathan T.; Huang, Weiliang; Kane, Maureen; Tang, Keqi; Bieberich, Charles J.

2016-12-06

Reversible protein phosphorylation regulates essentially all cellular activities. Aberrant protein phosphorylation is an etiological factor in a wide array of diseases, including cancer1, diabetes2, and Alzheimer’s3. Given the broad impact of protein phosphorylation on cellular biology and organismal health, understanding how protein phosphorylation is regulated and the consequences of gain and loss of phosphoryl moieties from proteins is of primary importance. Advances in instrumentation, particularly in mass spectrometry, coupled with high throughput approaches have recently yielded large datasets cataloging tens of thousands of protein phosphorylation sites in multiple organisms4-6. While these studies are seminal in term of data collection, our understanding of protein phosphorylation regulation remains largely one-dimensional.

Phosphorylation of myelin basic proteins and its relevance to myelin biogenesis

Energy Technology Data Exchange (ETDEWEB)

Ulmer, J.B.

1985-01-01

Age-related differences in the in vivo incorporation of (32-P) into mouse myelin basic proteins (MBPs) of the central nervous system were observed. The resulting specific radioactivity (S.A.) of the MBPs appeared to be related to the S.A. of the acid-soluble pool of phosphates of myelin. In development, MBPs were phosphorylated in vivo prior to the onset of myelination in the brain, indicating that MBPs are phosphorylated prior to their deposition in the myelin sheath. The incorporation of (32-P) into MBPs and the turnover rates of MBP phosphates were studied in vivo in developmentally-related myelin compartments. The results suggest that there are two separate events in MBP phosphorylation and that the turnover rates of the MBP phosphates derived from these two events are different. A model for MBP phosphorylation, that could explain in these observations, is postulated and discussed in the light of existing information.

Tyrosine phosphorylation in signal transduction

International Nuclear Information System (INIS)

Roberts, T.M.; Kaplan, D.; Morgan, W.; Keller, T.; Mamon, H.; Piwnica-Worms, H.; Druker, B.; Whitman, M.; Morrison, D.; Cohen, B.; Schaffhausen, B.; Cantley, L.; Rapp, U.

1988-01-01

Recent work has focused on the elucidation of the mechanisms by which membrane-bound tyrosine kinases transmit signals within the cell. To examine the role of tyrosine phosphorylation the authors have employed the following strategy. First, they have utilized antibodies to phosphotyrosine (anti-P.Tyr) to identify candidate substrates of various tyrosine kinases, such as pp60 c-src , the CSF- receptor, or the platelet-derived growth factor (PDGF) receptor. Second, they have attempted to characterize the biochemical properties of the putative substrates and to determine in what manner these properties are modified by phosphorylation on tyrosine residues. In this endeavor, they are recapitulating the classic biochemical analysis used to study the effect of kinases on metabolism. The final portion of our work consists of using modern molecular biological strategies to clone the genes or cDNAs for the substrates and overproduce the relevant proteins for studies in vitro in defined systems. This paper describes the first and second aspects of this strategy, the identification and characterization of novel substrate molecules

Conformational Clusters of Phosphorylated Tyrosine.

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Abdelrasoul, Maha; Ponniah, Komala; Mao, Alice; Warden, Meghan S; Elhefnawy, Wessam; Li, Yaohang; Pascal, Steven M

2017-12-06

Tyrosine phosphorylation plays an important role in many cellular and intercellular processes including signal transduction, subcellular localization, and regulation of enzymatic activity. In 1999, Blom et al., using the limited number of protein data bank (PDB) structures available at that time, reported that the side chain structures of phosphorylated tyrosine (pY) are partitioned into two conserved conformational clusters ( Blom, N.; Gammeltoft, S.; Brunak, S. J. Mol. Biol. 1999 , 294 , 1351 - 1362 ). We have used the spectral clustering algorithm to cluster the increasingly growing number of protein structures with pY sites, and have found that the pY residues cluster into three distinct side chain conformations. Two of these pY conformational clusters associate strongly with a narrow range of tyrosine backbone conformation. The novel cluster also highly correlates with the identity of the n + 1 residue, and is strongly associated with a sequential pYpY conformation which places two adjacent pY side chains in a specific relative orientation. Further analysis shows that the three pY clusters are associated with distinct distributions of cognate protein kinases.

Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes.

Science.gov (United States)

Van Schaftingen, E; Vandercammen, A

1989-01-15

The phosphorylation of glucose was measured by the formation of [3H]H2O from [2-3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2-4-fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system. A half-maximal stimulatory effect was observed at about 50 microM fructose. Stimulation was maximal 5 min after addition of the ketose and was stable for at least 40 min, during which period 60% of the fructose was consumed. The effect of fructose was reversible upon removal of the ketose. Sorbitol and tagatose were as potent as fructose in stimulating the phosphorylation of 5 mM glucose. D-Glyceraldehyde also had a stimulatory effect but at tenfold higher concentrations. In contrast, dihydroxyacetone had no significant effect and glycerol inhibited the detritiation of glucose. Oleate did not affect the phosphorylation of glucose, even in the presence of fructose, although it stimulated the formation of ketone bodies severalfold, indicating that it was converted to its acyl-CoA derivative. These results allow the conclusion that fructose stimulates glucokinase in the intact hepatocyte. They also suggest that this effect is mediated through the formation of fructose 1-phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long-chain acyl-CoAs.

Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis

DEFF Research Database (Denmark)

Confalonieri, S; Salcini, A E; Puri, C

2000-01-01

for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function....... Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization...... of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular...

Developing CNS mitochondria oxidative phosphorylation P/O/ADP/O index for rats

International Nuclear Information System (INIS)

Egana, E.; Diaz, G.

1975-01-01

The effect of whole-body-gamma irradiation on developing CNS mitochondria oxidative phosphorylation was studied through the P/O/ADP/O index; three irradiation doses (5, 50 and 500 R) were employed at neonatal stage and both 'prompt' (10 min approx,) and 'delayed' (7 days for 500 R exposure, 21 days for 5 and 50 R) effects were observed. In the 'prompt' effects investigated after 500 R exposure, the oxidative phosphorylation diminished; the same occurred at 7 days with this dose ('delayed' effect). With doses of 5 and 50 R there was no alteration of oxidative phosphorylation as a 'prompt' effect, but it diminished at 21 days post irradiation. The uncoupling between respiration and oxidative phosphorylation should explain - at least, in part -these results. (author)

Developing CNS mitochondria oxidative phosphorylation P/O/ADP/O index for rats

Energy Technology Data Exchange (ETDEWEB)

Egana, E; Diaz, G [Institute of Experimental Medicine, Santiago (Chile). Lab. of Neurochemistry

1975-11-01

The effect of whole-body-gamma irradiation on developing CNS mitochondria oxidative phosphorylation was studied through the P/O/ADP/O index; three irradiation doses (5, 50 and 500 R) were employed at neonatal stage and both 'prompt' (10 min approx,) and 'delayed' (7 days for 500 R exposure, 21 days for 5 and 50 R) effects were observed. In the 'prompt' effects investigated after 500 R exposure, the oxidative phosphorylation diminished; the same occurred at 7 days with this dose ('delayed' effect). With doses of 5 and 50 R there was no alteration of oxidative phosphorylation as a 'prompt' effect, but it diminished at 21 days post irradiation. The uncoupling between respiration and oxidative phosphorylation should explain - at least, in part -these results.

Proteomic Analysis of Calcium- and Phosphorylation-dependentCalmodulin Complexes in Mammalian Cells

Energy Technology Data Exchange (ETDEWEB)

Jang, Deok-Jin; Wang, Daojing

2006-05-26

Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca{sup 2+}) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca{sup 2+}/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed 'Dynamic Phosphoprotein Complex Trapping', 'DPPC Trapping' for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulin-binding proteins are then trapped in vitro in a Ca{sup 2+}-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitin-protein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP{sub 3}R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP{sub 3}R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca{sup 2+}-dependent CaM binding. However, pSer/Thr of IP{sub 3}R1 did not substantially affect its Ca{sup 2+}-independent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B

Cdc15 Phosphorylates the C-terminal Domain of RNA Polymerase II for Transcription during Mitosis.

Science.gov (United States)

Singh, Amit Kumar; Rastogi, Shivangi; Shukla, Harish; Asalam, Mohd; Rath, Srikanta Kumar; Akhtar, Md Sohail

2017-03-31

In eukaryotes, the basal transcription in interphase is orchestrated through the regulation by kinases (Kin28, Bur1, and Ctk1) and phosphatases (Ssu72, Rtr1, and Fcp1), which act through the post-translational modification of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD comprises the repeated Tyr-Ser-Pro-Thr-Ser-Pro-Ser motif with potential epigenetic modification sites. Despite the observation of transcription and periodic expression of genes during mitosis with entailing CTD phosphorylation and dephosphorylation, the associated CTD specific kinase(s) and its role in transcription remains unknown. Here we have identified Cdc15 as a potential kinase phosphorylating Ser-2 and Ser-5 of CTD for transcription during mitosis in the budding yeast. The phosphorylation of CTD by Cdc15 is independent of any prior Ser phosphorylation(s). The inactivation of Cdc15 causes reduction of global CTD phosphorylation during mitosis and affects the expression of genes whose transcript levels peak during mitosis. Cdc15 also influences the complete transcription of clb2 gene and phosphorylates Ser-5 at the promoter and Ser-2 toward the 3' end of the gene. The observation that Cdc15 could phosphorylate Ser-5, as well as Ser-2, during transcription in mitosis is in contrast to the phosphorylation marks put by the kinases in interphase (G 1 , S, and G 2 ), where Cdck7/Kin28 phosphorylates Ser-5 at promoter and Bur1/Ctk1 phosphorylates Ser-2 at the 3' end of the genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Reconstitution of Biosynthetic Machinery for the Synthesis of the Highly Elaborated Indole Diterpene Penitrem

DEFF Research Database (Denmark)

Liu, Chengwei; Tagami, Koichi; Minami, Atsushi

2015-01-01

KULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products....

Molecular mechanism of APC/C activation by mitotic phosphorylation.

Science.gov (United States)

Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

2016-05-12

In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

Testing whether Metazoan Tyrosine Loss Was Driven by Selection against Promiscuous Phosphorylation

Science.gov (United States)

Pandya, Siddharth; Struck, Travis J.; Mannakee, Brian K.; Paniscus, Mary; Gutenkunst, Ryan N.

2015-01-01

Protein tyrosine phosphorylation is a key regulatory modification in metazoans, and the corresponding kinase enzymes have diversified dramatically. This diversification is correlated with a genome-wide reduction in protein tyrosine content, and it was recently suggested that this reduction was driven by selection to avoid promiscuous phosphorylation that might be deleterious. We tested three predictions of this intriguing hypothesis. 1) Selection should be stronger on residues that are more likely to be phosphorylated due to local solvent accessibility or structural disorder. 2) Selection should be stronger on proteins that are more likely to be promiscuously phosphorylated because they are abundant. We tested these predictions by comparing distributions of tyrosine within and among human and yeast orthologous proteins. 3) Selection should be stronger against mutations that create tyrosine versus remove tyrosine. We tested this prediction using human population genomic variation data. We found that all three predicted effects are modest for tyrosine when compared with the other amino acids, suggesting that selection against deleterious phosphorylation was not dominant in driving metazoan tyrosine loss. PMID:25312910

Phosphorylation of carbonic anhydrase IX controls its ability to mediate extracellular acidification in hypoxic tumors.

Science.gov (United States)

Ditte, Peter; Dequiedt, Franck; Svastova, Eliska; Hulikova, Alzbeta; Ohradanova-Repic, Anna; Zatovicova, Miriam; Csaderova, Lucia; Kopacek, Juraj; Supuran, Claudiu T; Pastorekova, Silvia; Pastorek, Jaromir

2011-12-15

In the hypoxic regions of a tumor, carbonic anhydrase IX (CA IX) is an important transmembrane component of the pH regulatory machinery that participates in bicarbonate transport. Because tumor pH has implications for growth, invasion, and therapy, determining the basis for the contributions of CA IX to the hypoxic tumor microenvironment could lead to new fundamental and practical insights. Here, we report that Thr443 phosphorylation at the intracellular domain of CA IX by protein kinase A (PKA) is critical for its activation in hypoxic cells, with the fullest activity of CA IX also requiring dephosphorylation of Ser448. PKA is activated by cAMP, which is elevated by hypoxia, and we found that attenuating PKA in cells disrupted CA IX-mediated extracellular acidification. Moreover, following hypoxia induction, CA IX colocalized with the sodium-bicarbonate cotransporter and other PKA substrates in the leading edge membranes of migrating tumor cells, in support of the concept that bicarbonate metabolism is spatially regulated at cell surface sites with high local ion transport and pH control. Using chimeric CA IX proteins containing heterologous catalytic domains derived from related CA enzymes, we showed that CA IX activity was modulated chiefly by the intracellular domain where Thr443 is located. Our findings indicate that CA IX is a pivotal mediator of the hypoxia-cAMP-PKA axis, which regulates pH in the hypoxic tumor microenvironment.

Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery.

Science.gov (United States)

Murphy, Anar K; Fitzgerald, Michael; Ro, Teresa; Kim, Jee Hyun; Rabinowitsch, Ariana I; Chowdhury, Dipanjan; Schildkraut, Carl L; Borowiec, James A

2014-08-18

Phosphorylation of replication protein A (RPA) by Cdk2 and the checkpoint kinase ATR (ATM and Rad3 related) during replication fork stalling stabilizes the replisome, but how these modifications safeguard the fork is not understood. To address this question, we used single-molecule fiber analysis in cells expressing a phosphorylation-defective RPA2 subunit or lacking phosphatase activity toward RPA2. Deregulation of RPA phosphorylation reduced synthesis at forks both during replication stress and recovery from stress. The ability of phosphorylated RPA to stimulate fork recovery is mediated through the PALB2 tumor suppressor protein. RPA phosphorylation increased localization of PALB2 and BRCA2 to RPA-bound nuclear foci in cells experiencing replication stress. Phosphorylated RPA also stimulated recruitment of PALB2 to single-strand deoxyribonucleic acid (DNA) in a cell-free system. Expression of mutant RPA2 or loss of PALB2 expression led to significant DNA damage after replication stress, a defect accentuated by poly-ADP (adenosine diphosphate) ribose polymerase inhibitors. These data demonstrate that phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress. © 2014 Murphy et al.

Proteomic analysis of tyrosine phosphorylation during human liver transplantation

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Boutros Tarek

2007-01-01

Full Text Available Abstract Background Ischemia-reperfusion (I/R causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. Results Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. Conclusion Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

Study of ATM Phosphorylation by Cdk5 in Neuronal Cells.

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She, Hua; Mao, Zixu

2017-01-01

The phosphatidylinositol-3-kinase-like kinase ATM (ataxia-telangiectasia mutated) plays a central role in coordinating the DNA damage responses including cell cycle checkpoint control, DNA repair, and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. We previously showed that Cdk5 (cyclin-dependent kinase 5) is activated by DNA damage and directly phosphorylates ATM at serine 794 in postmitotic neurons. Phosphorylation at serine 794 precedes and is required for ATM autophosphorylation at serine 1981, and activates ATM kinase activity. Cdk5-ATM pathway plays a crucial role in DNA damage-induced neuronal injury. This chapter describes protocols used in analyzing ATM phosphorylation by Cdk5 in CGNs (cerebellar granule neurons) and its effects on neuronal survival.

A CK2 site is reversibly phosphorylated in the photosystem II subunit CP29.

Science.gov (United States)

Testi, M G; Croce, R; Polverino-De Laureto, P; Bassi, R

1996-12-16

Protein phosphorylation is a major mechanism in the regulation of protein function. In chloroplast thylakoids several photosystem II subunits, including the major antenna light-harvesting complex II and several core complex components, are reversibly phosphorylated depending on the redox state of the electron carriers. A previously unknown reversible phosphorylation event has recently been described on the CP29 subunit which leads to conformational changes and protection from cold stress (Bergantino, E., Dainese, P., Cerovic, Z. Sechi, S. and Bassi, R. (1995) J. Biol Chem. 270, 8474-8481). In this study, we have identified the phosphorylation site on the N-terminal, stroma-exposed domain, showing that it is located in a sequence not homologous to the other members of the Lhc family. The phosphorylated sequence is unique in chloroplast membranes since it meets the requirements for CK2 (casein kinase II) kinases. The possibility that this phosphorylation is involved in a signal transduction pathway is discussed.

Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+.

Science.gov (United States)

Villalobo, A; Lehninger, A L

1980-03-25

Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.

Specific mixing facilitates the comparative quantification of phosphorylation sites with significant dysregulations

Energy Technology Data Exchange (ETDEWEB)

Liu, Jing [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xu, Bo [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); Liu, Zheyi; Dong, Mingming; Mao, Jiawei; Zhou, Ye; Chen, Jin [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Wang, Fangjun, E-mail: wangfj@dicp.ac.cn [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); Zou, Hanfa [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China)

2017-01-15

Mass spectrometry (MS) based quantitative analyses of proteome and proteome post-translational modifications (PTMs) play more and more important roles in biological, pharmaceutical and clinical studies. However, it is still a big challenge to accurately quantify the proteins or proteins PTM sites with extreme relative abundances in comparative protein samples, such as the significantly dysregulated ones. Herein, a novel quantification strategy, Mixing at Specific Ratio (MaSR) before isotope labeling, had been developed to improve the quantification accuracy and coverage of extreme proteins and protein phosphorylation sites. Briefly, the comparative protein samples were firstly mixed together at specific ratios of 9:1 and 1:9 (w/w), followed with mass differentiate light and heavy isotope labeling, respectively. The extreme proteins and protein phosphorylation sites, even if the newly expressed or disappeared ones, could be accurately quantified due to all of the proteins' relative abundances had been adjusted to 2 orders of magnitude (1/9-9) by this strategy. The number of quantified phosphorylation sites with more than 20 folds changes was improved about 10 times in comparative quantification of pervanadate stimulated phosphoproteome of HeLa cells, and 134 newly generated and 21 disappeared phosphorylation sites were solely quantified by the MaSR strategy. The significantly up-regulated phosphorylation sites were mainly involved in the key phosphoproteins regulating the insulin-related pathways, such as PI3K-AKT and RAS-MAPK pathways. Therefore, the MaSR strategy exhibits as a promising way in elucidating the biological processes with significant dysregulations. - Highlights: • All the proteins' relative abundances were adjusted into 2 orders of magnitude (1/9-9). • The quantification accuracy and coverage of extreme proteins and protein phosphorylation sites had been improved. • The newly expressed or disappeared proteins and protein

Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S. cerevisiae

DEFF Research Database (Denmark)

Rudashevskaya, Elena; Ye, Juanying; Young, Clifford

 It is known, that phosphorylation of both plant and yeast plasma membrane H+-ATPase results in enzyme activation or inhibition. Several sites at the regulatory C-terminus of the enzyme have been found to undergo phosphorylation in vivo in both plant and yeast. The C-termini of plant H...... of heterologous system of yeast cells, expressing plant proton pump. Therefore identification of possible regulatory effects by phosphorylation events in plant H+-ATPase in the system is significant. A number of putative phosphorylation sites at regulatory C-domain of H+-ATPase (AHA2) have been point...... functioning of the residues and suggests, that plant H+-ATPase could be regulated by phosphorylation at several sites being in yeast cells. Plant H+-ATPase purified from yeast cells by his-tag affinity chromatography was subjected to IMAC and TiO2 for enrichment of phosphopeptides. The phosphopeptides were...

Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, Bombyx mori

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Shi-Hong Gu

2018-02-01

Full Text Available In this study, phosphorylation of c-Jun N-terminal kinase (JNK by the prothoracicotropic hormone (PTTH was investigated in prothoracic glands (PGs of the silkworm, Bombyx mori. Results showed that JNK phosphorylation was stimulated by the PTTH in time- and dose-dependent manners. In vitro activation of JNK phosphorylation in PGs by the PTTH was also confirmed in an in vivo experiment, in which a PTTH injection greatly increased JNK phosphorylation in PGs of day-6 last instar larvae. JNK phosphorylation caused by PTTH stimulation was greatly inhibited by U73122, a potent and specific inhibitor of phospholipase C (PLC and an increase in JNK phosphorylation was also detected when PGs were treated with agents (either A23187 or thapsigargin that directly elevated the intracellular Ca2+ concentration, thereby indicating involvement of PLC and Ca2+. Pretreatment with an inhibitor (U0126 of mitogen-activated protein kinase (MAPK/extracellular signal-regulated kinase (ERK kinase (MEK and an inhibitor (LY294002 of phosphoinositide 3-kinase (PI3K failed to significantly inhibit PTTH-stimulated JNK phosphorylation, indicating that ERK and PI3K were not related to JNK. We further investigated the effect of modulation of the redox state on JNK phosphorylation. In the presence of either an antioxidant (N-acetylcysteine, NAC or diphenylene iodonium (DPI, PTTH-stimulated JNK phosphorylation was blocked. The JNK kinase inhibitor, SP600125, markedly inhibited PTTH-stimulated JNK phosphorylation and ecdysteroid synthesis. The kinase assay of JNK in PGs confirmed its stimulation by PTTH and inhibition by SP600125. Moreover, PTTH treatment did not affect JNK or Jun mRNA expressions. Based on these findings, we concluded that PTTH stimulates JNK phosphorylation in Ca2+- and PLC-dependent manners and that the redox-regulated JNK signaling pathway is involved in PTTH-stimulated ecdysteroid synthesis in B. mori PGs.

Petri net-based prediction of therapeutic targets that recover abnormally phosphorylated proteins in muscle atrophy.

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Jung, Jinmyung; Kwon, Mijin; Bae, Sunghwa; Yim, Soorin; Lee, Doheon

2018-03-05

Muscle atrophy, an involuntary loss of muscle mass, is involved in various diseases and sometimes leads to mortality. However, therapeutics for muscle atrophy thus far have had limited effects. Here, we present a new approach for therapeutic target prediction using Petri net simulation of the status of phosphorylation, with a reasonable assumption that the recovery of abnormally phosphorylated proteins can be a treatment for muscle atrophy. The Petri net model was employed to simulate phosphorylation status in three states, i.e. reference, atrophic and each gene-inhibited state based on the myocyte-specific phosphorylation network. Here, we newly devised a phosphorylation specific Petri net that involves two types of transitions (phosphorylation or de-phosphorylation) and two types of places (activation with or without phosphorylation). Before predicting therapeutic targets, the simulation results in reference and atrophic states were validated by Western blotting experiments detecting five marker proteins, i.e. RELA, SMAD2, SMAD3, FOXO1 and FOXO3. Finally, we determined 37 potential therapeutic targets whose inhibition recovers the phosphorylation status from an atrophic state as indicated by the five validated marker proteins. In the evaluation, we confirmed that the 37 potential targets were enriched for muscle atrophy-related terms such as actin and muscle contraction processes, and they were also significantly overlapping with the genes associated with muscle atrophy reported in the Comparative Toxicogenomics Database (p-value net. We generated a list of the potential therapeutic targets whose inhibition recovers abnormally phosphorylated proteins in an atrophic state. They were evaluated by various approaches, such as Western blotting, GO terms, literature, known muscle atrophy-related genes and shortest path analysis. We expect the new proposed strategy to provide an understanding of phosphorylation status in muscle atrophy and to provide assistance towards

Flux control through protein phosphorylation in yeast

DEFF Research Database (Denmark)

Chen, Yu; Nielsen, Jens

2016-01-01

Protein phosphorylation is one of the most important mechanisms regulating metabolism as it can directly modify metabolic enzymes by the addition of phosphate groups. Attributed to such a rapid and reversible mechanism, cells can adjust metabolism rapidly in response to temporal changes. The yeast...... as well as identify mechanisms underlying human metabolic diseases. Here we collect functional phosphorylation events of 41 enzymes involved in yeast metabolism and demonstrate functional mechanisms and the application of this information in metabolic engineering. From a systems biology perspective, we...... describe the development of phosphoproteomics in yeast as well as approaches to analysing the phosphoproteomics data. Finally, we focus on integrated analyses with other omics data sets and genome-scale metabolic models. Despite the advances, future studies improving both experimental technologies...

A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

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Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

2016-04-01

In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

Energy Technology Data Exchange (ETDEWEB)

Adi, Y. A., E-mail: yudi.adi@math.uad.ac.id [Department of Mathematic Faculty of MIPA Universitas Ahmad Dahlan (Indonesia); Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Kusumo, F. A.; Aryati, L. [Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Hardianti, M. S. [Department of Internal Medicine, Faculty of Medicine, Universitas Gadjah Mada (Indonesia)

2016-04-06

In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

The role of glucocorticoid receptor phosphorylation in Mcl-1 and NOXA gene expression

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Demonacos Constantinos

2010-02-01

Full Text Available Abstract Background The cyclin-dependent kinase (CDK and mitogen-activated protein kinase (MAPK mediated phosphorylation of glucocorticoid receptor (GR exerts opposite effects on GR transcriptional activity and affects other posttranslational modifications within this protein. The major phosphorylation site of human GR targeted by MAPK family is the serine 226 and multiple kinase complexes phosphorylate receptor at the serine 211 residue. We hypothesize that GR posttranslational modifications are involved in the determination of the cellular fate in human lymphoblastic leukemia cells. We investigated whether UV signalling through alternative GR phosphorylation determined the cell type specificity of glucocorticoids (GCs mediated apoptosis. Results We have identified putative Glucocorticoid Response Elements (GREs within the promoter regulatory regions of the Bcl-2 family members NOXA and Mcl-1 indicating that they are direct GR transcriptional targets. These genes were differentially regulated in CEM-C7-14, CEM-C1-15 and A549 cells by glucocorticoids and JNK pathway. In addition, our results revealed that the S211 phosphorylation was dominant in CEM-C7-14, whereas the opposite was the case in CEM-C1-15 where prevalence of S226 GR phosphorylation was observed. Furthermore, multiple GR isoforms with cell line specific patterns were identified in CEM-C7-14 cells compared to CEM-C1-15 and A549 cell lines with the same antibodies. Conclusions GR phosphorylation status kinetics, and site specificity as well as isoform variability differ in CEM-C7-14, CEM-C1-15, and A549 cells. The positive or negative response to GCs induced apoptosis in these cell lines is a consequence of the variable equilibrium of NOXA and Mcl-1 gene expression potentially mediated by alternatively phosphorylated GR, as well as the balance of MAPK/CDK pathways controlling GR phosphorylation pattern. Our results provide molecular base and valuable knowledge for improving the GC

OPTIMIZATION MODEL FOR VEHICLE ROUTING AND EQUIPMENT REPLACEMENT IN FARM MACHINERY

OpenAIRE

Grano, Carolina; Abensur, Eder

2017-01-01

ABSTRACT: An equipment replacement decision takes into account economic engineering models based on discounted cash flow (DCF) such as the Annual Equivalent Cost (AEC). Despite a large number of researches on industrial assets replacement, there is a lack of studies applied to farm goods. This study aimed at assessing an alternative model for economic decision analysis on farm machinery replacement, with no restrictions on the number of replacements and assessed goods during a defined timelin...

Triple Recycling Channel Strategies for Remanufacturing of Construction Machinery in a Retailer-Dominated Closed-Loop Supply Chain

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Min Huang

2017-12-01

Full Text Available Firms engaged in remanufacturing activities generally adopt more than one recycling channel to collect more used products and gain more profits. This paper explores the optimal strategies for a retailer-dominated closed-loop supply chain (CLSC with a triple recycling channel in the construction machinery remanufacturing context. In this special system, the retailer is the leader and authorized by the original equipment manufacturer (OEM to remanufacture. Moreover, the OEM, the retailer, and the secondary market all take part in the used products collection activities. Considering the differentiation of the OEM, the retailer, and the secondary market in collecting the used construction machinery, a mathematical model of the CLSC system based on reasonable assumptions is built, the closed-form optimal pricing decisions are derived, and the optimal collection efforts allocation strategies are explored within the framework of the game theory. In addition, the impacts of the reverse logistics cost coefficient, the competing coefficient, and the buy-back price coefficient on the supply chain performance are elaborately analyzed. These achievements provide decision makers with managerial insights and offer efficient guidelines for the construction machinery remanufacturing firms to solve similar puzzles.

Myosin light chain phosphorylation is critical for adaptation to cardiac stress.

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Warren, Sonisha A; Briggs, Laura E; Zeng, Huadong; Chuang, Joyce; Chang, Eileen I; Terada, Ryota; Li, Moyi; Swanson, Maurice S; Lecker, Stewart H; Willis, Monte S; Spinale, Francis G; Maupin-Furlowe, Julie; McMullen, Julie R; Moss, Richard L; Kasahara, Hideko

2012-11-27

Cardiac hypertrophy is a common response to circulatory or neurohumoral stressors as a mechanism to augment contractility. When the heart is under sustained stress, the hypertrophic response can evolve into decompensated heart failure, although the mechanism(s) underlying this transition remain largely unknown. Because phosphorylation of cardiac myosin light chain 2 (MLC2v), bound to myosin at the head-rod junction, facilitates actin-myosin interactions and enhances contractility, we hypothesized that phosphorylation of MLC2v plays a role in the adaptation of the heart to stress. We previously identified an enzyme that predominantly phosphorylates MLC2v in cardiomyocytes, cardiac myosin light-chain kinase (cMLCK), yet the role(s) played by cMLCK in regulating cardiac function in health and disease remain to be determined. We found that pressure overload induced by transaortic constriction in wild-type mice reduced phosphorylated MLC2v levels by ≈40% and cMLCK levels by ≈85%. To examine how a reduction in cMLCK and the corresponding reduction in phosphorylated MLC2v affect function, we generated Mylk3 gene-targeted mice and transgenic mice overexpressing cMLCK specifically in cardiomyocytes. Pressure overload led to severe heart failure in cMLCK knockout mice but not in mice with cMLCK overexpression in which cMLCK protein synthesis exceeded degradation. The reduction in cMLCK protein during pressure overload was attenuated by inhibition of ubiquitin-proteasome protein degradation systems. Our results suggest the novel idea that accelerated cMLCK protein turnover by the ubiquitin-proteasome system underlies the transition from compensated hypertrophy to decompensated heart failure as a result of reduced phosphorylation of MLC2v.

Phosphorylated form of adrenocorticotropin and corticotropin-like intermediary lobe peptide in human tumors

International Nuclear Information System (INIS)

Massias, J.F.; Hardouin, S.; Vieau, D.; Lenne, F.; Bertagna, X.

1994-01-01

Many peptides contribute to the heterogeneity of immunoreactive adrenocorticotropin (ACTH) in man. The use of a radioimmunoassay (RIA) specifically directed against the C-terminal end of ACTH allowed the precise study of the following four peptides: ACTH itself, corticotropin-like intermediary lobe peptide (CLIP) or ACTH and their phosphorylated forms on SeR 31 . The authors have set up a high-performance liquid chromatography system that separates these four molecules in a single run, to establish their relative distributions in tumors responsible for Cushing's disease or for the ectopic ACTH syndrome, and to evaluate the possible interference of phospho-Ser 31 on various RIA or immuno-radiometric assay (IRMA) recognition systems for ACTH. In this system, alkaline phosphatase treatment shifted the retention time of the phosphorylated peptides to that of their non-phosphorylated counterparts. In three tumors responsible for the ectopic ACTH syndrome, CLIP peptides were predominant in two and phosphorylated molecules represented between 22% and 50% of immuno-reactive materials. In five pituitary tumors responsible for Cushing's disease, ACTH peptides were predominant and the phosphorylated molecules varied between 35% and 75% in four of them. In the same tumor the ratios of phosphorylated to non-phosphorylated CLIP or ACTH were identical. The presence of phospho-Ser 31 did not affect the recognition ability of two mid-ACTH and two C-terminal ACTH RIA's, nor of the ACTH IRMA. 15 refs., 5 figs., 2 tabs

Neuroinflammation is not a prerequisite for diabetes-induced tau phosphorylation

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Judith M Van Der Harg

2015-11-01

Full Text Available Abnormal phosphorylation and aggregation of tau is a key hallmark of Alzheimer's disease (AD. AD is a multifactorial neurodegenerative disorder for which Diabetes Mellitus (DM is a risk factor. In animal models for DM, the phosphorylation and aggregation of tau is induced or exacerbated, however the underlying mechanism is unknown. In addition to the metabolic dysfunction, DM is characterized by chronic low-grade inflammation. This was reported to be associated with a neuroinflammatory response in the hypothalamus of DM animal models. Neuroinflammation is also implicated in the development and progression of AD. It is unknown whether DM also induces neuroinflammation in brain areas affected in AD, the cortex and hippocampus. Here we investigated whether neuroinflammation could be the mechanistic trigger to induce tau phosphorylation in the brain of DM animals. Two distinct diabetic animal models were used; rats on free-choice high-fat high-sugar (fcHFHS diet that are insulin resistant and streptozotocin-treated rats that are insulin deficient. The streptozotocin-treated animals demonstrated increased tau phosphorylation in the brain as expected, whereas the fcHFHS diet fed animals did not. Remarkably, neither of the diabetic animal models showed reactive microglia or increased GFAP and COX-2 levels in the cortex or hippocampus. From this, we conclude: 1. DM does not induce neuroinflammation in brain regions affected in AD, and 2. Neuroinflammation is not a prerequisite for tau phosphorylation. Neuroinflammation is therefore not the mechanism that explains the close connection between DM and AD.

Protein Kinase B/Akt Binds and Phosphorylates PED/PEA-15, Stabilizing Its Antiapoptotic Action

OpenAIRE

Trencia, Alessandra; Perfetti, Anna; Cassese, Angela; Vigliotta, Giovanni; Miele, Claudia; Oriente, Francesco; Santopietro, Stefania; Giacco, Ferdinando; Condorelli, Gerolama; Formisano, Pietro; Beguinot, Francesco

2003-01-01

The antiapoptotic protein PED/PEA-15 features an Akt phosphorylation motif upstream from Ser116. In vitro, recombinant PED/PEA-15 was phosphorylated by Akt with a stoichiometry close to 1. Based on Western blotting with specific phospho-Ser116 PED/PEA-15 antibodies, Akt phosphorylation of PED/PEA-15 occurred mainly at Ser116. In addition, a mutant of PED/PEA-15 featuring the substitution of Ser116→Gly (PEDS116→G) showed 10-fold-decreased phosphorylation by Akt. In intact 293 cells, Akt also i...

The Regulation of NF-κB Subunits by Phosphorylation

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Frank Christian

2016-03-01

Full Text Available The NF-κB transcription factor is the master regulator of the inflammatory response and is essential for the homeostasis of the immune system. NF-κB regulates the transcription of genes that control inflammation, immune cell development, cell cycle, proliferation, and cell death. The fundamental role that NF-κB plays in key physiological processes makes it an important factor in determining health and disease. The importance of NF-κB in tissue homeostasis and immunity has frustrated therapeutic approaches aimed at inhibiting NF-κB activation. However, significant research efforts have revealed the crucial contribution of NF-κB phosphorylation to controlling NF-κB directed transactivation. Importantly, NF-κB phosphorylation controls transcription in a gene-specific manner, offering new opportunities to selectively target NF-κB for therapeutic benefit. This review will focus on the phosphorylation of the NF-κB subunits and the impact on NF-κB function.

Trihydrophobin 1 Phosphorylation by c-Src Regulates MAPK/ERK Signaling and Cell Migration

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Wu, Weibin; Sun, Zhichao; Wu, Jingwen; Peng, Xiaomin; Gan, Huacheng; Zhang, Chunyi; Ji, Lingling; Xie, Jianhui; Zhu, Haiyan; Ren, Shifang

2012-01-01

c-Src activates Ras-MAPK/ERK signaling pathway and regulates cell migration, while trihydrophobin 1 (TH1) inhibits MAPK/ERK activation and cell migration through interaction with A-Raf and PAK1 and inhibiting their kinase activities. Here we show that c-Src interacts with TH1 by GST-pull down assay, coimmunoprecipitation and confocal microscopy assay. The interaction leads to phosphorylation of TH1 at Tyr-6 in vivo and in vitro. Phosphorylation of TH1 decreases its association with A-Raf and PAK1. Further study reveals that Tyr-6 phosphorylation of TH1 reduces its inhibition on MAPK/ERK signaling, enhances c-Src mediated cell migration. Moreover, induced tyrosine phosphorylation of TH1 has been found by EGF and estrogen treatments. Taken together, our findings demonstrate a novel mechanism for the comprehensive regulation of Ras/Raf/MEK/ERK signaling and cell migration involving tyrosine phosphorylation of TH1 by c-Src. PMID:22238675

Histone phosphorylation during radiation-induced mitotic delay in synchronous plasmodia of Physarum polycephalum

International Nuclear Information System (INIS)

Brewer, E.N.; Oleinick, N.L.

1980-01-01

Using the nearly perfect synchrony of the mitotic stages in Physarum plasmodia, and making use of 32 P as a tracer, studies were made to define the time course of histone phosphorylation during the late G2 and prophase and the alterations in that time course accompanying radiation-induced mitotic delay. Histone H1 was phosphorylated throughout the last 2-3 hours of the mitotic cycle coincident with the early stages of chromosome condensation. H1 phosphorylation appeared to be reduced in irradiated plasmodia. It is postulated that a longer time period, i.e. the mitotic delay, may be required to obtain the same eventual level of H1-phosphate. In normal cultures, nucleosome core histones were phosphorylated late in G2 and prophase, the peak corresponding closely with the γ-transition point. In irradiated plasmodia, phosphorylation of the core histones had an extended time course similar to H1. (U.K.)

Topographical distribution of phosphorylation sites of phosvitins by mass spectrometry.

Science.gov (United States)

Czernick, Drew; Liu, Jess; Serge, Dibart; Salih, Erdjan

2013-05-27

Phosvitin, derived from the vitellogenin II gene protein, is a highly phosphorylated protein found in egg yolk. A second hypothetical protein has been predicted based on the vitellogenin I gene, but has not been defined at the protein level. Mass spectrometric analysis was used to identify the phosphopeptide sequences and the precise sites of phosphorylation of two phosvitins, phosvitin 1 and phosvitin 2 derived from vitellogenins I and II, respectively. Samples of native phosvitin were subjected to tryptic digestion followed by mass spectrometric analysis: (i) native phosvitin peptides, (ii) after treatment with NaOH, and (iii) after chemical derivatization of P-Ser/P-Thr residues by dithiothreitol under base-catalyzed conditions. A combination of these approaches led to the identification of 68 and 35 phosphopeptides with 89 (81 P-Ser and 8 P-Thr residues) and 62 (57 P-Ser and 5 P-Thr residues) phosphorylation sites of phosvitin 1 and phosvitin 2, respectively. These data for the first time documented on a large scale the major states and sites of phosphorylation of phosvitins with a total of 151 phosphorylation sites. Importantly, the present work also provided the first direct de novo protein amino-acid sequence data for phosvitin 1 protein and evidence for the full expression of vitellogenin I gene. We have for the first time generated a large number of phosphopeptides (~100) and identified 151 phosphorylation sites of phosvitin 1 and phosvitin 2, respectively. Importantly, this study also led to the discovery of a novel phosvitin 1 and provided the first direct de novo protein amino-acid sequence data for the full expression of vitellogenin I gene. There is considerable interest in naturally occurring phosphopeptides/phosphoproteins and their application in biomedical fields and in the food industry because of their molecular characteristics and non-toxic nature, hence, our work opens new avenues to pursue such endeavors. In addition, the results provide

77 FR 64462 - Conference on International Trade Issues Facing U.S. Machinery Manufacturing Industries

Science.gov (United States)

2012-10-22

... manufacturing is one of the largest and most competitive sectors of the American manufacturing economy. The... billion in 2011. The economic impact of machinery manufacturing extends throughout the American economy... service industries. Process control and other automation technologies enable end-users to maximize the...

Legal regulations of restrictions of air pollution made by non-road mobile machinery-the case study for Europe: a review.

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Waluś, Konrad J; Warguła, Łukasz; Krawiec, Piotr; Adamiec, Jarosław M

2018-02-01

The high awareness of intensification and frequency of smog phenomenon all over the world in XXI age makes for detailed analyses of the reasons of its formation and prevention. The governments of the developed countries and conscious of real hazards, including many European countries, aim to restrict the emission of harmful gases. In literature, we can find the discussions on the influence of this phenomenon on the health and life of inhabitants of contaminated areas. Some elaborations of prognostic models, descriptions of pollution sources, the manner of their restriction, and the analysis of causal-consecutive correlation are also popular. The influence of pollutions resulting from the operation of vehicles, planes, and the industry are well described. However, every machine and device which is driven with a combustion engine has the effect on the general level of anthropogenic pollutions. These drives are subject of different regulations limiting their emission for service conditions and applications. One of the groups of such machines described in European and American regulations is non-road mobile machinery. The aim of this paper is the presentation of the problem of weak analysis and application of engineering and technological tools for machinery drive emission, despite of many publications on hazards and problems of emission. These machines have the influence on both the increase of global contamination and the machine users. The regulations of the European Union take into consideration the generated hazards and restrict the emission of machine exhaust gases by approval tests-these regulations are continually improved, and the effects of these works are new emission limits in 2019. However, these activities seem to be liberal as opposed to limits of the emission for passenger and goods vehicles where the technological development of the construction is greater and the regulations are the most rigorous. During the analysis of the development of non

Platelet-derived growth factor induces phosphorylation of a 64-kDa nuclear protein

International Nuclear Information System (INIS)

Shawver, L.K.; Pierce, G.F.; Kawahara, R.S.; Deuel, T.F.

1989-01-01

The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase

Science.gov (United States)

Bachmann, M.; Shiraishi, N.; Campbell, W. H.; Yoo, B. C.; Harmon, A. C.; Huber, S. C.; Davies, E. (Principal Investigator)

1996-01-01

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

P³DB 3.0: From plant phosphorylation sites to protein networks.

Science.gov (United States)

Yao, Qiuming; Ge, Huangyi; Wu, Shangquan; Zhang, Ning; Chen, Wei; Xu, Chunhui; Gao, Jianjiong; Thelen, Jay J; Xu, Dong

2014-01-01

In the past few years, the Plant Protein Phosphorylation Database (P(3)DB, http://p3db.org) has become one of the most significant in vivo data resources for studying plant phosphoproteomics. We have substantially updated P(3)DB with respect to format, new datasets and analytic tools. In the P(3)DB 3.0, there are altogether 47 923 phosphosites in 16 477 phosphoproteins curated across nine plant organisms from 32 studies, which have met our multiple quality standards for acquisition of in vivo phosphorylation site data. Centralized by these phosphorylation data, multiple related data and annotations are provided, including protein-protein interaction (PPI), gene ontology, protein tertiary structures, orthologous sequences, kinase/phosphatase classification and Kinase Client Assay (KiC Assay) data--all of which provides context for the phosphorylation event. In addition, P(3)DB 3.0 incorporates multiple network viewers for the above features, such as PPI network, kinase-substrate network, phosphatase-substrate network, and domain co-occurrence network to help study phosphorylation from a systems point of view. Furthermore, the new P(3)DB reflects a community-based design through which users can share datasets and automate data depository processes for publication purposes. Each of these new features supports the goal of making P(3)DB a comprehensive, systematic and interactive platform for phosphoproteomics research.

Towards the systematic discovery of signal transduction networks using phosphorylation dynamics data

Directory of Open Access Journals (Sweden)

Yachie Nozomu

2010-05-01

Full Text Available Abstract Background Phosphorylation is a ubiquitous and fundamental regulatory mechanism that controls signal transduction in living cells. The number of identified phosphoproteins and their phosphosites is rapidly increasing as a result of recent mass spectrometry-based approaches. Results We analyzed time-course phosphoproteome data obtained previously by liquid chromatography mass spectrometry with the stable isotope labeling using amino acids in cell culture (SILAC method. This provides the relative phosphorylation activities of digested peptides at each of five time points after stimulating HeLa cells with epidermal growth factor (EGF. We initially calculated the correlations between the phosphorylation dynamics patterns of every pair of peptides and connected the strongly correlated pairs to construct a network. We found that peptides extracted from the same intracellular fraction (nucleus vs. cytoplasm tended to be close together within this phosphorylation dynamics-based network. The network was then analyzed using graph theory and compared with five known signal-transduction pathways. The dynamics-based network was correlated with known signaling pathways in the NetPath and Phospho.ELM databases, and especially with the EGF receptor (EGFR signaling pathway. Although the phosphorylation patterns of many proteins were drastically changed by the EGF stimulation, our results suggest that only EGFR signaling transduction was both strongly activated and precisely controlled. Conclusions The construction of a phosphorylation dynamics-based network provides a useful overview of condition-specific intracellular signal transduction using quantitative time-course phosphoproteome data under specific experimental conditions. Detailed prediction of signal transduction based on phosphoproteome dynamics remains challenging. However, since the phosphorylation profiles of kinase-substrate pairs on the specific pathway were localized in the dynamics

Structural Modulation of Phosducin by Phosphorylation and 14-3-3 Protein Binding

Science.gov (United States)

Rezabkova, Lenka; Kacirova, Miroslava; Sulc, Miroslav; Herman, Petr; Vecer, Jaroslav; Stepanek, Miroslav; Obsilova, Veronika; Obsil, Tomas

2012-01-01

Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the Gtβγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function. PMID:23199924

Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals.

Science.gov (United States)

Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

2004-05-01

The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.

Insulin increases phosphorylation of mitochondrial proteins in human skeletal muscle in vivo

DEFF Research Database (Denmark)

Zhao, Xiaolu; Bak, Steffen; Pedersen, Andreas James Thestrup

2014-01-01

, we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. Using a combination of TiO2 phosphopeptide-enrichment, HILIC fractionation, and LC−MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples...... obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number...... of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, p = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, p = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (n = 94) were exclusively identified in the insulin-stimulated state and included...

Effect of some organic solvents on oxidative phosphorylation in rat liver mitochondria

DEFF Research Database (Denmark)

Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

2013-01-01

The effect of acetone, acetonitrile, dimethyl sulfoxide (DMSO), ethanol and methanol on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria has been studied. All the organic solvents inhibited the oxidative phosphorylation in a concentration dependent manner, but with differences...... in potencies. Among the tested organic solvents, acetonitrile and acetone were more potent than ethanol, methanol, and DMSO. There was no significant difference in oxidative phosphorylation, compared to controls, when the concentrations of acetone was below 1% (v/v), of acetonitrile below 2% (v/v), of DMSO...... below 10% (v/v), of ethanol below 5% or of methanol below 2%, respectively. There was complete inhibition of oxidative phosphorylation at 50% (v/v) of acetone, acetonitrile and ethanol. But in the case of DMSO and methanol there were some residual activities observed at the 50% concentration level. DMSO...

Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

International Nuclear Information System (INIS)

Stratton, K.R.; Worley, P.F.; Huganir, R.L.; Baraban, J.M.

1989-01-01

The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipid system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation

Phenobarbital Meets Phosphorylation of Nuclear Receptors.

Science.gov (United States)

Negishi, Masahiko

2017-05-01

Phenobarbital was the first therapeutic drug to be characterized for its induction of hepatic drug metabolism. Essentially at the same time, cytochrome P450, an enzyme that metabolizes drugs, was discovered. After nearly 50 years of investigation, the molecular target of phenobarbital induction has now been delineated to phosphorylation at threonine 38 of the constitutive androstane receptor (NR1I3), a member of the nuclear receptor superfamily. Determining this mechanism has provided us with the molecular basis to understand drug induction of drug metabolism and disposition. Threonine 38 is conserved as a phosphorylation motif in the majority of both mouse and human nuclear receptors, providing us with an opportunity to integrate diverse functions of nuclear receptors. Here, I review the works and accomplishments of my laboratory at the National Institutes of Health National Institute of Environmental Health Sciences and the future research directions of where our study of the constitutive androstane receptor might take us. U.S. Government work not protected by U.S. copyright.

The Machinery Of Climate Anti-Science, Its Efforts Against Education, Top To Bottom

Science.gov (United States)

Mashey, J. R.

2014-12-01

"There's always one every year" a fine local science teacher said after once again being hassled by a vocal parent for teaching appropriate climate science in school. How does that happen? The machinery of climate anti-science starts from the top with funders working through a maze of money paths, think tanks and front groups, employing spokespeople who can be portrayed as experts. While much of the money flows are still dark, some have been exposed over the last few years, and the effects finally filter down to the state and local levels of education. Among others, the Heartland Institute has a long history of trying to inject anti-science into K-12 and college education, having sent books, DVDs or brochures to teachers or school boards, as well as monthly newsletters to state legislators. Such are aimed at the top of the state or local organizations that affect education. For a vocal subset of the citizenry, a constant flow of misinformation from books, blogs, newsletters and some newspapers and magazines stirs action such as writing letters to editors, complaining to schools, calling on pseudo-experts and demanding equal time for pseudoscience. As the teacher said, it only takes one person to cause trouble at the local level. After a brief review of the overall machinery, this focuses on examples of anti-education tactics seen already, with some brief advice for climate scientists and educators who need to understand the machinery that supports such tactics. Some earlier history is included in http://www.desmogblog.com/2012/10/23/fakery-2-more-funny-finances-free-tax, but other attempts have surfaced in last few years, including state-wide efforts to reject Common Core Educational standard to avoid teaching climate science. Fortunately, school boards sometimes respond quite well, including one just recently in Pennsylvania.

Kinase-loaded magnetic beads for sequential in vitro phosphorylation of peptides and proteins.

Science.gov (United States)

Hromadkova, Lenka; Kupcik, Rudolf; Vajrychova, Marie; Prikryl, Petr; Charvatova, Andrea; Jankovicova, Barbora; Ripova, Daniela; Bilkova, Zuzana; Slovakova, Marcela

2018-01-15

Post-translational modifications, including phosphorylation, greatly impact the physiological function of proteins, especially those that are natively unfolded and implicated in many neurodegenerative diseases. However, structural and functional studies of such proteins require fully defined phosphorylation, including those that are not physiological. Thus, the kinases ERK2 and GSK-3β were immobilized to various superparamagnetic beads with carboxylic, aldehyde, Ni 2+ , or Co 3+ functional groups, with a view to efficiently phosphorylate peptides and proteins in vitro. Full phosphorylation of specific synthetic peptides confirmed that beads were successfully loaded with kinases. Remarkably, enzymes covalently immobilized on carboxylated SeraMag beads remained active upon reuse, with residual activity after 10 uses 99.5 ± 0.34% for GSK-3β and 36.2 ± 2.01% for ERK2. The beads were also used to sequentially phosphorylate recombinant tau, which in vivo is a biomarker of Alzheimer's disease. Thus, a system consisting of two fully active kinases immobilized to magnetic beads is demonstrated for the first time. In comparison to soluble enzymes, the beads are easier to handle, reusable, and thus low-cost. Importantly, these beads are also convenient to remove from reactions to minimize contamination of phosphorylated products or to exchange with other kinases.

Human Machine Interaction by Simulation of Dynamics of Construction Machinery

DEFF Research Database (Denmark)

Langer, Thomas Heegaard

-body vibration exposure was more than 20 percent and at the same time the fuel consumption was reduced significant. Training of operators is hence beneficial for both employees and employers of the construction industry. The whole-body vibration exposure on operators of dump trucks are dominated by off-road......This industrial Ph.D. project concerns whole-body vibrations in human operated construction machinery. The emissions of these vibrations is closely related to the subjective experience of comfort and in some cases these vibrations can occur in a level which can cause the operator back disorders...

WAVELETS AND PRINCIPAL COMPONENT ANALYSIS METHOD FOR VIBRATION MONITORING OF ROTATING MACHINERY

OpenAIRE

Bendjama, Hocine; S. Boucherit, Mohamad

2017-01-01

Fault diagnosis is playing today a crucial role in industrial systems. To improve reliability, safety and efficiency advanced monitoring methods have become increasingly important for many systems. The vibration analysis method is essential in improving condition monitoring and fault diagnosis of rotating machinery. Effective utilization of vibration signals depends upon effectiveness of applied signal processing techniques. In this paper, fault diagnosis is performed using a com...

Physical and Mechanical Properties of Palm Oil Frond and Stem Bunch for Developing Pruner and Harvester Machinery Design

Directory of Open Access Journals (Sweden)

Yazid Ismi Intara

2013-06-01

Full Text Available A development of oil palm pruner and harvester machinery design implemented in the field still faces a problem due to the lack of effective and efficient design which is need to be solved. It was noted that in order to develop the design, an early data and information of physical and mechanical properties of palm oil frond and stem fruits is critically important. The objective of the research was to obtain the physical and mechanical properties of palm oil frond and stem in order to develop the design of pruner and harvester machinery. The result showed that tool machinery was been advantageous by the physical properties of the plant i.e. the total weight of frond and leaf which enable to support the cutting process. The average of total weight of frond and leaf was 16.8 kg. The diagonal cutting trajectory was been more advantageous because of total weight and frond shape toward to the different of the plant tissue area. The measurement result shows that cutting curve follows the time required for cutting. The comparison among cutting curve shows differences in cutting thickness or length. In this case, the thickness is linear with cutting time. Besides, those curves show differences at the height which determine the maximum value of tested material cutting resistance. Alternative solution for machinery development design is pruner-harvester for height plant below 6 m and among 6 to 12 m. For below 6 m, pruner-harvester was designed by incorporating motor as power source and cutter-disc as the knife cutter. That condition was relied on that estate which was maintenance intensively commonly used cutter-disc. Pruner-harvester above 6 m and up to 12 m was improved based on manual egrek-designed by adding fresh fruit bunch alley supply glide in order to keep the fruits still in intact form. The consideration was based on affectivity and efficiency. It also considers homogenous ecological of palm oil plant which should be maintained to reduce global