Factor XIII in plasma, but not in platelets, mediates red blood cell retention in clots and venous thrombus size in mice.

PubMed

Kattula, Sravya; Byrnes, James R; Martin, Sara M; Holle, Lori A; Cooley, Brian C; Flick, Matthew J; Wolberg, Alisa S

2018-01-09

The transglutaminase factor XIII (FXIII) stabilizes clots against mechanical and biochemical disruption and is essential for hemostasis. In vitro and in vivo models of venous thrombosis demonstrate that FXIII mediates clot size by promoting red blood cell (RBC) retention. However, the key source of FXIII and whether FXIII activity can be reduced to suppress thrombosis without imposing deleterious hemostatic consequences are 2 critical unresolved questions. FXIII is present in multiple compartments, including plasma (FXIII plasma ) as a heterotetramer of A 2 and B 2 subunits and platelets (FXIII plt ) as an A 2 homodimer. We determined the role of the FXIII compartment and level in clot contraction, composition, and size in vitro and using in vivo models of hemostasis and venous thrombosis. Reducing overall FXIII levels decreased whole blood clot weight but did not alter thrombin generation or contraction of platelet-rich plasma clots. In reconstituted platelet-rich plasma and whole blood clot contraction assays, FXIII plasma , but not FXIII plt , produced high-molecular-weight fibrin crosslinks, promoted RBC retention, and increased clot weights. Genetically imposed reduction of FXIII delayed FXIII activation and fibrin crosslinking, suggesting FXIII levels mediate the kinetics of FXIII activation and activity and that the timing of these processes is a critical determinant of RBC retention during clot formation and contraction. A 50% reduction in FXIII plasma produced significantly smaller venous thrombi but did not increase bleeding in tail transection or saphenous vein puncture models in vivo. Collectively, these findings suggest that partial FXIII reduction may be a therapeutic strategy for reducing venous thrombosis.

Recombinant Factor XIII Mitigates Hemorrhagic Shock-Induced Organ Dysfunction

PubMed Central

Zaets, Sergey B.; Xu, Da-Zhong; Lu, Qi; Feketova, Eleonora; Berezina, Tamara L.; Malinina, Inga V.; Deitch, Edwin A.; Olsen, Eva H.

2012-01-01

Background Plasma factor XIII (FXIII) is responsible for stabilization of fibrin clot at the final stage of blood coagulation. Since FXIII has also been shown to modulate inflammation, endothelial permeability, as well as diminish multiple organ dysfunction (MOD) after gut ischemia-reperfusion injury, we hypothesized that FXIII would reduce MOD caused by trauma-hemorrhagic shock (THS). Materials and methods Rats were subjected to a 90 min THS or trauma sham shock (TSS) and treated with either recombinant human FXIII A2 subunit (rFXIII) or placebo immediately after resuscitation with shed blood or at the end of the TSS period. Lung permeability, lung and gut myeloperoxidase (MPO) activity, gut histology, neutrophil respiratory burst, microvascular blood flow in the liver and muscles, and cytokine levels were measured 3 h after the THS or TSS. FXIII levels were measured before THS or TSS and after the 3-h post-shock period. Results THS-induced lung permeability as well as lung and gut MPO activity was significantly lower in rFXIII-treated than in placebo-treated animals. Similarly, rFXIII-treated rats had lower neutrophil respiratory burst activity and less ileal mucosal injury. rFXIII-treated rats also had a higher liver microvascular blood flow compared with the placebo group. Cytokine response was more favorable in rFXIII-treated animals. Trauma-hemorrhagic shock did not cause a drop in FXIII activity during the study period. Conclusions Administration of rFXIII diminishes THS-induced MOD in rats, presumably by preservation of the gut barrier function, limitation of polymorphonuclear leukocyte (PMN) activation, and modulation of the cytokine response. PMID:21276979

RECOMBINANT FACTOR XIII DIMINISHES MULTIPLE ORGAN DYSFUNCTION IN RATS CAUSED BY GUT ISCHEMIA-REPERFUSION INJURY

PubMed Central

Zaets, Sergey B.; Xu, Da-Zhong; Lu, Qi; Feketova, Eleonora; Berezina, Tamara L.; Gruda, Maryann; Malinina, Inga V.; Deitch, Edwin A.; Olsen, Eva H. N.

2010-01-01

Plasma factor XIII (FXIII) is responsible for stabilization of fibrin clot at the final stage of blood coagulation. Because FXIII has also been shown to modulate inflammation and endothelial permeability, we hypothesized that FXIII diminishes multiple organ dysfunction caused by gut I/R injury. A model of superior mesenteric artery occlusion (SMAO) was used to induce gut I/R injury. Rats were subjected to 45-min SMAO or sham SMAO and treated with recombinant human FXIII A2 subunit (rFXIII) or placebo at the beginning of the reperfusion period. Lung permeability, lung and gut myeloperoxidase activity, gut histology, neutrophil respiratory burst, and microvascular blood flow in the liver and muscles were measured after a 3-h reperfusion period. The effect of activated rFXIII on transendothelial resistance of human umbilical vein endothelial cells was tested in vitro. Superior mesenteric artery occlusion–induced lung permeability as well as lung and gut myeloperoxidase activity was significantly lower in rFXIII-treated versus untreated animals. Similarly, rFXIII-treated rats had lower neutrophil respiratory burst activity and ileal mucosal injury. Rats treated with rFXIII also had higher liver microvascular blood flow compared with the placebo group. Superior mesenteric artery occlusion did not cause FXIII consumption during the study period. In vitro, activated rFXIII caused a dose-dependent increase in human umbilical vein endothelial cell monolayer resistance to thrombin-induced injury. Thus, administration of rFXIII diminishes SMAO-induced multiple organ dysfunction in rats, presumably by preservation of endothelial barrier function and the limitation of polymorphonuclear leukocyte activation. PMID:18948851

A comparison of the mechanical, kinetic, and biochemical properties of fibrin clots formed with two different fibrin sealants.

PubMed

Hickerson, William L; Nur, Israel; Meidler, Roberto

2011-01-01

The objective of the present study was to compare the mechanical, kinetic, and biochemical properties of fibrin clots produced using EVICEL Fibrin Sealant (Human) and TISSEEL Fibrin Sealant. The stiffness/elasticity and strength of fibrin clots formed with EVICEL and TISSEEL were assessed using applied mechanical force and thromboelastography (TEG). The factor XIII content of the fibrin clots was also evaluated. Mean Young modulus and tensile strength of the fibrin clots produced by EVICEL were significantly higher than those of clots produced by TISSEEL (P < 0.05 for both). The mean time to initial clot formation and mean time to the predefined level of clot formation were numerically shorter for EVICEL compared with TISSEEL. Furthermore, mean maximal amplitude of the clots formed with EVICEL was significantly greater than that for the clots formed with TISSEEL. Mean concentration of factor XIII for the EVICEL fibrinogen samples tested was 9 IU/ml compared with undetectable concentrations of factor XIII for the TISSEEL fibrinogen samples. Fibrin clots formed with EVICEL have a much higher resistance to stretching and tensile strength and are more capable of maintaining their structure against applied force than those formed with TISSEEL. EVICEL also allows more rapid development of fibrin clots than TISSEEL. This superior clot strength and resilience obtained with EVICEL relative to TISSEEL may be due in large part to the presence of factor XIII.

Free factor XIII activation peptide (fAP-FXIII) is a regulator of factor XIII activity via factor XIII-B.

PubMed

Dodt, Johannes; Pasternack, Ralf; Seitz, Rainer; Volkers, Peter

2016-02-01

In a factor XIIIa (FXIIIa) generation assay with recombinant FXIII-A2 (rFXIII-A2 ) free FXIII activation peptide (fAP-FXII) prolonged the time to peak (TTP) but did not affect the area under the curve (AUC) or concentration at peak (CP). Addition of recombinant factorXIII-B2 (rFXIII-B2 ) restored the characteristics of the FXIIIa generation parameters (AUC, TTP and CP) to those observed for plasma FXIII (FXIII-A2 B2 ). FXIII-A2 B2 reconstituted from rFXIII-A2 and rFXIII-B2 showed a similar effect on AUC, TTP and CP in the presence of fAP-FXII as observed for plasma FXIII-A2 B2 , indicating a role for FXIII-B in this observation. An effect of fAP-FXIII on thrombin, the proteolytic activator of FXIII, was excluded by thrombin generation assays and clotting experiments. In a purified system, fAP-FXIII did not interfere with the FXIIIa activity development of thrombin-cleaved rFXIII-A2 (rFXIII-A2 ') also excluding direct inhibition of FXIIIa. However, FXIIIa activity development of FXIII-A2 'B2 was inhibited in a concentration-dependent manner by fAP-FXIII, indicating that an interaction between AP-FXIII and FXIII-B2 contributes to the overall stability of FXIII-A2 'B2 . In addition to its well-known role, FXIII-B also contributes to FXIII-A2 B2 stability or dissociation depending on fAP-FXIII and calcium concentrations. © 2015 John Wiley & Sons Ltd.

Platelet factor 4 (CXCL4) seals blood clots by altering the structure of fibrin.

PubMed

Amelot, Aymeric A; Tagzirt, Madjid; Ducouret, Guylaine; Kuen, René Lai; Le Bonniec, Bernard F

2007-01-05

Platelet factor-4 (PF4/CXCL4) is an orphan chemokine released in large quantities in the vicinity of growing blood clots. Coagulation of plasma supplemented with a matching amount of PF4 results in a translucent jelly-like clot. Saturating amounts of PF4 reduce the porosity of the fibrin network 4.4-fold and decrease the values of the elastic and loss moduli by 31- and 59-fold, respectively. PF4 alters neither the cleavage of fibrinogen by thrombin nor the cross-linking of protofibrils by activated factor XIII but binds to fibrin and dramatically transforms the structure of the ensuing network. Scanning electron microscopy showed that PF4 gives rise to a previously unreported pattern of polymerization where fibrin assembles to form a sealed network. The subunits constituting PF4 form a tetrahedron having at its corners a RPRH motif that mimics (in reverse orientation) the Gly-His-Arg-Pro-amide peptides that co-crystallize with fibrin. Molecular modeling showed that PF4 could be docked to fibrin with remarkable complementarities and absence of steric clashes, allowing the assembly of irregular polymers. Consistent with this hypothesis, as little as 50 microm the QVRPRHIT peptide derived from PF4 affects the polymerization of fibrin.

Muscle-derived collagen XIII regulates maturation of the skeletal neuromuscular junction.

PubMed

Latvanlehto, Anne; Fox, Michael A; Sormunen, Raija; Tu, Hongmin; Oikarainen, Tuomo; Koski, Anu; Naumenko, Nikolay; Shakirzyanova, Anastasia; Kallio, Mika; Ilves, Mika; Giniatullin, Rashid; Sanes, Joshua R; Pihlajaniemi, Taina

2010-09-15

Formation, maturation, stabilization, and functional efficacy of the neuromuscular junction (NMJ) are orchestrated by transsynaptic and autocrine signals embedded within the synaptic cleft. Here, we demonstrate that collagen XIII, a nonfibrillar transmembrane collagen, is another such signal. We show that collagen XIII is expressed by muscle and its ectodomain can be proteolytically shed into the extracellular matrix. The collagen XIII protein was found present in the postsynaptic membrane and synaptic basement membrane. To identify a role for collagen XIII at the NMJ, mice were generated lacking this collagen. Morphological and ultrastructural analysis of the NMJ revealed incomplete adhesion of presynaptic and postsynaptic specializations in collagen XIII-deficient mice of both genders. Strikingly, Schwann cells erroneously enwrapped nerve terminals and invaginated into the synaptic cleft, resulting in a decreased contact surface for neurotransmission. Consistent with morphological findings, electrophysiological studies indicated both postsynaptic and presynaptic defects in Col13a1(-/-) mice, such as decreased amplitude of postsynaptic potentials, diminished probabilities of spontaneous release and reduced readily releasable neurotransmitter pool. To identify the role of collagen XIII at the NMJ, shed ectodomain of collagen XIII was applied to cultured myotubes, and it was found to advance acetylcholine receptor (AChR) cluster maturation. Together with the delay in AChR cluster development observed in collagen XIII-deficient mutants in vivo, these results suggest that collagen XIII plays an autocrine role in postsynaptic maturation of the NMJ. Altogether, the results presented here reveal that collagen XIII is a novel muscle-derived cue necessary for the maturation and function of the vertebrate NMJ.

Newly-recognized roles of factor XIII in thrombosis

PubMed Central

Byrnes, James R.; Wolberg, Alisa S.

2017-01-01

Arterial and venous thrombosis are major contributors to coagulation-associated morbidity and mortality. Greater understanding of mechanisms leading to thrombus formation and stability is expected to lead to improved treatment strategies. Factor XIII (FXIII) is a transglutaminase found in plasma and platelets. During thrombosis, activated FXIII crosslinks fibrin and promotes thrombus stability. Recent studies have provided new information about FXIII activity during coagulation and its effects on clot composition and function. These findings reveal newly-recognized roles for FXIII in thrombosis. Herein, we review published literature on FXIII biology and effects on fibrin structure and stability, epidemiologic data associating FXIII with thrombosis, and evidence from animal models indicating FXIII has an essential role in determining thrombus stability, composition, and size. PMID:27056150

A calorimetric study on the low temperature dynamics of doped ice V and its reversible phase transition to hydrogen ordered ice XIII.

PubMed

Salzmann, Christoph G; Radaelli, Paolo G; Finney, John L; Mayer, Erwin

2008-11-07

Doped ice V samples made from solutions containing 0.01 M HCl (DCl), HF (DF), or KOH (KOD) in H(2)O (D(2)O) were slow-cooled from 250 to 77 K at 0.5 GPa. The effect of the dopant on the hydrogen disorder --> order transition and formation of hydrogen ordered ice XIII was studied by differential scanning calorimetry (DSC) with samples recovered at 77 K. DSC scans of acid-doped samples are consistent with a reversible ice XIII <--> ice V phase transition at ambient pressure, showing an endothermic peak on heating due to the hydrogen ordered ice XIII --> disordered ice V phase transition, and an exothermic peak on subsequent cooling due to the ice V --> ice XIII phase transition. The equilibrium temperature (T(o)) for the ice V <--> ice XIII phase transition is 112 K for both HCl doped H(2)O and DCl doped D(2)O. From the maximal enthalpy change of 250 J mol(-1) on the ice XIII --> ice V phase transition and T(o) of 112 K, the change in configurational entropy for the ice XIII --> ice V transition is calculated as 2.23 J mol(-1) K(-1) which is 66% of the Pauling entropy. For HCl, the most effective dopant, the influence of HCl concentration on the formation of ice XIII was determined: on decreasing the concentration of HCl from 0.01 to 0.001 M, its effectiveness is only slightly lowered. However, further HCl decrease to 0.0001 M drastically lowered its effectiveness. HF (DF) doping is less effective in inducing formation of ice XIII than HCl (DCl) doping. On heating at a rate of 5 K min(-1), kinetic unfreezing starts in pure ice V at approximately 132 K, whereas in acid doped ice XIII it starts at about 105 K due to acceleration of reorientation of water molecules. KOH doping does not lead to formation of hydrogen ordered ice XIII, a result which is consistent with our powder neutron diffraction study (C. G. Salzmann, P. G. Radaelli, A. Hallbrucker, E. Mayer, J. L. Finney, Science, 2006, 311, 1758). We further conjecture whether or not ice XIII has a stable region in the water/ice phase diagram, and on a metastable triple point where ice XIII, ice V and ice II are in equilibrium.

Simulation of Aircraft Sortie Generation Under an Autonomic Logistics System

DTIC Science & Technology

2016-12-01

56 Design of Experiment...Figure 8. Pre -flight Operations ......................................................................................... 40 Figure 9. Sortie...Critical Factors and Their Associated Levels ................................................... 57 xiii Table 18. Design of Experiment

Evaluation of a Salmonella Strain Lacking the Secondary Messenger C-di-GMP and RpoS as a Live Oral Vaccine

PubMed Central

García, Begoña; Gil, Carmen; García-Ona, Enrique; Burgui, Saioa; Casares, Noelia; Hervás-Stubbs, Sandra; Lasarte, Juan José; Lasa, Iñigo

2016-01-01

Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. C-di-GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ΔXIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ΔXIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF-α, IL-2, IL-17 and IL-10. ΔXIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ΔXIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ΔXIII as a safe and effective live DIVA vaccine. PMID:27537839

[Best time to administer coagulation factor XIII( Fibrogammin P) for postoperative intractable pancreatic fistula following gastrectomy for gastric cancer].

PubMed

Shoda, Katsutoshi; Komatsu, Shuhei; Ichikawa, Daisuke; Kubota, Takeshi; Okamoto, Kazuma; Arita, Tomohiro; Konishi, Hirotaka; Murayama, Yasutoshi; Shiozaki, Atsushi; Kuriu, Yoshiaki; Ikoma, Hisashi; Nakanishi, Masayoshi; Fujiwara, Hitoshi; Otsuji, Eigo

2013-11-01

Coagulation factor XIII( Fibrogammin P, F XIII) has been used to treat postoperative pancreatic fistulas following gastrectomy for gastric cancer in Japan. However, little is known about the best timing to start this treatment for early recovery. This study was designed to examine the appropriate time to start Fibrogammin P treatment for pancreatic fistulas, based on nutritional and inflammatory parameters. We retrospectively examined 27 consecutive patients with Grade B or C pancreatic fistulas as defined by the International Study Group of Pancreatic Fistula( ISGPF) classification who underwent gastrectomy at our institute between 1997 and 2011. We analyzed data on total protein( TP), albumin (Alb), C-reactive protein( CRP), and hemoglobin( Hb) concentrations and white blood cell( WBC) and lymphocyte counts. We used this information to determine laboratory cut-off values that indicate the most advantageous time to start the administration of Fibrogammin P in order to achieve early recovery within 2 weeks. When Fibrogammin P administration was based on more than 2 cut-off values such as Alb>2.6 g/dL and Hb>9.0 g/dL and WBC<9,000/μL (p= 0.1563), early cure of pancreatic fistulas was achieved. The use of nutritional and inflammatory parameter values to determine the best time to administer Fibrogammin P may shorten the treatment period.

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbβ3 and transglutaminase factor XIII

PubMed Central

Mattheij, Nadine J.A.; Swieringa, Frauke; Mastenbroek, Tom G.; Berny-Lang, Michelle A.; May, Frauke; Baaten, Constance C.F.M.J.; van der Meijden, Paola E.J.; Henskens, Yvonne M.C.; Beckers, Erik A.M.; Suylen, Dennis P.L.; Nolte, Marc W.; Hackeng, Tilman M.; McCarty, Owen J.T.; Heemskerk, Johan W.M.; Cosemans, Judith M.E.M.

2016-01-01

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin αIIbβ3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin αIIbβ3. Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin αIIbβ3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in αIIbβ3 (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial αIIbβ3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin αIIbβ3. PMID:26721892

40 CFR Appendix Xiii to Part 86 - State Requirements Incorporated by Reference in Part 86 of the Code of Federal Regulations

Code of Federal Regulations, 2012 CFR

2012-07-01

... AND IN-USE HIGHWAY VEHICLES AND ENGINES (CONTINUED) Pt. 86, App. XIII Appendix XIII to Part 86—State...-Line Test Procedures for 1983 Through 1997 Model-Year Passenger Cars, Light-Duty Trucks and Medium-Duty...: California Assembly-Line Test Procedures for 1998 and Subsequent Model-Year Passenger Cars, Light-Duty Trucks...

40 CFR Appendix Xiii to Part 86 - State Requirements Incorporated by Reference in Part 86 of the Code of Federal Regulations

Code of Federal Regulations, 2013 CFR

2013-07-01

... AND IN-USE HIGHWAY VEHICLES AND ENGINES (CONTINUED) Pt. 86, App. XIII Appendix XIII to Part 86—State...-Line Test Procedures for 1983 Through 1997 Model-Year Passenger Cars, Light-Duty Trucks and Medium-Duty...: California Assembly-Line Test Procedures for 1998 and Subsequent Model-Year Passenger Cars, Light-Duty Trucks...

40 CFR Appendix Xiii to Part 86 - State Requirements Incorporated by Reference in Part 86 of the Code of Federal Regulations

Code of Federal Regulations, 2011 CFR

2011-07-01

... AND IN-USE HIGHWAY VEHICLES AND ENGINES (CONTINUED) Pt. 86, App. XIII Appendix XIII to Part 86—State...-Line Test Procedures for 1983 Through 1997 Model-Year Passenger Cars, Light-Duty Trucks and Medium-Duty...: California Assembly-Line Test Procedures for 1998 and Subsequent Model-Year Passenger Cars, Light-Duty Trucks...

Agreement between factor XIII activity and antigen assays in measurement of factor XIII: A French multicenter study of 147 human plasma samples.

PubMed

Caron, C; Meley, R; Le Cam Duchez, V; Aillaud, M F; Lavenu-Bombled, C; Dutrillaux, F; Flaujac, C; Ryman, A; Ternisien, C; Lasne, D; Galinat, H; Pouplard, C

2017-06-01

Factor XIII (FXIII) deficiency is a rare hemorrhagic disorder whose early diagnosis is crucial for appropriate treatment and prophylactic supplementation in cases of severe deficiency. International guidelines recommend a quantitative FXIII activity assay as first-line screening test. FXIII antigen measurement may be performed to establish the subtype of FXIII deficiency (FXIIID) when activity is decreased. The aim of this multicenter study was to evaluate the analytical and diagnostic levels of performance of a new latex immunoassay, K-Assay ® FXIII reagent from Stago, for first-line measurement of FXIII antigen. Results were compared to those obtained with the Berichrom ® FXIII chromogenic assay for measurement of FXIII activity. Of the 147 patient plasma samples, 138 were selected for analysis. The accuracy was very good, with intercenter reproducibility close to 7%. Five groups were defined on FXIII activity level (<5% (n = 5), 5%-30% (n = 23), 30%-60% (n = 17), 60%-120% (n = 69), above 120% (n = 24)), without statistical differences between activity and antigen levels (P value >0.05). Correlation of the K-Assay ® with the Berichrom ® FXIII activity results was excellent (r = 0.919). Good agreement was established by the Bland and Altman method, with a bias of +9.4% on all samples, and of -1.4% for FXIII levels lower than 30%. One patient with afibrinogenemia showed low levels of Berichrom ® FXIII activity but normal antigen level and clot solubility as expected. The measurement of FXIII antigen using the K-Assay ® is a reliable first-line tool for detection of FXIII deficiency when an activity assay is not available. © 2017 John Wiley & Sons Ltd.

[A case of pancreatic and duodenal fistula after total gastrectomy successfully treated with coagulation factor XIII].

PubMed

Nishino, Hitoe; Kojima, Kazuhiro; Oshima, Hirokazu; Nakagawa, Koji; Fumura, Masao; Kikuchi, Norio

2013-11-01

Pancreatic fistula( PF) is a challenging postoperative complication. We report a case of PF following gastrectomy successfully treated using intravenous coagulation factor XIII( FXIII).A 78-year-old man with early gastric cancer underwent total gastrectomy with Roux-en-Y reconstruction. PF developed postoperatively, following which, leakage from the duodenal stump was observed. Percutaneous drainage and re-operative surgery were performed. A somatostatin analogue, antibiotic drugs, and gabexate mesilate were administrated along with nutritional support. The pancreatic and duodenal fistula had been producing duodenal juice for over 30 days since the re-operative surgery. As suspected, reduced FXIII activity was confirmed in the patient. After administering FXIII for 5 days, the amount of duodenal juice from the fistula markedly reduced, and the fistula closed immediately afterwards. The results of our study suggest that administration of FXIII could be a reasonable and effective treatment for patients with pancreatic or/and enterocutaneous fistula who are resistant to standard treatments.

The predictive value of markers of fibrinolysis and endothelial dysfunction in the post thrombotic syndrome. A systematic review.

PubMed

Rabinovich, Anat; Cohen, Jacqueline M; Kahn, Susan R

2014-06-01

The post thrombotic syndrome (PTS) develops in 20-40% of deep venous thrombosis (DVT) patients. Risk factors for PTS have not been well elucidated. Identification of risk factors would facilitate individualised risk assessment for PTS. We conducted a systematic review to determine whether biomarkers of fibrinolysis or endothelial dysfunction can predict the risk for PTS among DVT patients. Studies were identified by searching the electronic databases PubMed, EMBASE, Scopus and Web of science. We included studies published between 1990 and 2013, measured biomarker levels in adult DVT patients, and reported rates of PTS development. Fourteen studies were included: 11 investigated the association between D-dimer and PTS; three examined fibrinogen; two measured von Willebrand factor; one measured plasminogen activator inhibitor-1; one assessed ADAMTS-13 (A Disintegrin and Metalloprotease with Thrombospondin type 1 repeats) and one measured factor XIII activity. Studies varied with regards to inclusion criteria, definition of PTS, time point and method of biomarker measurement. We were unable to meta-analyse results due to marked clinical heterogeneity. Descriptively, a significant association with PTS was found for D-dimer in four studies and factor XIII in one study. Further prospective research is needed to elucidate whether these markers might be useful to predict PTS development.

Characterization of carbonic anhydrase XIII in the erythrocytes of the Burmese python, Python molurus bivittatus.

PubMed

Esbaugh, A J; Secor, S M; Grosell, M

2015-09-01

Carbonic anhydrase (CA) is one of the most abundant proteins found in vertebrate erythrocytes with the majority of species expressing a low activity CA I and high activity CA II. However, several phylogenetic gaps remain in our understanding of the expansion of cytoplasmic CA in vertebrate erythrocytes. In particular, very little is known about isoforms from reptiles. The current study sought to characterize the erythrocyte isoforms from two squamate species, Python molurus and Nerodia rhombifer, which was combined with information from recent genome projects to address this important phylogenetic gap. Obtained sequences grouped closely with CA XIII in phylogenetic analyses. CA II mRNA transcripts were also found in erythrocytes, but found at less than half the levels of CA XIII. Structural analysis suggested similar biochemical activity as the respective mammalian isoforms, with CA XIII being a low activity isoform. Biochemical characterization verified that the majority of CA activity in the erythrocytes was due to a high activity CA II-like isoform; however, titration with copper supported the presence of two CA pools. The CA II-like pool accounted for 90 % of the total activity. To assess potential disparate roles of these isoforms a feeding stress was used to up-regulate CO2 excretion pathways. Significant up-regulation of CA II and the anion exchanger was observed; CA XIII was strongly down-regulated. While these results do not provide insight into the role of CA XIII in the erythrocytes, they do suggest that the presence of two isoforms is not simply a case of physiological redundancy. Copyright © 2015. Published by Elsevier Inc.

Traffic analysis toolbox volume XIII : integrated corridor management analysis, modeling, and simulation guide

DOT National Transportation Integrated Search

2017-02-01

As part of the Federal Highway Administration (FHWA) Traffic Analysis Toolbox (Volume XIII), this guide was designed to help corridor stakeholders implement the Integrated Corridor Management (ICM) Analysis, Modeling, and Simulation (AMS) methodology...

Traffic analysis toolbox volume XIII : integrated corridor management analysis, modeling, and simulation guide.

DOT National Transportation Integrated Search

2017-02-01

As part of the Federal Highway Administration (FHWA) Traffic Analysis Toolbox (Volume XIII), this guide was designed to help corridor stakeholders implement the Integrated Corridor Management (ICM) Analysis, Modeling, and Simulation (AMS) methodology...

In-situ AFM measurement of single fibrin fiber stiffness before and after addition of Factor XIII

NASA Astrophysics Data System (ADS)

Houser, John; O'Brien, E. Timothy; Lord, Susan T.; Superfine, Richard; Falvo, Michael R.

2008-10-01

Fibrin fibers are the main structural component of blood clots. Ligation of fibrin by native Factor XIII (FXIII) serves to fine tune the mechanical properties of the clot. Mechanical alteration is important because a clot must be stiff enough to resist forces from blood flow but compliant enough to prevent embolism (fracture). Cone and Plate measurements of fibrin gels, which represent the vast majority of mechanical measurements on fibrin, show that FXIII increases clot stiffness. More recently, measurements on individual fibrin fibers show that they exhibit remarkable extensibility, breaking at strains up to 300%. As of yet, the origin of this extensibility is not fully understood. The different responses of ligated and unligated fibrin fibers can give us clues as to it's mechanism of extension. We use a combined fluorescence/atomic force microscope to stretch individual, isolated, fibrin fibers and then compare force extension curves of the same fiber before and after addition of FXIII. We found up to a 3.5-fold increase in fiber stiffness after addition of FXIII. We also show stiffening of individual fibrin fibers after crosslinking by gluteraldehyde.

Dirac R-matrix calculations of photoionization cross sections of Ni XII and atomic structure data of Ni XIII

NASA Astrophysics Data System (ADS)

Nazir, R. T.; Bari, M. A.; Bilal, M.; Sardar, S.; Nasim, M. H.; Salahuddin, M.

2017-02-01

We performed R-matrix calculations for photoionization cross sections of the two ground state configuration 3s23p5 (^2P^o3/2,1/2) levels and 12 excited states of Ni XII using relativistic Dirac Atomic R-matrix Codes (DARC) across the photon energy range between the ionizations thresholds of the corresponding states and well above the thresholds of the last level of the Ni XIII target ion. Generally, a good agreement is obtained between our results and the earlier theoretical photoionization cross sections. Moreover, we have used two independent fully relativistic GRASP and FAC codes to calculate fine-structure energy levels, wavelengths, oscillator strengths, transitions rates among the lowest 48 levels belonging to the configuration (3s23p4, 3s3p5, 3p6, 3s23p33d) in Ni XIII. Additionally, radiative lifetimes of all the excited states of Ni XIII are presented. Our results of the atomic structure of Ni XIII show good agreement with other theoretical and experimental results available in the literature. A good agreement is found between our calculated lifetimes and the experimental ones. Our present results are useful for plasma diagnostic of fusion and astrophysical plasmas.

Biomimetic Delivery of Keratinocyte Growth Factor upon Cellular Demand for Accelerated Wound Healing in Vitro and in Vivo

PubMed Central

Geer, David J.; Swartz, Daniel D.; Andreadis, Stelios T.

2005-01-01

Exogenous keratinocyte growth factor (KGF) significantly enhances wound healing, but its use is hampered by a short biological half-life and lack of tissue selectivity. We used a biomimetic approach to achieve cell-controlled delivery of KGF by covalently attaching a fluorescent matrix-binding peptide that contained two domains: one recognized by factor XIII and the other by plasmin. Modified KGF was incorporated into the fibrin matrix at high concentration in a factor XIII-dependent manner. Cell-mediated activation of plasminogen to plasmin degraded the fibrin matrix and cleaved the peptides, releasing active KGF to the local microenvironment and enhancing epithelial cell proliferation and migration. To demonstrate in vivo effectiveness, we used a hybrid model of wound healing that involved transplanting human bioengineered skin onto athymic mice. At 6 weeks after grafting, the transplanted tissues underwent full thickness wounding and treatment with fibrin gels containing bound KGF. In contrast to topical KGF, fibrin-bound KGF persisted in the wounds for several days and was released gradually, resulting in significantly enhanced wound closure. A fibrinolytic inhibitor prevented this healing, indicating the requirement for cell-mediated fibrin degradation to release KGF. In conclusion, this biomimetic approach of localized, cell-controlled delivery of growth factors may accelerate healing of large full-thickness wounds and chronic wounds that are notoriously difficult to heal. PMID:16314471

FE-XIII Infrared / FE-XIV Green Line Ratio Diagnostics (P55)

NASA Astrophysics Data System (ADS)

Srivastava, A. K.; et al.

2006-11-01

aks.astro.itbhu@gmail.com We consider the first 27-level atomic model of Fe XIII (5.9 < log Te < 6.4 K) to estimate its ground level populations, taking account of electron as well as proton collisional excitations and de-excitations, radiative cascades, radiative excitations and de-excitations. Radiative cascade is important but the effect of dilution factor is negligible at higher electron densities. The 3 P1-3P0 and 3P2-3P1 transitions in the ground configuration 3s2 3p2 of Fe XIII result in two forbidden coronal emission lines in the infrared region, namely 10747 Å and 10798 Å., while the 5303 Å green line is formed in the 3s2 3p 2 2 ground configuration of Fe XIV as a result of P3 / 2 - P1 / 2 magnetic dipole transition. The line-widths of appropriate pair of forbidden coronal emission lines observed simultaneously can be useful diagnostic tool to deduce temperature and non-thermal velocity in the large scale coronal structures using intensity ratios of the lines as the temperature signature, instead of assuming ion temperature to be equal to the electron temperature. Since the line intensity ratios IG5303/IIR10747 and IG5303/IIR10798 have very week density dependence, they are ideal monitors of temperature mapping in the solar corona.

Association of the F13A1 Val34Leu polymorphism and recurrent pregnancy loss: A meta-analysis.

PubMed

Jung, Jae Hyun; Kim, Jae-Hoon; Song, Gwan Gyu; Choi, Sung Jae

2017-08-01

Factor XIII (FXIII) plays role in stabilizing the linkage between fibrins during blood clotting and has been implicated in recurrent pregnancy loss (RPL). The relationship between the Val34Leu polymorphism in F13A1, which encodes the enzymatic subunit of FXIII, and RPL is unclear. The aim of this meta-analysis was to evaluate the association betweenF13A1 Val34Leu and the risk of RPL. We performed a meta-analysis of 11 studies involving 1092 cases and 678 controls using published literature from PubMed and Embase. We detected an association in recessive (Val/Val vs. Val/Leu+Leu/Leu; OR=0.71, 95% CI=0.51-0.99, P=0.04), and one of the two co-dominant (Val/Val vs. Val/Leu; OR=0.71, 95% CI=0.52-0.98, P=0.03) models of in heritance. Subgroup analysis revealed that the F13A1 Val34Leu polymorphism was associated with RPL in Asians (Val vs. Leu; OR=0.53, CI=0.33-0.85, P=0.01). However, there was no association between F13A1 Val34Leu and RPL in Europeans and South Americans. Our meta-analysis supports an association between F13A1 Val34Leu and RPL. Copyright © 2017 Elsevier B.V. All rights reserved.

Rare coagulation disorders: fibrinogen, factor VII and factor XIII.

PubMed

de Moerloose, P; Schved, J-F; Nugent, D

2016-07-01

Rare coagulation disorders (RCDs) include the inherited deficiencies of fibrinogen, factor (F) II, FV, combined FV and VIII, FVII, FX, combined FVII and X, FXI, FXIII and combined congenital deficiency of vitamin K-dependent factors (VKCFDs). Despite their rarity, a deep comprehension of all these disorders is essential to really understand haemostasis. Indeed, even if they share some common features each RCD has some particularity which makes it unique. In this review, we focus on three disorders: fibrinogen, FVII and FXIII. © 2016 John Wiley & Sons Ltd.

76 FR 48563 - Medicare and Medicaid Programs; Quarterly Listing of Program Issuances-January Through March 2011...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-08

...-7205 Ventricular Assist Device (Destination Therapy) Facilities. XIII Medicare-Approved Lung JoAnna...-Approved Ventricular Assist Device (Destination Therapy) Facilities, Addendum XIII: Lung Volume Reduction...-Approved Ventricular Assist Device (Destination Therapy) Facilities (January Through March 2011) Addendum...

Basic Needs and Education in Portugal. The Portugal Project. Document No. XIII.

ERIC Educational Resources Information Center

Erasmie, Thord

The aim of this study was to describe and compare the socio-economic factors that can be expected to influence the education system in Portugal, where urbanization has been very slow. The report examines basic needs in Portugal's 18 districts and makes recommendations about resource allocation to planners of adult education programs. Extensive…

Phylogenetic analysis of Newcastle disease viruses from Bangladesh suggests continuing evolution of genotype XIII.

PubMed

Barman, Lalita Rani; Nooruzzaman, Mohammed; Sarker, Rahul Deb; Rahman, Md Tazinur; Saife, Md Rajib Bin; Giasuddin, Mohammad; Das, Bidhan Chandra; Das, Priya Mohan; Chowdhury, Emdadul Haque; Islam, Mohammad Rafiqul

2017-10-01

A total of 23 Newcastle disease virus (NDV) isolates from Bangladesh taken between 2010 and 2012 were characterized on the basis of partial F gene sequences. All the isolates belonged to genotype XIII of class II NDV but segregated into three sub-clusters. One sub-cluster with 17 isolates aligned with sub-genotype XIIIc. The other two sub-clusters were phylogenetically distinct from the previously described sub-genotypes XIIIa, XIIIb and XIIIc and could be candidates of new sub-genotypes; however, that needs to be validated through full-length F gene sequence data. The results of the present study suggest that genotype XIII NDVs are under continuing evolution in Bangladesh.

25 CFR 36.40 - Standard XIII-Library/media program.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 25 Indians 1 2010-04-01 2010-04-01 false Standard XIII-Library/media program. 36.40 Section 36.40... § 36.40 Standard XIII—Library/media program. (a) Each school shall provide a library/media program... objectives have been met. (2) A written policy for the selection of materials and equipment shall be...

25 CFR 36.40 - Standard XIII-Library/media program.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 25 Indians 1 2012-04-01 2011-04-01 true Standard XIII-Library/media program. 36.40 Section 36.40... § 36.40 Standard XIII—Library/media program. (a) Each school shall provide a library/media program... developed by a library committee in collaboration with the librarian and be approved by the school board...

25 CFR 36.40 - Standard XIII-Library/media program.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 25 Indians 1 2014-04-01 2014-04-01 false Standard XIII-Library/media program. 36.40 Section 36.40... § 36.40 Standard XIII—Library/media program. (a) Each school shall provide a library/media program... developed by a library committee in collaboration with the librarian and be approved by the school board...

25 CFR 36.40 - Standard XIII-Library/media program.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 25 Indians 1 2013-04-01 2013-04-01 false Standard XIII-Library/media program. 36.40 Section 36.40... § 36.40 Standard XIII—Library/media program. (a) Each school shall provide a library/media program... developed by a library committee in collaboration with the librarian and be approved by the school board...

Thermal Characterization of a Hall Effect Thruster

DTIC Science & Technology

2008-03-01

View Factor A = Area θ = Angle R = Distance xiii J = Radiosity q = Heat Transfer Rate W = Radiated Power U = Voltage C...summation rule. 1 1 N ij j F = =∑ (18) Radiosity (Ji) takes into account both radiation emitted and reflected from a surface. Analyzing radiation...exchanges between two surfaces is made easier with a few assumptions. Each surface is assumed isothermal and characterized by a uniform radiosity

Enhanced lysis and accelerated establishment of viscoelastic properties of fibrin clots are associated with pulmonary embolism.

PubMed

Martinez, Marissa R; Cuker, Adam; Mills, Angela M; Crichlow, Amanda; Lightfoot, Richard T; Chernysh, Irina N; Nagaswami, Chandrasekaran; Weisel, John W; Ischiropoulos, Harry

2014-03-01

The factors that contribute to pulmonary embolism (PE), a potentially fatal complication of deep vein thrombosis (DVT), remain poorly understood. Whereas fibrin clot structure and functional properties have been implicated in the pathology of venous thromboembolism and the risk for cardiovascular complications, their significance in PE remains uncertain. Therefore, we systematically compared and quantified clot formation and lysis time, plasminogen levels, viscoelastic properties, activated factor XIII cross-linking, and fibrin clot structure in isolated DVT and PE subjects. Clots made from plasma of PE subjects showed faster clot lysis times with no differences in lag time, rate of clot formation, or maximum absorbance of turbidity compared with DVT. Differences in lysis times were not due to alterations in plasminogen levels. Compared with DVT, clots derived from PE subjects showed accelerated establishment of viscoelastic properties, documented by a decrease in lag time and an increase in the rate of viscoelastic property formation. The rate and extent of fibrin cross-linking by activated factor XIII were similar between clots from DVT and PE subjects. Electron microscopy revealed that plasma fibrin clots from PE subjects exhibited lower fiber density compared with those from DVT subjects. These data suggest that clot structure and functional properties differ between DVT and PE subjects and provide insights into mechanisms that may regulate embolization.

Enhanced lysis and accelerated establishment of viscoelastic properties of fibrin clots are associated with pulmonary embolism

PubMed Central

Martinez, Marissa R.; Cuker, Adam; Mills, Angela M.; Crichlow, Amanda; Lightfoot, Richard T.; Chernysh, Irina N.; Nagaswami, Chandrasekaran; Weisel, John W.

2014-01-01

The factors that contribute to pulmonary embolism (PE), a potentially fatal complication of deep vein thrombosis (DVT), remain poorly understood. Whereas fibrin clot structure and functional properties have been implicated in the pathology of venous thromboembolism and the risk for cardiovascular complications, their significance in PE remains uncertain. Therefore, we systematically compared and quantified clot formation and lysis time, plasminogen levels, viscoelastic properties, activated factor XIII cross-linking, and fibrin clot structure in isolated DVT and PE subjects. Clots made from plasma of PE subjects showed faster clot lysis times with no differences in lag time, rate of clot formation, or maximum absorbance of turbidity compared with DVT. Differences in lysis times were not due to alterations in plasminogen levels. Compared with DVT, clots derived from PE subjects showed accelerated establishment of viscoelastic properties, documented by a decrease in lag time and an increase in the rate of viscoelastic property formation. The rate and extent of fibrin cross-linking by activated factor XIII were similar between clots from DVT and PE subjects. Electron microscopy revealed that plasma fibrin clots from PE subjects exhibited lower fiber density compared with those from DVT subjects. These data suggest that clot structure and functional properties differ between DVT and PE subjects and provide insights into mechanisms that may regulate embolization. PMID:24414255

Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

USDA-ARS?s Scientific Manuscript database

Background: The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to better u...

Molecular cloning of human protein 4.2: a major component of the erythrocyte membrane.

PubMed Central

Sung, L A; Chien, S; Chang, L S; Lambert, K; Bliss, S A; Bouhassira, E E; Nagel, R L; Schwartz, R S; Rybicki, A C

1990-01-01

Protein 4.2 (P4.2) comprises approximately 5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. We now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-pair insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4- and 2.5-kb cDNAs predict proteins of approximately 77 and approximately 80 kDa, respectively, and the authenticity was confirmed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4.2. Northern blot analysis detected a major 2.4-kb RNA species in reticulocytes. Isolation of two P4.2 cDNAs implies existence of specific regulation of P4.2 expression in human RBCs. Human RBC P4.2 has significant homology with human factor XIII subunit a and guinea pig liver transglutaminase. Sequence alignment of P4.2 with these two transglutaminases, however, revealed that P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates. Images PMID:1689063

Activation of tissue kallikrein-kininogen-kinin system in rabbit skin by a fraction isolated from Phoneutria nigriventer (armed spider) venom.

PubMed

Antunes, E; Marangoni, R A; Giglio, J R; Brain, S D; de Nucci, G

1993-11-01

Phoneutria nigriventer venom was fractionated by gel filtration followed by ion-exchange chromatography from which 16 fractions (I-XVI) were obtained and assayed in rabbit skin in order to identify those responsible for the increased vascular permeability observed with the whole venom. The fractions, and control mediators (tissue kallikrein, bradykinin and histamine) were intradermally injected in male New Zealand white rabbits. Local oedema formation was measured as the local accumulation of i.v. injected 125I-human serum albumin into skin sites. Fraction XIII was the only fraction assayed which significantly induced oedema formation. Fraction XIII-induced oedema was greatly reduced by either the protease inhibitor aprotinin or the bradykinin B2 receptor antagonist D-Arg,[Hyp3,Thi5,8D-Phe7]-Bk, whereas the plasma kallikrein inhibitor soybean trypsin inhibitor failed to significantly affect this oedematogenic response. The kininase II inhibitor captopril markedly potentiated fraction XIII-induced oedema. Our results indicate that the increased vascular permeability induced by fraction XIII is due to local generation of kinins in response to tissue (but not plasma) kallikrein-kinin system activation.

Migraine and genetic polymorphisms: an overview.

PubMed

Pizza, Vincenzo; Agresta, Anella; Agresta, Antonio; Lamaida, Eros; Lamaida, Norman; Infante, Francesco; Capasso, Anna

2012-01-01

The relationship between genetic polymorphisms and migraine as a cause of an increased risk of thrombotic disorders development is still debated In this respect, factor V Leiden, factor V (H1299R), prothrombin G20210A, factor XIII (V34L), β-fibrinogen, MTHFR (C677T), MTHFR (A1298C), APO E, PAI-1, HPA-1 and ACE I/D seem to play a determinant role in vascular diseases related to migraine. The present review analyzes both the incidence of the above genetic vascular mutations in migraineurs and the most re-cent developments related to genetic polymorphisms and migraine.

Migraine and Genetic Polymorphisms: An Overview

PubMed Central

Pizza, Vincenzo; Agresta, Anella; Agresta, Antonio; Lamaida, Eros; Lamaida, Norman; Infante, Francesco; Capasso, Anna

2012-01-01

The relationship between genetic polymorphisms and migraine as a cause of an increased risk of thrombotic disorders development is still debated In this respect, factor V Leiden, factor V (H1299R), prothrombin G20210A, factor XIII (V34L), β-fibrinogen, MTHFR (C677T), MTHFR (A1298C), APO E, PAI-1, HPA-1 and ACE I/D seem to play a determinant role in vascular diseases related to migraine. The present review analyzes both the incidence of the above genetic vascular mutations in migraineurs and the most re-cent developments related to genetic polymorphisms and migraine. PMID:22962564

Clotting of mammalian fibrinogens by papain: a re-examination.

PubMed

Doolittle, Russell F

2014-10-28

Papain has long been known to cause the gelation of mammalian fibrinogens. It has also been reported that papain-fibrin is insoluble in dispersing solvents like strong urea or sodium bromide solutions, similar to what is observed with thrombin-generated clots in the presence of factor XIIIa and calcium. In those old studies, both the gelation and subsequent clot stabilization were attributed to papain, although the possibility that the second step might be due to contaminating factor XIII in fibrinogen preparations was considered. I have revisited this problem in light of knowledge acquired over the past half-century about thiol proteases like papain, which mostly cleave peptide bonds, and transglutaminases like factor XIIIa that catalyze the formation of ε-lysyl-γ-glutamyl cross-links. Recombinant fibrinogen, inherently free of factor XIII and other plasma proteins, formed a stable gel when treated with papain alone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the intermolecular cross-linking in papain-fibrin leads to γ-chain dimers, trimers, and tetramers, just as is the case with thrombin-factor XIIIa-stabilized fibrin. Mass spectrometry of bands excised from gels showed that the cross-linked material is quite different from what occurs with factor XIIIa, however. With papain, the cross-linking occurs between γ chains in neighboring protofibrils becoming covalently linked in a "head-to-tail" fashion by a transpeptidation reaction involving the α-amino group of γ-Tyr1 and a papain cleavage site at γ-Gly403 near the carboxy terminus, rather than by the (reciprocal) "tail-to-tail" manner that occurs with factor XIIIa and that depends on cross-links between γ-Lys406 and γ-Gln398.

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network*

PubMed Central

Miszta, Adam; Pelkmans, Leonie; Lindhout, Theo; Krishnamoorthy, Ganeshram; de Groot, Philip G.; Hemker, Coenraad H.; Heemskerk, Johan W. M.; Kelchtermans, Hilde; de Laat, Bas

2014-01-01

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate. PMID:25381443

Correlation between thyroidal and peripheral blood total T cells, CD8+ T cells, and CD8+ T- regulatory cells and T-cell reactivity to calsequestrin and collagen XIII in patients with Graves' ophthalmopathy.

PubMed

Al-Ansari, Farah; Lahooti, Hooshang; Stokes, Leanne; Edirimanne, Senarath; Wall, Jack

2018-05-22

Purpose/aim of the study: Graves' ophthalmopathy (GO) is closely related to the thyroid autoimmune disorder Graves' disease. Previous studies have suggested roles for thyroidal CD8 +  T cells and autoimmunity against calsequestrin-1 (CASQ)-1 in the link between thyroidal and orbital autoimmune reactions in GO. A role for autoimmunity against CollXIII has also been suggested. In this study, we aimed to investigate correlations between some thyroidal and peripheral blood T-cell subsets and thyroidal T-cell reactivity against CASQ1 and CollXIII in patients with GO. Fresh thyroid tissues were processed by enzyme digestion and density gradient to isolate mononuclear cells (MNCs). Peripheral blood MNCs were also isolated using density gradient. Flow-cytometric analysis was used to identify the various T-cell subsets. T -cell reactivity to CASQ1 and CollXIII was measured by a 5-day culture of the MNCs and BrdU uptake method. We found a positive correlation between thyroidal CD8 +  T cells and CD8 +  T-regulatory (T-reg) cells in patients with GO. Thyroidal T cells from two out of the three patients with GO tested (66.7%) showed a positive response to CASQ1, while thyroidal T cells from none of the six Graves' Disease patients without ophthalmopathy (GD) tested showed a positive response to this antigen. Thyroidal T cells from these patient groups however, showed no significant differences in their response to CollXIII. Our observations provide further evidence for a possible role of thyroidal CD8 +  T cells, CD8 +  T-reg cells and the autoantigen CASQ1 in the link between thyroidal and orbital autoimmune reactions of GO.

Proceedings of the XIII International Symposium on Biological Control of Weeds; September 11-16, 2011; Waikoloa, Hawaii, USA

Treesearch

Yun Wu; Tracy Johnson; Sharlene Sing; S. Raghu; Greg Wheeler; Paul Pratt; Keith Warner; Ted Center; John Goolsby; Richard Reardon

2013-01-01

A total of 208 participants from 78 organizations in 19 countries gathered at the Waikoloa Beach Marriott on the Big Island of Hawaii on September 11-16, 2011 for the XIII International Symposium on Biological Control of Weeds. Following a reception on the first evening, Symposium co-chairs Tracy Johnson and Pat Conant formally welcomed the attendees on the morning of...

Safety of recombinant human factor XIII in a cynomolgus monkey model of extracorporeal blood circulation.

PubMed

Ponce, R; Armstrong, K; Andrews, K; Hensler, J; Waggie, K; Heffernan, J; Reynolds, T; Rogge, M

2005-01-01

Factor XIII (FXIII) is a thrombin-activated plasma coagulation factor critical for blood clot stabilization and longevity. Administration of exogenous FXIII to replenish depleted stores after major surgery, including cardiopulmonary bypass, may reduce bleeding complications and transfusion requirements. Thus, a model of extracorporeal circulation (ECC) was developed in adult male cynomolgus monkeys (Macaca fascicularis) to evaluate the nonclinical safety of recombinant human FXIII (rFXIII). The hematological and coagulation profile in study animals during and after 2 h of ECC was similar to that reported for humans during and after cardiopulmonary bypass, including observations of anemia, thrombocytopenia, and activation of coagulation and platelets. Intravenous slow bolus injection of 300 U/kg (2.1 mg/kg) or 1000 U/kg (7 mg/kg) rFXIII after 2 h of ECC was well tolerated in study animals, and was associated with a dose-dependent increase in FXIII activity. No clinically significant effects in respiration, ECG, heart rate, blood pressure, body temperature, clinical chemistry, hematology (including platelet counts), or indicators of thrombosis (thrombin:anti-thrombin complex and D-Dimer) or platelet activation (platelet factor 4 and beta-thromboglobulin) were related to rFXIII administration. Specific examination of brain, heart, lung, liver, and kidney from rFXIII-treated animals provided no evidence of histopathological alterations suggestive of subclinical hemorrhage or thrombosis. Taken as a whole, the results demonstrate the ECC model suitably replicated the clinical presentation reported for humans during and after cardiopulmonary bypass surgery, and do not suggest significant concerns regarding use of rFXIII in replacement therapy after extracorporeal circulation.

Factor XIII Val34Leu polymorphism and the risk of myocardial infarction under the age of 36 years.

PubMed

Rallidis, Loukianos S; Politou, Marianna; Komporozos, Christoforos; Panagiotakos, Demosthenes B; Belessi, Chrisoula I; Travlou, Anthi; Lekakis, John; Kremastinos, Dimitrios T

2008-06-01

There are limited and controversial data regarding the impact of factor XIII (FXIII) Val34Leu polymorphism in the pathogenesis of premature myocardial infarction (MI). We examined whether FXIII Val34Leu polymorphism is associated with the development of early MI. We recruited 159 consecutive patients who had survived their first acute MI under the age of 36 years (mean age = 32.1 +/- 3.6 years, 138 were men). The control group consisted of 121 healthy individuals matched with cases for age and sex, without a family history of premature coronary heart disease (CHD). FXIII Val34Leu polymorphism was tested with polymerase chain reaction and reverse hybridization. There was a lower prevalence of carriers of the Leu34 allele in patients than in controls (30.2 vs. 47.1%, p = 0.006). FXIII Val34Leu polymorphism was associated with lower risk for acute MI after adjusting for major cardiovascular risk factors (odds ratio [OR] = 0.51, 95% confidence interval [CI] 0.27-0.95, p = 0.03). Subgroup analysis according to angiographic findings ("normal" coronary arteries [n = 29] or significant CHD [n = 130]) showed that only patients with MI and significant CHD had lower prevalence of carriers of the Leu34 allele compared to controls after adjusting for major cardiovascular risk factors (OR = 0.42, 95% CI 0.22-0.83, p = 0.01). Our data indicate that FXIII Val34Leu polymorphism has a protective effect against the development of MI under the age of 36 years, particularly in the setting of significant CHD.

Evaluation of Factor V G1691A, prothrombin G20210A, Factor XIII V34L, MTHFR A1298C, MTHFR C677T and PAI-1 4G/5G genotype frequencies of patients subjected to cardiovascular disease (CVD) panel in south-east region of Turkey.

PubMed

Oztuzcu, Serdar; Ergun, Sercan; Ulaşlı, Mustafa; Nacarkahya, Gülper; Iğci, Yusuf Ziya; Iğci, Mehri; Bayraktar, Recep; Tamer, Ali; Çakmak, Ecir Ali; Arslan, Ahmet

2014-06-01

Cardiovascular disease (CVD) risk factors, such as arterial hypertension, obesity, dyslipidemia or diabetes mellitus, as well as CVDs, including myocardial infarction, coronary artery disease or stroke, are the most prevalent diseases and account for the major causes of death worldwide. In the present study, 4,709 unrelated patients subjected to CVD panel in south-east part of Turkey between the years 2010 and 2013 were enrolled and DNA was isolated from the blood samples of these patients. Mutation analyses were conducted using the real-time polymerase chain reaction method to screen six common mutations (Factor V G1691A, PT G20210A, Factor XIII V34L, MTHFR A1298C and C677T and PAI-1 -675 4G/5G) found in CVD panel. The prevalence of these mutations were 0.57, 0.25, 2.61, 13.78, 9.34 and 24.27 % in homozygous form, respectively. Similarly, the mutation percent of them in heterozygous form were 7.43, 3.44, 24.91, 44.94, 41.09 and 45.66%, respectively. No mutation was detected in 92 (1.95%) patients in total. Because of the fact that this is the first study to screen six common mutations in CVD panel in south-east region of Turkey, it has a considerable value on the diagnosis and treatment of these diseases. Upon the results of the present and previous studied a careful examination for these genetic variants should be carried out in thrombophilia screening programs, particularly in Turkish population.

Identification of a 'Candidatus Phytoplasma hispanicum'-related strain, associated with yellows-type diseases, in smoke-tree sharpshooter (Homalodisca liturata Ball).

PubMed

Servín-Villegas, Rosalía; Caamal-Chan, Maria Goretty; Chavez-Medina, Alicia; Loera-Muro, Abraham; Barraza, Aarón; Medina-Hernández, Diana; Holguín-Peña, Ramón Jaime

2018-04-11

The 16SrXIII group from phytoplasma bacteria were identified in salivary glands from Homalodisca liturata, which were collected in El Comitán on the Baja California peninsula in Mexico. We were able to positively identify 15 16S rRNA gene sequences with the corresponding signature sequence of 'CandidatusPhytoplasma' (CAAGAYBATKATGTKTAGCYGGDCT) and in silico restriction fragment length polymorphism (RFLP) profiles (F value estimations) coupled with a phylogenetic analysis to confirm their relatedness to 'CandidatusPhytoplasma hispanicum', which in turn belongs to the 16SrXIII group. A restriction analysis was carried out with AluI and EcoRI to confirm that the five sequences belongs to subgroup D. The rest of the sequences did not exhibit any known RFLP profile related to a subgroup reported in the 16SrXIII group.

Relative ion expansion velocity in laser-produced plasmas

NASA Technical Reports Server (NTRS)

Goldsmith, S.; Moreno, J. C.; Griem, H. R.; Cohen, Leonard; Richardson, M. C.

1988-01-01

The spectra of highly ionized titanium, Ti XIII through Ti XXI, and C VI Lyman lines were excited in laser-produced plasmas. The plasma was produced by uniformly irradiating spherical glass microballoons coated with thin layers of titanium and parylene. The 24-beam Omega laser system produced short, 0.6 ns, and high-intensity, 4 x 10 to the 14th W/sq cm, laser pulses at a wavelength of 351 nm. The measured wavelength for the 2p-3s Ti XIII resonance lines had an average shift of + 0.023 A relative to the C VI and Ti XX spectral lines. No shift was found between the C VI, Ti XIX, and Ti XX lines. The shift is attributed to a Doppler effect, resulting from a difference of (2.6 + or - 0.2) x 10 to the 7th cm/s in the expansion velocities of Ti XIX and Ti XX ions compared to Ti XIII ions.

Evaluating Factor XIII Specificity for Glutamine-Containing Substrates Using a MALDI-TOF Mass Spectrometry Assay

PubMed Central

Doiphode, Prakash G.; Malovichko, Marina V.; Mouapi, Kelly Njine; Maurer, Muriel C.

2014-01-01

Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase. PMID:24751466

Local activation of coagulation factor XIII reduces systemic complications and improves the survival of mice after Streptococcus pyogenes M1 skin infection.

PubMed

Deicke, Christin; Chakrakodi, Bhavya; Pils, Marina C; Dickneite, Gerhard; Johansson, Linda; Medina, Eva; Loof, Torsten G

2016-11-01

Coagulation is a mechanism for wound healing after injury. Several recent studies delineate an additional role of the intrinsic pathway of coagulation, also known as the contact system, in the early innate immune response against bacterial infections. In this study, we investigated the role of factor XIII (FXIII), which is activated upon coagulation induction, during Streptococcus pyogenes-mediated skin and soft tissue infections. FXIII has previously been shown to be responsible for the immobilization of bacteria within a fibrin network which may prevent systemic bacterial dissemination. In order to investigate if the FXIII-mediated entrapment of S. pyogenes also influences the disease outcome we used a murine S. pyogenes M1 skin and soft tissue infection model. Here, we demonstrate that a lack of FXIII leads to prolonged clotting times, increased signs of inflammation, and elevated bacterial dissemination. Moreover, FXIII-deficient mice show an impaired survival when compared with wildtype animals. Additionally, local reconstitution of FXIII-deficient mice with a human FXIII-concentrate (Fibrogammin ® P) could reduce the systemic complications, suggesting a protective role for FXIII during early S. pyogenes skin infection. FXIII therefore might be a possible therapeutically application to support the early innate immune response during skin infections caused by S. pyogenes. Copyright © 2016 Elsevier GmbH. All rights reserved.

Functional factor XIII-A is exposed on the stimulated platelet surface

PubMed Central

Mitchell, Joanne L.; Lionikiene, Ausra S.; Fraser, Steven R.; Whyte, Claire S.; Booth, Nuala A.

2014-01-01

Factor XIII (FXIII) stabilizes thrombi against fibrinolysis by cross-linking α2-antiplasmin (α2AP) to fibrin. Cellular FXIII (FXIII-A) is abundant in platelets, but the extracellular functions of this pool are unclear because it is not released by classical secretion mechanisms. We examined the function of platelet FXIII-A using Chandler model thrombi formed from FXIII-depleted plasma. Platelets stabilized FXIII-depleted thrombi in a transglutaminase-dependent manner. FXIII-A activity on activated platelets was unstable and was rapidly lost over 1 hour. Inhibiting platelet activation abrogated the ability of platelets to stabilize thrombi. Incorporating a neutralizing antibody to α2AP into FXIII-depleted thrombi revealed that the stabilizing effect of platelet FXIII-A on lysis was α2AP dependent. Platelet FXIII-A activity and antigen were associated with the cytoplasm and membrane fraction of unstimulated platelets, and these fractions were functional in stabilizing FXIII-depleted thrombi against lysis. Fluorescence confocal microscopy and flow cytometry revealed exposure of FXIII-A on activated membranes, with maximal signal detected with thrombin and collagen stimulation. FXIII-A was evident in protruding caps on the surface of phosphatidylserine-positive platelets. Our data show a functional role for platelet FXIII-A through exposure on the activated platelet membrane where it exerts antifibrinolytic function by cross-linking α2AP to fibrin. PMID:25331118

Targeted inactivation of the mouse locus encoding coagulation factor XIII-A: hemostatic abnormalities in mutant mice and characterization of the coagulation deficit.

PubMed

Lauer, Peter; Metzner, Hubert J; Zettlmeissl, Gerd; Li, Meng; Smith, Austin G; Lathe, Richard; Dickneite, Gerhard

2002-12-01

Blood coagulation factor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin gamma-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-A deficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.

Stability of Beriplast P fibrin sealant: storage and reconstitution.

PubMed

Eberhard, Ulrich; Broder, Martin; Witzke, Günther

2006-04-26

This study was performed to investigate the stability of Beriplast P fibrin sealant (FS) across a range of storage conditions, both pre- and post-reconstitution. Storage stability of the FS was evaluated during long-term refrigeration (24 months) with or without interim storage at elevated temperatures (40 degrees C for 1 week and 25 degrees C for 1 and 3 months). Stability of individual FS components was assessed by measuring: fibrinogen content, Factor XIII activity (FXIII), thrombin activity and aprotinin potency. The package integrity of each component was also checked (sterility testing, moisture content and pH). Storage stability was also evaluated by testing the reconstituted product for adhesion (tearing force testing after mixing the solutions) and sterility. Reconstitution stability was evaluated following 3-months' storage, for up to 50 h post-reconstitution using the same tests as for the storage stability investigations. Pre-defined specifications were met for fibrinogen content, Factor XIII activity, and thrombin activity, demonstrating storage stability. Package integrity and the functionality and sterility of the reconstituted product were confirmed throughout. Reconstitution stability was demonstrated for up to 50 h following reconstitution, in terms of both tearing force and sterility tests. In conclusion, the storage stability of Beriplast P was demonstrated over a range of 24-month storage schedules including interim exposure to elevated temperature, and the reconstituted product was stable for up to 50 h.

[Aldose reductase gene polymorphism and rate of appearance of retinopathy in non insulin dependent diabetics].

PubMed

Olmos, P; Acosta, A M; Schiaffino, R; Díaz, R; Alvarado, D; O'Brien, A; Muñoz, X; Arriagada, P; Claro, J C; Vega, R; Vollrath, V; Velasco, S; Emmerich, M; Maiz, A

1999-04-01

Recent studies suggest that polymorphisms associated to the aldose reductase gene could be related to early retinopathy in noninsulin dependent diabetics (NIDDM). There is also new interest on the genetic modulation of coagulation factors in relation to this complication. To look for a possible relationship between the rate of appearance of retinopathy and the genotype of (AC)n polymorphic marker associated to aldose reductase gene. A random sample of 27 NIDDM, aged 68.1 +/- 10.6 years, with a mean diabetes duration of 20.7 +/- 4.8 years and a mean glycosilated hemoglobin of 10.6 +/- 1.6%, was studied. The genotype of the (AC)n, polymorphic marker associated to the 5' end of the aldose reductase (ALR2) gene was determined by 32P-PCR plus sequenciation. Mutations of the factor XIII-A gene were studied by single stranded conformational polymorphism, sequenciation and restriction fragment length polymorphism. Four patients lacked the (AC)24 and had a higher rate of appearance of retinopathy than patients with the (AC)24 allele (0.0167 and 0.0907 score points per year respectively, p = 0.047). Both groups had similar glycosilated hemoglobin (11.7 +/- 0.2 and 10.5 +/- 1.6% respectively). Factor XIII gene mutations were not related to the rate of appearance of retinopathy. Our data suggest that the absence of the (AC)24 allele of the (AC)n polymorphic marker associated to the 5' end of the aldose reductase gene, is associated to a five fold reduction of retinopathy appearance rate.

Relative effects of plasma, fibrinogen concentrate, and factor XIII on ROTEM coagulation profiles in an in vitro model of massive transfusion in trauma.

PubMed

Schmidt, David E; Halmin, Märit; Wikman, Agneta; Östlund, Anders; Ågren, Anna

2017-10-01

Massive traumatic haemorrhage is aggravated through the development of trauma-induced coagulopathy, which is managed by plasma transfusion and/or fibrinogen concentrate administration. It is yet unclear whether these treatments are equally potent in ensuring adequate haemostasis, and whether additional factor XIII (FXIII) administration provides further benefits. In this study, we compared ROTEM whole blood coagulation profiles after experimental massive transfusion with different transfusion regimens in an in vitro model of dilution- and transfusion-related coagulopathy. Healthy donor blood was mixed 1 + 1 with six different transfusion regimens. Each regimen contained RBC, platelet concentrate, and either fresh frozen plasma (FFP) or Ringer's acetate (RA). The regimens were further augmented through addition of a low- or medium-dose fibrinogen concentrate and FXIII. Transfusion with FFP alone was insufficient to maintain tissue-factor activated clot strength, coincidental with a deficiency in fibrin-based clot strength. Fibrinogen concentrate conserved, but did not improve coagulation kinetics and overall clot strength. Only combination therapy with FFP and low-dose fibrinogen concentrate improved both coagulation kinetics and fibrin-based clot strength. Administration of FXIII did not result in an improvement of clot strength. In conclusion, combination therapy with both FFP and low-dose fibrinogen concentrate improved clotting time and produced firm clots, representing a possible preferred first-line regimen to manage trauma-induced coagulopathy when RBC and platelets are also transfused. Further research is required to identify optimal first-line transfusion fluids for massive traumatic haemorrhage.

Factors Affecting Nuclear Export of the 60S Ribosomal Subunit In Vivo

PubMed Central

Stage-Zimmermann, Tracy; Schmidt, Ute; Silver, Pamela A.

2000-01-01

In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit in S. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the ∼45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b–GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNA-processing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b–GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b–GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b–GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120. PMID:11071906

Raman spectroscopic study of hydrogen ordered ice XIII and of its reversible phase transition to disordered ice V.

PubMed

Salzmann, Christoph G; Hallbrucker, Andreas; Finney, John L; Mayer, Erwin

2006-07-14

Raman spectra of recovered ordered H(2)O (D(2)O) ice XIII doped with 0.01 M HCl (DCl) recorded in vacuo at 80 K are reported in the range 3600-200 cm(-1). The bands are assigned to the various types of modes on the basis of isotope ratios. On thermal cycling between 80 and 120 K, the reversible phase transition to disordered ice V is observed. The remarkable effect of HCl (DCl) on orientational ordering in ice V and its phase transition to ordered ice XIII, first reported in a powder neutron diffraction study of DCl doped D(2)O ice V (C. G. Salzmann, P. G. Radaelli, A. Hallbrucker, E. Mayer, J. L. Finney, Science, 2006, 311, 1758), is demonstrated by Raman spectroscopy and discussed. The dopants KOH and HF have only a minor effect on hydrogen ordering in ice V, as shown by the Raman spectra.

Tabulation of comet observations.

NASA Astrophysics Data System (ADS)

Concerning comets: 1962 VIII Humason, 1971 V Toba, 1975 XI Bradfield, 1979 X Bradfield, 1980 X P/Stephan-Oterma, 1980 XI P/Encke, 1980 XIII P/Tuttle, 1981 II Panther, 1982 VI Austin, 1982 VIII P/Churyumov-Gerasimenko, 1983 V Sugano-Saigusa-Fujikawa, 1983 VII IRAS-Araki-Alcock, 1983 XIII P/Kopff, 1984 III P/Hartley-IRAS, 1985 XIII P/Giacobini-Zinner, 1985 XVII Hartley-Good, 1985 XIX Thiele, 1986 III P/Halley, 1986h P/Schwassmann-Wachmann 2, 1986j P/Comas Solá, 1986k P/Kohoutek, 1986l Wilson, 1986m P/Grigg-Skjellerup, 1986n Sorrells, 1987h P/Howell, 1987l P/Reinmuth 2, 1987m P/Brooks 2, 1987n P/Harrington, 1987p P/Borrelly, 1987r P/Reinmuth 1, 1987s Bradfield, 1987u Rudenko, 1987y Levy, 1987z P/Shoemaker-Holt, 1987b1 McNaught, 1987d1 Ichimura, 1987f1 Furuyama, 1988a Liller, 1988b Shoemaker, 1988c Maury-Phinney, 1988e Levy, P/Schwassmann-Wachmann 1.

Expression of accessory colonization factor subunit A (ACFA) of Vibrio cholerae and ACFA fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

PubMed

Sharma, Manoj Kumar; Jani, Dewal; Thungapathra, M; Gautam, J K; Meena, L S; Singh, Yogendra; Ghosh, Amit; Tyagi, Akhilesh Kumar; Sharma, Arun Kumar

2008-05-20

In earlier study from our group, cholera toxin B subunit had been expressed in tomato for developing a plant-based vaccine against cholera. In the present investigation, gene for accessory colonization factor (acf) subunit A, earlier reported to be essential for efficient colonization in the intestine, has been expressed in Escherichia coli as well as tomato plants. Gene encoding for a chimeric protein having a fusion of cholera toxin B subunit and accessory colonization factor A was also expressed in tomato to generate more potent combinatorial antigen. CaMV35S promoter with a duplicated enhancer sequence was used for expression of these genes in tomato. Integration of transgenes into tomato genome was confirmed by PCR and Southern hybridization. Expression of the genes was confirmed at transcript and protein levels. Accessory colonization factor A and cholera toxin B subunit fused to this protein accumulated up to 0.25% and 0.08% of total soluble protein, respectively, in the fruits of transgenic plants. Whereas protein purified from E. coli, in combination with cholera toxin B subunit can be used for development of conventional subunit vaccine, tomato fruits expressing these proteins can be used together with tomato plants expressing cholera toxin B subunit for development of oral vaccine against cholera.

Artesanias Mexico - Americanas. Programa Piloto de Entrenamiento Para El Asociado Bilingue y Bicultural En El Desarrollo del Nino: Guia XIII [Arts and Crafts of Mexico and the Americas. Pilot Program for the Training of Bilingual and Bicultural Teachers for the Cognitive Development of the Child: Guide XIII].

ERIC Educational Resources Information Center

de Celis, Margarita

This Child Development Associate (CDA) training module, the thirteenth in a series of 16, provides creative experiences with arts and crafts for young children. Designed for preschool teachers and paraprofessional trainees, the Spanish text offers a variety of craft activities. A list of materials necessary, step-by-step directions and…

Factor XIII deficiency in Iran: a comprehensive review of the literature.

PubMed

Dorgalaleh, Akbar; Naderi, Majid; Hosseini, Maryam Sadat; Alizadeh, Shaban; Hosseini, Soudabeh; Tabibian, Shadi; Eshghi, Peyman

2015-04-01

Factor XIII deficiency (FXIIID) is a rare bleeding disorder with an estimated prevalence of 1 in 2-million population worldwide. In Iran, a Middle Eastern country with a high rate of consanguineous marriages, there are approximately 473 patients afflicted with FXIIID. An approximately 12-fold higher prevalence of FXIIID is estimated in Iran in comparison with overall worldwide frequency. In this study, we have undertaken a comprehensive review on different aspects of FXIIID in the Iranian population. The distribution of this disease in different regions of Iran reveals that Sistan and Baluchestan Province has not only the highest number of patients with FXIIID in Iran but the highest global incidence of this condition. Among Iranian patients, umbilical cord bleeding, hematoma, and prolonged wound bleeding are the most frequent clinical manifestations. There are several disease causing mutations in Iranian patients with FXIIID, with Trp187Arg being the most common mutation in FXIIID in Iran. Traditionally, the management of FXIIID in Iran was only based on administration of fresh frozen plasma or cryoprecipitate, until 2009 when FXIII concentrate became available for patient management. Various studies have evaluated the efficacy and safety of prophylactic regimens in different situations with valuable findings. Although the focus of this study is on Iran, it offers considerable insight into FXIIID, which can be applied more extensively to improve the management and quality of life in all affected patients. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Phosphorylation of Wheat Germ Initiation Factors and Ribosomal Proteins 1

PubMed Central

Browning, Karen S.; Yan, Tyan Fuh J.; Lauer, Stephen J.; Aquino, Lu Ann; Tao, Mariano; Ravel, Joanne M.

1985-01-01

The ability of the wheat germ initiation factors and ribosomes to serve as substrates for a wheat germ protein kinase (Yan and Tao 1982 J Biol Chem 257: 7037-7043) has been investigated. The wheat germ kinase catalyzes the phosphorylation of the 42,000 dalton subunit of eukaryotic initiation factor (eIF)-2 and the 107,000 dalton subunit of eIF-3. Other initiation factors, eIF-4B and eIF-4A, and elongation factors, EF-1 and EF-2, are not phosphorylated by the kinase. Quantitative analysis indicates that the kinase catalyzes the incorporation of about 0.5 to 0.6 mole of phosphate per mole of the 42,000 dalton subunit of eIF-2 and about 6 moles of phosphate per mole of the 107,000 dalton subunit of eIF-3. Three proteins (Mr = 38,000, 14,800, and 12,600) of the 60S ribosomal subunit are phosphorylated by the kinase, but none of the 40S ribosomal proteins are substrates of the kinase. No effects of phosphorylation on the activities of eIF-2, eIF-3, or 60S ribosomal subunits could be demonstrated in vitro. Images Fig. 1 Fig. 3 Fig. 4 PMID:16664060

Structure and Biophysics of CBFβ/RUNX and Its Translocation Products.

PubMed

Tahirov, Tahir H; Bushweller, John

2017-01-01

The core binding factor (CBF) transcription factor is somewhat unique in that it is composed of a DNA binding RUNX subunit (RUNX1, 2, or 3) and a non-DNA binding CBFβ subunit, which modulates RUNX protein activity by modulating the auto-inhibition of the RUNX subunits. Since the discovery of this fascinating transcription factor more than 20 years ago, there has been a robust effort to characterize the structure as well as the biochemical properties of CBF. More recently, these efforts have also extended to the fusion proteins that arise from the subunits of CBF in leukemia. This chapter highlights the work of numerous labs which has provided a detailed understanding of the structure and function of this transcription factor and its fusion proteins.

Identification of a Kinase in Wheat Germ that Phosphorylates the Large Subunit of Initiation Factor 4F 1

PubMed Central

Humphreys, Jean; Browning, Karen S.; Ravel, Joanne M.

1988-01-01

A kinase has been isolated from wheat (Triticum aestivum) germ that phosphorylates the 220 kilodaltons (kD) subunit of wheat germ initiation factor (eIF) 4F, the 80 kD subunit of eIF-4B (an isozyme form of eIF-4F) and eIF-4G (the functional equivalent to mammalian eIF-4B). The kinase elutes from Sephacryl S-200 slightly in front of ovalbumin. The kinase phosphorylates casein and histone IIA to a small extent, but does not phosphorylate phosvitin. Of the wheat germ initiation factors, elongation factors, and small and large ribosomal subunits, only eIF-4F, eIF-4B, and eIF-4G are phosphorylated to a significant extent. The kinase phosphorylates eIF-4F to the extent of two phosphates per mole of the 220 kD subunit and phosphorylates eIF-4B to the extent of one phosphate per mole of the 80 kD subunit. The 26 kD subunit of eIF-4F and the 28 kD subunit of eIF-4B are not phosphorylated by the kinase. The kinase phosphorylates the 59 kD component of eIF-4G to the extent of 0.25 phosphate per mole of eIF-4G. Phosphorylation of eIF-4F and eIF-4B does not affect their ability to support the binding of mRNA to small ribosomal subunits in vitro. Images Fig. 2 Fig. 3 PMID:16666331

The in vivo effect of fibrinogen and factor XIII on clot formation and fibrinolysis in Glanzmann's thrombasthenia.

PubMed

Shenkman, Boris; Livnat, Tami; Misgav, Mudi; Budnik, Ivan; Einav, Yulia; Martinowitz, Uriel

2012-01-01

Glanzmann's thrombasthenia (GT) is characterized by increased bleeding risk. The treatment options in GT are limited. The aim of this study was to test the effect of GT blood supplementation with fibrinogen and factor XIII on thrombin generation, blood clotting, and fibrinolysis. Whole blood samples of GT patients and normal donors treated with eptifibatide (GT model) were subjected to clotting by CaCl(2) and tissue factor. Thrombin generation was measured in platelet-rich plasma. Clot formation and tPA-induced fibrinolysis were evaluated in whole blood by rotation thromboelastometry (ROTEM). Blood was supplemented with fibrinogen (3 g/L) and/or FXIII (2 IU/mL). Thrombin generation analysis of blood derived from GT model and GT patients revealed decreased endogenous thrombin potential and peak height and extended lag time compared to control. However, this method was not sensitive to blood spiking with fibrinogen and FXIII. ROTEM revealed lower maximum clot firmness (MCF) and area under curve (AUC) in the blood of GT model and GT patients. In the absence of exogenous tPA, blood spiking with fibrinogen markedly enhanced clot quality while FXIII had no effect. Combination of fibrinogen and FXIII did not add to the effect of fibrinogen. In contrast, by the addition of tPA, both fibrinogen and FXIII separately and, to more extent, in combination enhanced clot quality as well as resistance against tPA-induced fibrinolysis (increasing MCF, AUC, and lysis onset time). In conclusion, fibrinogen and FXIII exerted stimulation of blood clotting and inhibition of fibrinolysis. Treating normal blood with eptifibatide mimics the changes of coagulopathy in GT blood.

Architecture of human translation initiation factor 3

PubMed Central

Querol-Audi, Jordi; Sun, Chaomin; Vogan, Jacob M.; Smith, Duane; Gu, Yu; Cate, Jamie; Nogales, Eva

2013-01-01

SUMMARY Eukaryotic translation initiation factor 3 (eIF3) plays a central role in protein synthesis by organizing the formation of the 43S preinitiation complex. Using genetic tag visualization by electron microscopy, we reveal the molecular organization of ten human eIF3 subunits, including an octameric core. The structure of eIF3 bears a close resemblance to that of the proteasome lid, with a conserved spatial organization of eight core subunits containing PCI and MPN domains that coordinate functional interactions in both complexes. We further show that eIF3 subunits a and c interact with initiation factors eIF1 and eIF1A, which control the stringency of start codon selection. Finally, we find that subunit j, which modulates messenger RNA interactions with the small ribosomal subunit, makes multiple independent interactions with the eIF3 octameric core. These results highlight the conserved architecture of eIF3 and how it scaffolds key factors that control translation initiation in higher eukaryotes, including humans. PMID:23623729

Transcriptional regulators of Na,K-ATPase subunits

PubMed Central

Li, Zhiqin; Langhans, Sigrid A.

2015-01-01

The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic α-subunit, the β-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids, and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits has been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease. PMID:26579519

Factor XIII stiffens fibrin clots by causing fiber compaction.

PubMed

Kurniawan, N A; Grimbergen, J; Koopman, J; Koenderink, G H

2014-10-01

Factor XIII-induced cross-linking has long been associated with the ability of fibrin blood clots to resist mechanical deformation, but how FXIII can directly modulate clot stiffness is unknown. We hypothesized that FXIII affects the self-assembly of fibrin fibers by altering the lateral association between protofibrils. To test this hypothesis, we studied the cross-linking kinetics and the structural evolution of the fibers and clots during the formation of plasma-derived and recombinant fibrins by using light scattering, and the response of the clots to mechanical stresses by using rheology. We show that the lateral aggregation of fibrin protofibrils initially results in the formation of floppy fibril bundles, which then compact to form tight and more rigid fibers. The first stage is reflected in a fast (10 min) increase in clot stiffness, whereas the compaction phase is characterized by a slow (hours) development of clot stiffness. Inhibition of FXIII completely abrogates the slow compaction. FXIII strongly increases the linear elastic modulus of the clots, but does not affect the non-linear response at large deformations. We propose a multiscale structural model whereby FXIII-mediated cross-linking tightens the coupling between the protofibrils within a fibrin fiber, thus making the fiber stiffer and less porous. At small strains, fiber stiffening enhances clot stiffness, because the clot response is governed by the entropic elasticity of the fibers, but once the clot is sufficiently stressed, the modulus is independent of protofibril coupling, because clot stiffness is governed by individual protofibril stretching. © 2014 International Society on Thrombosis and Haemostasis.

Therapeutic potential of Mediator complex subunits in metabolic diseases.

PubMed

Ranjan, Amol; Ansari, Suraiya A

2018-01-01

The multisubunit Mediator is an evolutionary conserved transcriptional coregulatory complex in eukaryotes. It is needed for the transcriptional regulation of gene expression in general as well as in a gene specific manner. Mediator complex subunits interact with different transcription factors as well as components of RNA Pol II transcription initiation complex and in doing so act as a bridge between gene specific transcription factors and general Pol II transcription machinery. Specific interaction of various Mediator subunits with nuclear receptors (NRs) and other transcription factors involved in metabolism has been reported in different studies. Evidences indicate that ligand-activated NRs recruit Mediator complex for RNA Pol II-dependent gene transcription. These NRs have been explored as therapeutic targets in different metabolic diseases; however, they show side-effects as targets due to their overlapping involvement in different signaling pathways. Here we discuss the interaction of various Mediator subunits with transcription factors involved in metabolism and whether specific interaction of these transcription factors with Mediator subunits could be potentially utilized as therapeutic strategy in a variety of metabolic diseases. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Energy Levels and Radiative Rates for Transitions in F-like Sc XIII and Ne-like Sc XII and Y XXX

NASA Astrophysics Data System (ADS)

Aggarwal, Kanti

2018-05-01

Energy levels, radiative rates and lifetimes are reported for F-like Sc~XIII and Ne-like Sc~XII and Y~XXX for which the general-purpose relativistic atomic structure package ({\\sc grasp}) has been adopted. For all three ions limited data exist in the literature but comparisons have been made wherever possible to assess the accuracy of the calculations. In the present work the lowest 102, 125 and 139 levels have been considered for the respective ions. Additionally, calculations have also been performed with the flexible atomic code ({\\sc fac}) to (particularly) confirm the accuracy of energy levels.

A Protocol for the Preparation of Cryoprecipitate and Cryo-depleted Plasma for Proteomic Studies.

PubMed

Sparrow, Rosemary L; Simpson, Richard J; Greening, David W

2017-01-01

Cryoprecipitate is a concentrate of high-molecular-weight plasma proteins that precipitate when frozen plasma is slowly thawed at 1-6 °C. The concentrate contains factor VIII (antihemophilic factor), von Willebrand factor (vWF), fibrinogen, factor XIII, fibronectin, and small amounts of other plasma proteins. Clinical grade preparations of cryoprecipitate are mainly used to treat fibrinogen deficiency caused by acute bleeding or functional abnormalities of the fibrinogen protein. In the past, cryoprecipitate was used to treat von Willebrand disease and hemophilia A (factor VIII deficiency), but the availability of more highly purified coagulation factor concentrates or recombinant protein preparations has superseded the use of cryoprecipitate for these coagulopathies. Cryo-depleted plasma ("cryosupernatant") is the plasma supernatant remaining following removal of the cryoprecipitate from frozen-thawed plasma. It contains all the other plasma proteins and clotting factors present in plasma that remain soluble during cold-temperature thawing of the plasma. This protocol describes the clinical-scale preparation of cryoprecipitate and cryo-depleted plasma for proteomic studies.

Aerodynamic Design of Axial-flow Compressors. Volume III

NASA Technical Reports Server (NTRS)

Johnson, Irving A; Bullock, Robert O; Graham, Robert W; Costilow, Eleanor L; Huppert, Merle C; Benser, William A; Herzig, Howard Z; Hansen, Arthur G; Jackson, Robert J; Yohner, Peggy L;

Establishment of a prenatal diagnosis schedule as part of a prophylaxis program of factor XIII deficiency in the southeast of Iran.

PubMed

Naderi, Majid; Reykande, Samira Esmaeili; Dorgalaleh, Akbar; Alizadeh, Shaban; Tabibian, Shadi; Einollahi, Nahid; Moghaddam, Ebrahim Miri

2016-01-01

Factor XIII deficiency (FXIIID) is an extremely rare bleeding disorder with a prevalence of 1 in 3 million in the general population. Compared to its global incidence, it has the greatest prevalence in Sistan and Baluchistan Province in the southeast of Iran. The high incidence of FXIIID in this region causes a high rate of morbidity and mortality among the affected individuals because of life-threatening episodes such as central nervous system (CNS) bleeding, umbilical cord bleeding, as well as miscarriage. CNS bleeding leads to a considerable number of neurological and behavioral complications. Therefore, we have designed an established prenatal diagnosis (PND) program to prevent the increasing incidence of life-threatening bleeding episodes and related complications among neonates with congenital FXIIID. This study was conducted from September 2013 to August 2014. A consent form was signed by the parents. Fetal sampling was done via abdominal chorionic villus sampling passage under local anesthesia and ultrasonic guidance within the first trimester of pregnancy. Fetal DNA was extracted, and PCR-restriction fragment length polymorphism was performed for the only reported mutation of FXIII (Trp187Arg) in the southeast of Iran. During the period of study, PND was performed on eight fetuses. Six fetuses were offspring of parental consanguineous marriages, and all of them had a positive family history of FXIIID. Seven out of the eight fetuses had a family member with CNS bleeding due to FXIIID. Four fetuses had a FXIIID-related death. One of the fetuses bore homozygous Trp187Arg mutation, whereas six were heterozygous, and one of the mothers gave birth to an unaffected fetus. To the best of our knowledge, PND is a possible solution to control high incidence of life-threatening episodes of FXIIID in southeast Iran.

Transglutaminases factor XIII-A and TG2 regulate resorption, adipogenesis and plasma fibronectin homeostasis in bone and bone marrow

PubMed Central

Mousa, Aisha; Cui, Cui; Song, Aimei; Myneni, Vamsee D; Sun, Huifang; Li, Jin Jin; Murshed, Monzur; Melino, Gerry; Kaartinen, Mari T

2017-01-01

Appropriate bone mass is maintained by bone-forming osteoblast and bone-resorbing osteoclasts. Mesenchymal stem cell (MSC) lineage cells control osteoclastogenesis via expression of RANKL and OPG (receptor activator of nuclear factor κB ligand and osteoprotegerin), which promote and inhibit bone resorption, respectively. Protein crosslinking enzymes transglutaminase 2 (TG2) and Factor XIII-A (FXIII-A) have been linked to activity of myeloid and MSC lineage cells; however, in vivo evidence has been lacking to support their function. In this study, we show in mice that TG2 and FXIII-A control monocyte-macrophage cell differentiation into osteoclasts as well as RANKL production in MSCs and in adipocytes. Long bones of mice lacking TG2 and FXIII-A transglutaminases, show compromised biomechanical properties and trabecular bone loss in axial and appendicular skeleton. This was caused by increased osteoclastogenesis, a cellular phenotype that persists in vitro. The increased potential of TG2 and FXIII-A deficient monocytes to form osteoclasts was reversed by chemical inhibition of TG activity, which revealed the presence of TG1 in osteoclasts and assigned different roles for the TGs as regulators of osteoclastogenesis. TG2- and FXIII-A-deficient mice had normal osteoblast activity, but increased bone marrow adipogenesis, MSCs lacking TG2 and FXIII-A showed high adipogenic potential and significantly increased RANKL expression as well as upregulated TG1 expression. Chemical inhibition of TG activity in the null cells further increased adipogenic potential and RANKL production. Altered differentiation of TG2 and FXIII-A null MSCs was associated with plasma fibronectin (FN) assembly defect in cultures and FN retention in serum and marrow in vivo instead of assembly into bone. Our findings provide new functions for TG2, FXIII-A and TG1 in bone cells and identify them as novel regulators of bone mass, plasma FN homeostasis, RANKL production and myeloid and MSC cell differentiation. PMID:28387755

Subunit architecture and functional modular rearrangements of the transcriptional Mediator complex

PubMed Central

Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C.; Conaway, Joan W.; Asturias, Francisco J.

2014-01-01

SUMMARY The multisubunit Mediator comprising ~30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism. PMID:24882805

The delta-subunit of murine guanine nucleotide exchange factor eIF-2B. Characterization of cDNAs predicts isoforms differing at the amino-terminal end.

PubMed

Henderson, R A; Krissansen, G W; Yong, R Y; Leung, E; Watson, J D; Dholakia, J N

1994-12-02

Protein synthesis in mammalian cells is regulated at the level of the guanine nucleotide exchange factor, eIF-2B, which catalyzes the exchange of eukaryotic initiation factor 2-bound GDP for GTP. We have isolated and sequenced cDNA clones encoding the delta-subunit of murine eIF-2B. The cDNA sequence encodes a polypeptide of 544 amino acids with molecular mass of 60 kDa. Antibodies against a synthetic polypeptide of 30 amino acids deduced from the cDNA sequence specifically react with the delta-subunit of mammalian eIF-2B. The cDNA-derived amino acid sequence shows significant homology with the yeast translational regulator Gcd2, supporting the hypothesis that Gcd2 may be the yeast homolog of the delta-subunit of mammalian eIF-2B. Primer extension studies and anchor polymerase chain reaction analysis were performed to determine the 5'-end of the transcript for the delta-subunit of eIF-2B. Results of these experiments demonstrate two different mRNAs for the delta-subunit of eIF-2B in murine cells. The isolation and characterization of two different full-length cDNAs also predicts the presence of two alternate forms of the delta-subunit of eIF-2B in murine cells. These differ at their amino-terminal end but have identical nucleotide sequences coding for amino acids 31-544.

Nuclear respiratory factor 2 regulates the expression of the same NMDA receptor subunit genes as NRF-1: both factors act by a concurrent and parallel mechanism to couple energy metabolism and synaptic transmission.

PubMed

Priya, Anusha; Johar, Kaid; Wong-Riley, Margaret T T

2013-01-01

Neuronal activity and energy metabolism are tightly coupled processes. Previously, we found that nuclear respiratory factor 1 (NRF-1) transcriptionally co-regulates energy metabolism and neuronal activity by regulating all 13 subunits of the critical energy generating enzyme, cytochrome c oxidase (COX), as well as N-methyl-d-aspartate (NMDA) receptor subunits 1 and 2B, GluN1 (Grin1) and GluN2B (Grin2b). We also found that another transcription factor, nuclear respiratory factor 2 (NRF-2 or GA-binding protein) regulates all subunits of COX as well. The goal of the present study was to test our hypothesis that NRF-2 also regulates specific subunits of NMDA receptors, and that it functions with NRF-1 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation of mouse neuroblastoma cells and rat visual cortical tissue, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate Grin1 and Grin2b genes, but not any other NMDA subunit genes. Grin1 and Grin2b transcripts were up-regulated by depolarizing KCl, but silencing of NRF-2 prevented this up-regulation. On the other hand, over-expression of NRF-2 rescued the down-regulation of these subunits by the impulse blocker TTX. NRF-2 binding sites on Grin1 and Grin2b are conserved among species. Our data indicate that NRF-2 and NRF-1 operate in a concurrent and parallel manner in mediating the tight coupling between energy metabolism and neuronal activity at the molecular level. Copyright © 2012 Elsevier B.V. All rights reserved.

Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

PubMed

Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

2013-11-01

Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons. © 2013 International Society for Neurochemistry.

Effects on coagulation factor production following primary hepatomitogen-induced direct hyperplasia.

PubMed

Tatsumi, Kohei; Ohashi, Kazuo; Taminishi, Sanae; Takagi, Soichi; Utoh, Rie; Yoshioka, Akira; Shima, Midori; Okano, Teruo

2009-11-14

To investigate the molecular mechanisms involved in coagulation factor expression and/or function during direct hyperplasia (DH)-mediated liver regeneration. Direct hyperplasia-mediated liver regeneration was induced in female C57BL/6 mice by administering 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a representative hepatomitogen. Mice were weighed and sacrificed at various time points [Day 0 (D0: prior to injection), 3 h, D1, D2, D3, and D10] after TCPOBOP administration to obtain liver and blood samples. Using the RNA samples extracted from the liver, a comprehensive analysis was performed on the hepatic gene expression profiling of coagulation-related factors by real-time RT-PCR (fibrinogen, prothrombin, factors V, VII, VIII, IX, X, XI, XII, XIIIbeta, plasminogen, antithrombin, protein C, protein S, ADAMTS13, and VWF). The corresponding plasma levels of coagulation factors (fibrinogen, prothrombin, factors V, VII, VIII, IX, X, XI, XII, XIII, and VWF) were also analyzed and compared with their mRNA levels. Gavage administration of TCPOBOP (3 mg/kg body weight) resulted in a marked and gradual increase in the weight of the mouse livers relative to the total body weight to 220% by D10 relative to the D0 (control) ratios. At the peak of liver regeneration (D1 and D2), the gene expression levels for most of the coagulation-related factors (fibrinogen, prothrombin, factors V, VII, VIII, IX, XI, XII, XIIIbeta, plasminogen, antithrombin, protein C, ADAMTS13, VWF) were found to be down-regulated in a time-dependent manner, and gradually recovered by D10 to the basal levels. Only mRNA levels of factor X and protein S failed to show any decrease during the regenerative phase. As for the plasma levels, 5 clotting factors (prothrombin, factors VIII, IX, XI, and XII) demonstrated a significant decrease (P<0.05) during the regeneration phase compared with D0. Among these 5 factors, factor IX and factor XI showed the most dramatic decline in their activities by about 50% at D2 compared to the basal levels, and these reductions in plasma activity for both factors were consistent with our RT-PCR findings. In contrast, the plasma activities of the other coagulation factors (fibrinogen, factors V, VII, XIII, and VWF) were not significantly reduced, despite the reduction in the liver mRNA levels. Unlike the other factors, FX showed a temporal increase in its plasma activity, with significant increases (P<0.05) detected at D1. Investigating the coagulation cascade protein profiles during liver regeneration by DH may help to better understand the basic biology of the liver under normal and pathological conditions.

Compaction of fibrin clots reveals the antifibrinolytic effect of factor XIII.

PubMed

Rijken, D C; Abdul, S; Malfliet, J J M C; Leebeek, F W G; Uitte de Willige, S

2016-07-01

Essentials Factor XIIIa inhibits fibrinolysis by forming fibrin-fibrin and fibrin-inhibitor cross-links. Conflicting studies about magnitude and mechanisms of inhibition have been reported. Factor XIIIa most strongly inhibits lysis of mechanically compacted or retracted plasma clots. Cross-links of α2-antiplasmin to fibrin prevent the inhibitor from being expelled from the clot. Background Although insights into the underlying mechanisms of the effect of factor XIII on fibrinolysis have improved considerably in the last few decades, in particular with the discovery that activated FXIII (FXIIIa) cross-links α2 -antiplasmin to fibrin, the topic remains a matter of debate. Objective To elucidate the mechanisms of the antifibrinolytic effect of FXIII. Methods and Results Platelet-poor plasma clot lysis, induced by the addition of tissue-type plasminogen activator, was measured in the presence or absence of a specific FXIIIa inhibitor. Both in a turbidity assay and in a fluorescence assay, the FXIIIa inhibitor had only a small inhibitory effect: 1.6-fold less tissue-type plasminogen activator was required for 50% clot lysis in the presence of the FXIIIa inhibitor. However, when the plasma clot was compacted by centrifugation, the FXIIIa inhibitor had a strong inhibitory effect, with 7.7-fold less tissue-type plasminogen activator being required for 50% clot lysis in the presence of the FXIIIa inhibitor. In both experiments, the effects of the FXIIIa inhibitor were entirely dependent on the cross-linking of α2 -antiplasmin to fibrin. The FXIIIa inhibitor reduced the amount of α2 -antiplasmin present in the compacted clots from approximately 30% to < 4%. The results were confirmed with experiments in which compaction was achieved by platelet-mediated clot retraction. Conclusions Compaction or retraction of fibrin clots reveals the strong antifibrinolytic effect of FXIII. This is explained by the cross-linking of α2 -antiplasmin to fibrin by FXIIIa, which prevents the plasmin inhibitor from being fully expelled from the clot during compaction/retraction. © 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Local dynamics measured by hydrogen/deuterium exchange and mass spectrometry of creatine kinase digested by two proteases.

PubMed

Mazon, Hortense; Marcillat, Olivier; Forest, Eric; Vial, Christian

2005-12-01

Hydrogen/deuterium exchange coupled to mass spectrometry has been used to investigate the structure and dynamics of native dimeric cytosolic muscle creatine kinase. The protein was incubated in D2O for various time. After H/D exchange and rapid quenching of the reaction, the partially deuterated protein was cleaved in parallel by two different proteases (pepsin or type XIII protease from Aspergillus saitoi) to increase the sequence coverage and spatial resolution of deuterium incorporation. The resulting peptides were analyzed by liquid chromatography coupled to mass spectrometry. In comparison with the 3D structure of MM-CK, the analysis of the two independent proteolysis deuteration patterns allowed us to get new insights into CK local dynamics as compared to a previous study using pepsin [Mazon et al. Protein Science 13 (2004) 476-486]. In particular, we obtained more information on the kinetics and extent of deuterium exchange in the N- and C-terminal extremities represented by the 1-22 and 362-380 pepsin peptides. Indeed, we observed a very different behaviour of the 1-12 and 13-22 type XIII protease peptides, and similarly for the 362-373 and 374-380 peptides. Moreover, comparison of the deuteration patterns of type XIII protease segments of the large 90-126 pepsin peptide led us to identify a small relatively dynamic region (108-114).

Leptospira Immunoglobulin-Like Protein B (LigB) Binds to Both the C-Terminal 23 Amino Acids of Fibrinogen αC Domain and Factor XIII: Insight into the Mechanism of LigB-Mediated Blockage of Fibrinogen α Chain Cross-Linking.

PubMed

Hsieh, Ching-Lin; Chang, Eric; Tseng, Andrew; Ptak, Christopher; Wu, Li-Chen; Su, Chun-Li; McDonough, Sean P; Lin, Yi-Pin; Chang, Yung-Fu

2016-09-01

The coagulation system provides a primitive but effective defense against hemorrhage. Soluble fibrinogen (Fg) monomers, composed of α, β and γ chains, are recruited to provide structural support for the formation of a hemostatic plug. Fg binds to platelets and is processed into a cross-linked fibrin polymer by the enzymatic clotting factors, thrombin and Factor XIII (FXIII). The newly formed fibrin-platelet clot can act as barrier to protect against pathogens from entering the bloodstream. Further, injuries caused by bacterial infections can be confined to the initial wound site. Many pathogenic bacteria have Fg-binding adhesins that can circumvent the coagulation pathway and allow the bacteria to sidestep containment. Fg expression is upregulated during lung infection providing an attachment surface for bacteria with the ability to produce Fg-binding adhesins. Fg binding by leptospira might play a crucial factor in Leptospira-associated pulmonary hemorrhage, the main factor contributing to lethality in severe cases of leptospirosis. The 12th domain of Leptospira immunoglobulin-like protein B (LigB12), a leptospiral adhesin, interacts with the C-terminus of FgαC (FgαCC). In this study, the binding site for LigB12 was mapped to the final 23 amino acids at the C-terminal end of FgαCC (FgαCC8). The association of FgαCC8 with LigB12 (ELISA, KD = 0.76 μM; SPR, KD = 0.96 μM) was reduced by mutations of both charged residues (R608, R611 and H614 from FgαCC8; D1061 from LigB12) and hydrophobic residues (I613 from FgαCC8; F1054 and A1065 from LigB12). Additionally, LigB12 bound strongly to FXIII and also inhibited fibrin formation, suggesting that LigB can disrupt coagulation by suppressing FXIII activity. Here, the detailed binding mechanism of a leptospiral adhesin to a host hemostatic factor is characterized for the first time and should provide better insight into the pathogenesis of leptospirosis.

Leptospira Immunoglobulin-Like Protein B (LigB) Binds to Both the C-Terminal 23 Amino Acids of Fibrinogen αC Domain and Factor XIII: Insight into the Mechanism of LigB-Mediated Blockage of Fibrinogen α Chain Cross-Linking

PubMed Central

Hsieh, Ching-Lin; Chang, Eric; Tseng, Andrew; Ptak, Christopher; Wu, Li-Chen; Su, Chun-Li; McDonough, Sean P.; Lin, Yi-Pin; Chang, Yung-Fu

2016-01-01

The coagulation system provides a primitive but effective defense against hemorrhage. Soluble fibrinogen (Fg) monomers, composed of α, β and γ chains, are recruited to provide structural support for the formation of a hemostatic plug. Fg binds to platelets and is processed into a cross-linked fibrin polymer by the enzymatic clotting factors, thrombin and Factor XIII (FXIII). The newly formed fibrin-platelet clot can act as barrier to protect against pathogens from entering the bloodstream. Further, injuries caused by bacterial infections can be confined to the initial wound site. Many pathogenic bacteria have Fg-binding adhesins that can circumvent the coagulation pathway and allow the bacteria to sidestep containment. Fg expression is upregulated during lung infection providing an attachment surface for bacteria with the ability to produce Fg-binding adhesins. Fg binding by leptospira might play a crucial factor in Leptospira-associated pulmonary hemorrhage, the main factor contributing to lethality in severe cases of leptospirosis. The 12th domain of Leptospira immunoglobulin-like protein B (LigB12), a leptospiral adhesin, interacts with the C-terminus of FgαC (FgαCC). In this study, the binding site for LigB12 was mapped to the final 23 amino acids at the C-terminal end of FgαCC (FgαCC8). The association of FgαCC8 with LigB12 (ELISA, KD = 0.76 μM; SPR, KD = 0.96 μM) was reduced by mutations of both charged residues (R608, R611 and H614 from FgαCC8; D1061 from LigB12) and hydrophobic residues (I613 from FgαCC8; F1054 and A1065 from LigB12). Additionally, LigB12 bound strongly to FXIII and also inhibited fibrin formation, suggesting that LigB can disrupt coagulation by suppressing FXIII activity. Here, the detailed binding mechanism of a leptospiral adhesin to a host hemostatic factor is characterized for the first time and should provide better insight into the pathogenesis of leptospirosis. PMID:27622634

77 FR 68155 - Sunshine Act Meeting Notice

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-15

...-Quality Control Compliance/3rd Party CPA XII. National Foreclosure Mitigation Counseling (NFMC) Compliance Update XIII. OHTS Watch List and Overview of Organizational Assessment Review Process XIV. Adjournment...

32 CFR 865.114 - Decisional document.

Code of Federal Regulations, 2013 CFR

2013-07-01

...) Awards and decorations. (ix) Educational level. (x) Aptitude test scores. (xi) Incidents of punishment... punishment). (xii) Conviction by court-martial. (xiii) Prior military service and type of discharge received...

32 CFR 865.114 - Decisional document.

Code of Federal Regulations, 2011 CFR

2011-07-01

...) Awards and decorations. (ix) Educational level. (x) Aptitude test scores. (xi) Incidents of punishment... punishment). (xii) Conviction by court-martial. (xiii) Prior military service and type of discharge received...

32 CFR 865.114 - Decisional document.

Code of Federal Regulations, 2014 CFR

2014-07-01

...) Awards and decorations. (ix) Educational level. (x) Aptitude test scores. (xi) Incidents of punishment... punishment). (xii) Conviction by court-martial. (xiii) Prior military service and type of discharge received...

32 CFR 865.114 - Decisional document.

Code of Federal Regulations, 2012 CFR

2012-07-01

...) Awards and decorations. (ix) Educational level. (x) Aptitude test scores. (xi) Incidents of punishment... punishment). (xii) Conviction by court-martial. (xiii) Prior military service and type of discharge received...

Dual function of Rpn5 in two PCI complexes, the 26S proteasome and COP9 signalosome.

PubMed

Yu, Zanlin; Kleifeld, Oded; Lande-Atir, Avigail; Bsoul, Maisa; Kleiman, Maya; Krutauz, Daria; Book, Adam; Vierstra, Richard D; Hofmann, Kay; Reis, Noa; Glickman, Michael H; Pick, Elah

2011-04-01

Subunit composition and architectural structure of the 26S proteasome lid is strictly conserved between all eukaryotes. This eight-subunit complex bears high similarity to the eukaryotic translation initiation factor 3 and to the COP9 signalosome (CSN), which together define the proteasome CSN/COP9/initiation factor (PCI) troika. In some unicellular eukaryotes, the latter two complexes lack key subunits, encouraging questions about the conservation of their structural design. Here we demonstrate that, in Saccharomyces cerevisiae, Rpn5 plays dual roles by stabilizing proteasome and CSN structures independently. Proteasome and CSN complexes are easily dissected, with Rpn5 the only subunit in common. Together with Rpn5, we identified a total of six bona fide subunits at roughly stoichiometric ratios in isolated, affinity-purified CSN. Moreover, the copy of Rpn5 associated with the CSN is required for enzymatic hydrolysis of Rub1/Nedd8 conjugated to cullins. We propose that multitasking by a single subunit, Rpn5 in this case, allows it to function in different complexes simultaneously. These observations demonstrate that functional substitution of subunits by paralogues is feasible, implying that the canonical composition of the three PCI complexes in S. cerevisiae is more robust than hitherto appreciated.

The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

PubMed

Hemsley, Piers A; Hurst, Charlotte H; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R; De Cothi, Elizabeth A; Steele, John F; Knight, Heather

2014-01-01

The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced.

Translational Control of Hemoglobin Synthesis by an Initiation Factor Required for Recycling of Ribosomes and for their Binding to Messenger RNA

PubMed Central

Kaempfer, Raymond; Kaufman, Jennifer

1972-01-01

The continued recycling of ribosomes during protein synthesis in rabbit reticulocyte lysates at 37° requires an initiation factor whose activity is rapidly lost in the absence of added heme. Partially purified factor (i) fully maintains the polysomes; (ii) inhibits the association of 40S and 60S ribosomal subunits into single ribosomes; (iii) promotes the quantitative entry of added 60S subunits into polysomes; (iv) allows the accumulation of ribosomal subunits, instead of single ribosomes, when initiation is blocked with aurin tricarboxylate; and (v) is absolutely required for the binding of globin messenger RNA to ribosomes. These properties suggest that this mammalian initiation factor functions analogously to bacterial IF-3. In addition, the translational control of globin synthesis by heme is exerted, directly or indirectly, through this factor. PMID:4508325

Subunit architecture and functional modular rearrangements of the transcriptional mediator complex.

PubMed

Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J

2014-06-05

The multisubunit Mediator, comprising ∼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

Department of Transportation Agency Semiannual Regulatory Agenda

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-20

... [The Regulatory Plan and Unified Agenda of Federal Regulatory and Deregulatory Actions] [Department of Transportation Agency Semiannual Regulatory Agenda ] Part XIII Department of Transportation Semiannual Regulatory Agenda [[Page 79812

A Nonparametric Approach to Segmentation of Ladar Images

DTIC Science & Technology

2012-12-01

82 5.3.4 Simulation ...91 6.2.1 Multi-Facet Board Simulation ...Pulse spreading effects are ignored. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 xiii Figure Page 5.4. Signal pulse energy with

Structure of ratcheted ribosomes with tRNAs in hybrid states

PubMed Central

Julián, Patricia; Konevega, Andrey L.; Scheres, Sjors H. W.; Lázaro, Melisa; Gil, David; Wintermeyer, Wolfgang; Rodnina, Marina V.; Valle, Mikel

2008-01-01

During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is accompanied by the displacement of the tRNAs on the large ribosomal subunit toward the hybrid A/P and P/E states and by a rotational movement (ratchet) of the ribosomal subunits relative to one another. So far, the structure of the ratcheted state has been observed only when translation factors were bound to the ribosome. Using cryo-electron microscopy and classification, we show here that ribosomes can spontaneously adopt a ratcheted conformation with tRNAs in their hybrid states. The peptidyl-tRNA molecule in the A/P state, which is visualized here, is not distorted compared with the A/A state except for slight adjustments of its acceptor end, suggesting that the displacement of the A-site tRNA on the 50S subunit is passive and is induced by the 30S subunit rotation. Simultaneous subunit ratchet and formation of the tRNA hybrid states precede and may promote the subsequent rapid and coordinated tRNA translocation on the 30S subunit catalyzed by elongation factor G. PMID:18971332

49 CFR 1572.103 - Disqualifying criminal offenses.

Code of Federal Regulations, 2010 CFR

2010-10-01

... controlled substance. (viii) Arson. (ix) Kidnapping or hostage taking. (x) Rape or aggravated sexual abuse. (xi) Assault with intent to kill. (xii) Robbery. (xiii) Fraudulent entry into a seaport as described...

49 CFR 1572.103 - Disqualifying criminal offenses.

Code of Federal Regulations, 2011 CFR

2011-10-01

... controlled substance. (viii) Arson. (ix) Kidnapping or hostage taking. (x) Rape or aggravated sexual abuse. (xi) Assault with intent to kill. (xii) Robbery. (xiii) Fraudulent entry into a seaport as described...

Analysis of the Antigenic and Prophylactic Properties of the Leishmania Translation Initiation Factors eIF2 and eIF2B in Natural and Experimental Leishmaniasis

PubMed Central

Garde, Esther; Ramírez, Laura; Corvo, Laura; Solana, José C.; Martín, M. Elena; González, Víctor M.; Gómez-Nieto, Carlos; Barral, Aldina; Barral-Netto, Manoel; Requena, José M.; Iborra, Salvador; Soto, Manuel

2018-01-01

Different members of intracellular protein families are recognized by the immune system of the vertebrate host infected by parasites of the genus Leishmania. Here, we have analyzed the antigenic and immunogenic properties of the Leishmania eIF2 and eIF2B translation initiation factors. An in silico search in Leishmania infantum sequence databases allowed the identification of the genes encoding the α, β, and γ subunits and the α, β, and δ subunits of the putative Leishmania orthologs of the eukaryotic initiation factors F2 (LieIF2) or F2B (LieIF2B), respectively. The antigenicity of these factors was analyzed by ELISA using recombinant versions of the different subunits. Antibodies against the different LieIF2 and LieIF2B subunits were found in the sera from human and canine visceral leishmaniasis patients, and also in the sera from hamsters experimentally infected with L. infantum. In L. infantum (BALB/c) and Leishmania major (BALB/c or C57BL/6) challenged mice, a moderate humoral response against these protein factors was detected. Remarkably, these proteins elicited an IL-10 production by splenocytes derived from infected mice independently of the Leishmania species employed for experimental challenge. When DNA vaccines based on the expression of the LieIF2 or LieIF2B subunit encoding genes were administered in mice, an antigen-specific secretion of IFN-γ and IL-10 cytokines was observed. Furthermore, a partial protection against murine CL development due to L. major infection was generated in the vaccinated mice. Also, in this work we show that the LieIF2α subunit and the LieIF2Bβ and δ subunits have the capacity to stimulate IL-10 secretion by spleen cells from naïve mice. B-lymphocytes were identified as the major producers of this anti-inflammatory cytokine. Taking into account the data found in this study, it may be hypothesized that these proteins act as virulence factors implicated in the induction of humoral responses as well as in the production of the down-regulatory IL-10 cytokine, favoring a pathological outcome. Therefore, these proteins might be considered markers of disease. PMID:29675401

FXIIIA and TGF-beta over-expression produces normal musculo-skeletal phenotype in TG2-/- mice.

PubMed

Tarantino, U; Oliva, F; Taurisano, G; Orlandi, A; Pietroni, V; Candi, E; Melino, G; Maffulli, N

2009-04-01

Transglutaminase (TGs) enzymes and proteins crosslinking have for long time been implicated in the formation of hard tissue development, matrix maturation and mineralization. Among the TGs family members, in the context of connective tissue formation, TG2 and Factor XIII are expressed in cartilage by hypertrophic chondrocytes. Here, we analyse the morphological consequences of TG2 deficiency, during the development of skeletal elements. When TG2 is absent, there are not gross abnormalities in the development of the skeletal system, probably from compensatory mechanisms resulting in increased expression of FXIIIA and TGF-beta 1. In vivo other TGs may be involved in promoting chondrocytes and osteoblast differentiation and matrix mineralisation.

The Arabidopsis Mediator Complex Subunits MED16, MED14, and MED2 Regulate Mediator and RNA Polymerase II Recruitment to CBF-Responsive Cold-Regulated Genes[C][W][OPEN

PubMed Central

Hemsley, Piers A.; Hurst, Charlotte H.; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R.; De Cothi, Elizabeth A.; Steele, John F.; Knight, Heather

2014-01-01

The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation–induced freezing tolerance. In addition, these three subunits are required for low temperature–induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced. PMID:24415770

INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

PubMed Central

Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

2012-01-01

SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

Characterisation of Translation Elongation Factor eEF1B Subunit Expression in Mammalian Cells and Tissues and Co-Localisation with eEF1A2

PubMed Central

Janikiewicz, Justyna; Doig, Jennifer; Abbott, Catherine M.

2014-01-01

Translation elongation is the stage of protein synthesis in which the translation factor eEF1A plays a pivotal role that is dependent on GTP exchange. In vertebrates, eEF1A can exist as two separately encoded tissue-specific isoforms, eEF1A1, which is almost ubiquitously expressed, and eEF1A2, which is confined to neurons and muscle. The GTP exchange factor for eEF1A1 is a complex called eEF1B made up of subunits eEF1Bα, eEF1Bδ and eEF1Bγ. Previous studies have cast doubt on the ability of eEF1B to interact with eEF1A2, suggesting that this isoform might use a different GTP exchange factor. We show that eEF1B subunits are all widely expressed to varying degrees in different cell lines and tissues, and at different stages of development. We show that ablation of any of the subunits in human cell lines has a small but significant impact on cell viability and cycling. Finally, we show that both eEF1A1 and eEF1A2 colocalise with all eEF1B subunits, in such close proximity that they are highly likely to be in a complex. PMID:25436608

Book review: Rogue waves in the ocean

USGS Publications Warehouse

Geist, Eric L.

2011-01-01

Review info: Rogue Waves in the Ocean. Advances in Geophysical and Environmental Mechanics and Mathematics. By Christian Kharif, Efim Pelinovsky and Alexey Slunyaev, 2009. ISBN: 978-3540884187, xiii, 216 pp.

19 CFR 122.183 - Denial of access.

Code of Federal Regulations, 2014 CFR

2014-04-01

...) Destruction of an aircraft or aircraft facility (18 U.S.C. 32); (xiii) Murder; (xiv) Assault with intent to murder; (xv) Espionage; (xvi) Sedition; (xvii) Kidnapping or hostage taking; (xviii) Treason; (xix) Rape...

19 CFR 122.183 - Denial of access.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) Destruction of an aircraft or aircraft facility (18 U.S.C. 32); (xiii) Murder; (xiv) Assault with intent to murder; (xv) Espionage; (xvi) Sedition; (xvii) Kidnapping or hostage taking; (xviii) Treason; (xix) Rape...

Diuretics: from classical carbonic anhydrase inhibitors to novel applications of the sulfonamides.

PubMed

Supuran, Claudiu T

2008-01-01

The widely clinically used benzothiadiazines and high ceiling diuretics, such as hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide and bumetanide, contain SO(2)NH(2) moieties acting as an effective zinc-binding function in carbonic anhydrases (CAs, EC 4.2.1.1) inhibitors. These drugs were launched in a period when only isoform CA II was known and considered physiologically/pharmacologically relevant. Although acting as moderate-weak inhibitors of CA II, all these drugs considerably inhibit other CA isozymes known nowadays to be involved in critical physiologic processes, among the 16 CAs present in vertebrates. Some low nanomolar (or even subnanomolar) inhibitors against such isoforms were recently detected, such as metholazone against CA VII, XII and XIII, chlorthalidone against CA VB, VII, IX, XII and XIII, indapamide against CA VII, IX, XII and XIII, furosemide against CA I, II and XIV, and bumethanide against CA IX and XII. The X-ray crystal structure of the CA II-indapamide adduct was also reported recently, revealing interesting aspects useful for the drug design of CA inhibitors. It has also been proposed that the recently observed beneficial effect of indapamide for the treatment of patients with hypertension and type 2 diabetes might be due to its potent inhibition of CA isoforms present in kidneys and blood vessels, which would thus explain both the blood pressure lowering effects as well as organ-protective activity of the drug. Thus, these old drugs may be useful as leads for new applications.

The multifaceted subunit interfaces of ionotropic glutamate receptors.

PubMed

Green, Tim; Nayeem, Naushaba

2015-01-01

The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

The Mediator complex of Caenorhabditis elegans: insights into the developmental and physiological roles of a conserved transcriptional coregulator

PubMed Central

Grants, Jennifer M.; Goh, Grace Y. S.; Taubert, Stefan

2015-01-01

The Mediator multiprotein complex (‘Mediator’) is an important transcriptional coregulator that is evolutionarily conserved throughout eukaryotes. Although some Mediator subunits are essential for the transcription of all protein-coding genes, others influence the expression of only subsets of genes and participate selectively in cellular signaling pathways. Here, we review the current knowledge of Mediator subunit function in the nematode Caenorhabditis elegans, a metazoan in which established and emerging genetic technologies facilitate the study of developmental and physiological regulation in vivo. In this nematode, unbiased genetic screens have revealed critical roles for Mediator components in core developmental pathways such as epidermal growth factor (EGF) and Wnt/β-catenin signaling. More recently, important roles for C. elegans Mediator subunits have emerged in the regulation of lipid metabolism and of systemic stress responses, engaging conserved transcription factors such as nuclear hormone receptors (NHRs). We emphasize instances where similar functions for individual Mediator subunits exist in mammals, highlighting parallels between Mediator subunit action in nematode development and in human cancer biology. We also discuss a parallel between the association of the Mediator subunit MED12 with several human disorders and the role of its C. elegans ortholog mdt-12 as a regulatory hub that interacts with numerous signaling pathways. PMID:25634893

Order XIII. Pseudonocardineae ord.nov.

USDA-ARS?s Scientific Manuscript database

The formal description of the actinobacterial higher taxon, the suborder Pseudonocardineae, is presented. The differential morphological and chemotaxonomic characteristics and diagnostic signature nucleotides from the 16S rRNA gene for the families Actinosynnemataceae and Pseudonocardiaceae in the ...

Comparison of the effect of dabigatran and dalteparin on thrombus stability in a murine model of venous thromboembolism.

PubMed

Shaya, S A; Saldanha, L J; Vaezzadeh, N; Zhou, J; Ni, R; Gross, P L

2016-01-01

ESSENTIALS: Does thrombus stability alter the presentation of venous thromboembolism and do anticoagulants alter this? In a murine model, we imaged a femoral vein thrombus and quantified emboli in the pulmonary arteries. Dabigatran decreases thrombus stability via factor XIII increasing embolization and pulmonary emboli. This cautions against the unapproved use of dabigatran for acute initial treatment of deep vein thrombosis. Venous thromboembolism (VTE) is a collective term for deep vein thrombosis (DVT) and pulmonary embolism (PE). Thrombus instability possibly contributes to progression of DVT to PE, and direct thrombin inhibitors (DTIs) may alter this. To develop a model to assess thrombus stability and its link to PE burden, and identify whether DTIs, in contrast to low-molecular-weight heparin (LMWH), alter this correlation. Twelve minutes after ferric chloride-induced thrombus formation in the femoral vein of female mice, saline, dalteparin (LMWH) or dabigatran (DTI) was administered. Thrombus size and embolic events breaking off from the thrombus were quantified before treatment and at 10-min intervals after treatment for 2 h using intravital videomicroscopy. Lungs were stained for the presence of PE. Thrombus size was similar over time and between treatment groups. Total and large embolic events and pulmonary emboli were highest after treatment with dabigatran. Variations in amounts of pulmonary embolic events were not attributed to variations in thrombus size. Large embolic events correlated with the number of emboli per lung slice independent of treatment. Embolization in factor XIII deficient (FXIII(-/-) ) saline-treated mice was greater than that in wild-type (WT) saline-treated mice, but was similar to WT dabigatran-treated mice. We have developed a mouse model of VTE that can quantify emboli and correlate this with PE burden. Consistent with clinical data, dabigatran, a DTI, acutely decreases thrombus stability and increases PE burden compared with LMWH or saline, which is a FXIII-dependent effect. © 2015 International Society on Thrombosis and Haemostasis.

Intracranial Hemorrhage: A Devastating Outcome of Congenital Bleeding Disorders-Prevalence, Diagnosis, and Management, with a Special Focus on Congenital Factor XIII Deficiency.

PubMed

Alavi, Seyed Ezatolla Rafiee; Jalalvand, Masumeh; Assadollahi, Vahideh; Tabibian, Shadi; Dorgalaleh, Akbar

2018-04-01

Intracranial hemorrhage (ICH) is a medical emergency. In congenital bleeding disorders, ICH is a devastating presentation accompanied with a high rate of morbidity and mortality. The prevalence of ICH is highly variable among congenital bleeding disorders, with the highest incidence observed in factor (F) XIII deficiency (FXIIID) (∼30%). This life-threatening presentation is less common in afibrinogenemia, FVIII, FIX, FVII, and FX deficiencies, and is rare in severe FV and FII deficiencies, type 3 von Willebrand disease and inherited platelet function disorders (IPFDs). In FXIIID, this diathesis most often occurs after trauma in children, whereas spontaneous ICH is more frequent in adults. About 15% of patients with FXIIID and ICH die; the bleeding causes 80% of deaths in this coagulopathy. Although in FXIIID, the bleed most commonly is intraparenchymal (> 90%), epidural, subdural, and subarachnoid hemorrhages also have been reported, albeit rarely. As this life-threatening bleeding causes neurological complications, early diagnosis can prevent further expansion of the hematoma and secondary damage. Neuroimaging plays a crucial role in the diagnosis of ICH, but signs and symptoms in patients with severe FXIIID should trigger replacement therapy even before establishment of the diagnosis. Although a high dose of FXIII concentrate can reduce the rate of morbidity and mortality of ICH in FXIIID, it may occasionally trigger inhibitor development, thus complicating ICH management and future prophylaxis. Nevertheless, replacement therapy is the mainstay of treatment for ICH in FXIIID. Neurosurgery is performed in patients with FXIIID and epidural hematoma and a hemorrhage diameter exceeding 2 cm or a volume of ICH is more than 30 cm 3 . Contact sports are not recommended in people with FXIIID as they can elicit ICH. However, a considerable number of safe sports and activities have been suggested to have more benefits than dangers for patients with congenital bleeding disorders, and are hence suitable for these patients. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Screening cleavage of Factor XIII V34X Activation Peptides by thrombin mutants: A strategy for controlling fibrin architecture.

PubMed

Jadhav, Madhavi A; Goldsberry, Whitney N; Zink, Sara E; Lamb, Kelsey N; Simmons, Katelyn E; Riposo, Carmela M; Anokhin, Boris A; Maurer, Muriel C

2017-10-01

In blood coagulation, thrombin converts fibrinogen into fibrin monomers that polymerize into a clot network. Thrombin also activates Factor XIII by cleaving the R37-G38 peptide bond of the Activation Peptide (AP) segment. The resultant transglutaminase introduces covalent crosslinks into the fibrin clot. A strategy to modify clot architecture would be to design FXIII AP sequences that are easier or more difficult to be thrombin-cleaved thus controlling initiation of crosslinking. To aid in this design process, FXIII V34X (28-41) Activation Peptides were kinetically ranked for cleavage by wild-type thrombin and several anticoagulant mutants. Thrombin-catalyzed hydrolysis of aromatic FXIII F34, W34, and Y34 APs was compared with V34 and L34. Cardioprotective FXIII L34 remained the variant most readily cleaved by wild-type thrombin. The potent anticoagulant thrombins W215A and W215A/E217A (missing a key substrate platform for binding fibrinogen) were best able to hydrolyze FXIII F34 and W34 APs. Thrombin I174A and L99A could effectively accommodate FXIII W34 and Y34 APs yielding kinetic parameters comparable to FXIII AP L34 with wild-type thrombin. None of the aromatic FXIII V34X APs could be hydrolyzed by thrombin Y60aA. FXIII F34 and W34 are promising candidates for FXIII - anticoagulant thrombin systems that could permit FXIII-catalyzed crosslinking in the presence of reduced fibrin formation. By contrast, FXIII Y34 with thrombin (Y60aA or W215A/E217A) could help assure that both fibrin clot formation and protein crosslinking are hindered. Regulating the activation of FXIII is predicted to be a strategy for helping to control fibrin clot architecture and its neighboring environments. Copyright © 2017 Elsevier B.V. All rights reserved.

Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR Is Essential for PROTEIN PHOSPHATASE 2A Holoenzyme Assembly and Plays Important Roles in Hormone Signaling, Salt Stress Response, and Plant Development1[W][OPEN

PubMed Central

Chen, Jian; Hu, Rongbin; Zhu, Yinfeng; Shen, Guoxin; Zhang, Hong

2014-01-01

PROTEIN PHOSPHATASE 2A (PP2A) is a major group of serine/threonine protein phosphatases in eukaryotes. It is composed of three subunits: scaffolding subunit A, regulatory subunit B, and catalytic subunit C. Assembly of the PP2A holoenzyme in Arabidopsis (Arabidopsis thaliana) depends on Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR (AtPTPA). Reduced expression of AtPTPA leads to severe defects in plant development, altered responses to abscisic acid, ethylene, and sodium chloride, and decreased PP2A activity. In particular, AtPTPA deficiency leads to decreased methylation in PP2A-C subunits (PP2Ac). Complete loss of PP2Ac methylation in the suppressor of brassinosteroid insensitive1 mutant leads to 30% reduction of PP2A activity, suggesting that PP2A with a methylated C subunit is more active than PP2A with an unmethylated C subunit. Like AtPTPA, PP2A-A subunits are also required for PP2Ac methylation. The interaction between AtPTPA and PP2Ac is A subunit dependent. In addition, AtPTPA deficiency leads to reduced interactions of B subunits with C subunits, resulting in reduced functional PP2A holoenzyme formation. Thus, AtPTPA is a critical factor for committing the subunit A/subunit C dimer toward PP2A heterotrimer formation. PMID:25281708

Assembly and mechanism of a group II ECF transporter.

PubMed

Karpowich, Nathan K; Wang, Da-Neng

2013-02-12

Energy-coupling factor (ECF) transporters are a recently discovered family of primary active transporters for micronutrients and vitamins, such as biotin, thiamine, and riboflavin. Found exclusively in archaea and bacteria, including the human pathogens Listeria, Streptococcus, and Staphylococcus, ECF transporters may be the only means of vitamin acquisition in these organisms. The subunit composition of ECF transporters is similar to that of ATP binding cassette (ABC) importers, whereby both systems share two homologous ATPase subunits (A and A'), a high affinity substrate-binding subunit (S), and a transmembrane coupling subunit (T). However, the S subunit of ECF transporters is an integral membrane protein, and the transmembrane coupling subunits do not share an obvious sequence homology between the two transporter families. Moreover, the subunit stoichiometry of ECF transporters is controversial, and the detailed molecular interactions between subunits and the conformational changes during substrate translocation are unknown. We have characterized the ECF transporters from Thermotoga maritima and Streptococcus thermophilus. Our data suggests a subunit stoichiometry of 2S:2T:1A:1A' and that S subunits for different substrates can be incorporated into the same transporter complex simultaneously. In the first crystal structure of the A-A' heterodimer, each subunit contains a novel motif called the Q-helix that plays a key role in subunit coupling with the T subunits. Taken together, these findings suggest a mechanism for coupling ATP binding and hydrolysis to transmembrane transport by ECF transporters.

Investigation of the anticoagulant and antithrombotic effects of chlorogenic acid.

PubMed

Choi, Jun-Hui; Kim, Seung

2017-03-01

Thrombosis is a leading cause of morbidity and mortality throughout the world. Thrombolytic agents are important for both the prevention and treatment of thrombosis. Fibrin clot and turbidity assays revealed that it was able to inhibit the formation of fibrin clot. Chlorogenic acid degraded blood clot and inhibited the enzymatic activity of procoagulant proteases, thrombin, activated factor X (FXa), and activated factor XIII (FXIIIa). Chlorogenic acid was found to delay activated partial thromboplastin time, prothrombin time, and thrombin time. PFA-100 assays showed that it prolonged the closure time of citrated whole human blood. It demonstrated the antithrombotic effect in collagen and epinephrine-induced acute thromboembolism mice model. These antithrombotic profiles together with its anticoagulant and platelet disaggregation properties, and lack of toxicity to NIH-3T3 and 3T3-L1 cells, make it a potential agent for thrombotic treatment and prevention. © 2016 Wiley Periodicals, Inc.

Novel Insights into the Role of Neurospora crassa NDUFAF2, an Evolutionarily Conserved Mitochondrial Complex I Assembly Factor

PubMed Central

Pereira, Bruno; Videira, Arnaldo

2013-01-01

Complex I deficiency is commonly associated with mitochondrial oxidative phosphorylation diseases. Mutations in nuclear genes encoding structural subunits or assembly factors of complex I have been increasingly identified as the cause of the diseases. One such factor, NDUFAF2, is a paralog of the NDUFA12 structural subunit of the enzyme, but the mechanism by which it exerts its function remains unknown. Herein, we demonstrate that the Neurospora crassa NDUFAF2 homologue, the 13.4L protein, is a late assembly factor that associates with complex I assembly intermediates containing the membrane arm and the connecting part but lacking the N module of the enzyme. Furthermore, we provide evidence that dissociation of the assembly factor is dependent on the incorporation of the putative regulatory module composed of the subunits of 13.4 (NDUFA12), 18.4 (NDUFS6), and 21 (NDUFS4) kDa. Our results demonstrate that the 13.4L protein is a complex I assembly factor functionally conserved from fungi to mammals. PMID:23648483

77 FR 29648 - Medicare and Medicaid Programs; Quarterly Listing of Program Issuances-January Through March 2012

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-18

... (Destination Therapy) Facilities. XIII Medicare-Approved Lung Volume Reduction Surgery JoAnna Baldwin, MS (410) 786-7205 Facilities. XIV Medicare-Approved Bariatric Surgery Facilities........ Kate Tillman, RN, MAS...

16 CFR 4.9 - The public record.

Code of Federal Regulations, 2010 CFR

2010-01-01

... to § 305.8 of this chapter; (xiii) Annual filings by professional boxing sanctioning organizations as required by the Muhammed Ali Boxing Reform Act, 15 U.S.C. 6301 note, 6307a-6307h; (xiv) Other documents...

HNF-1B specifically regulates the transcription of the {gamma}a-subunit of the Na{sup +}/K{sup +}-ATPase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ferre, Silvia; Veenstra, Gert Jan C.; Bouwmeester, Rianne

2011-01-07

Research highlights: {yields} Defects in HNF-1B transcription factor affect Mg{sup 2+} handling in the distal kidney. {yields} {gamma}a- and {gamma}b- subunits of the Na{sup +}/K{sup +}-ATPase colocalize in the distal convoluted tubule of the nephron. {yields} HNF-1B specifically activates {gamma}a expression. {yields} HNF-1B mutants have a dominant negative effect on wild type HNF-1B activity. {yields} Defective transcription of {gamma}a may promote renal Mg{sup 2+} wasting. -- Abstract: Hepatocyte nuclear factor-1B (HNF-1B) is a transcription factor involved in embryonic development and tissue-specific gene expression in several organs, including the kidney. Recently heterozygous mutations in the HNF1B gene have been identified inmore » patients with hypomagnesemia due to renal Mg{sup 2+} wasting. Interestingly, ChIP-chip data revealed HNF-1B binding sites in the FXYD2 gene, encoding the {gamma}-subunit of the Na{sup +}/K{sup +}-ATPase. The {gamma}-subunit has been described as one of the molecular players in the renal Mg{sup 2+} reabsorption in the distal convoluted tubule (DCT). Of note, the FXYD2 gene can be alternatively transcribed into two main variants, namely {gamma}a and {gamma}b. In the present study, we demonstrated via two different reporter gene assays that HNF-1B specifically acts as an activator of the {gamma}a-subunit, whereas the {gamma}b-subunit expression was not affected. Moreover, the HNF-1B mutations H69fsdelAC, H324S325fsdelCA, Y352finsA and K156E, previously identified in patients with hypomagnesemia, prevented transcription activation of {gamma}a-subunit via a dominant negative effect on wild type HNF1-B. By immunohistochemistry, it was shown that the {gamma}a- and {gamma}b-subunits colocalize at the basolateral membrane of the DCT segment of mouse kidney. On the basis of these data, we suggest that abnormalities involving the HNF-1B gene may impair the relative abundance of {gamma}a and {gamma}b, thus affecting the transcellular Mg{sup 2+} reabsorption in the DCT.« less

Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shigeno, Yuta; Uchiumi, Toshio; Nomura, Takaomi, E-mail: nomurat@shinshu-u.ac.jp

Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly,more » cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.« less

Diagnosing the Magnetic Field Structure of a Coronal Cavity Observed during the 2017 Total Solar Eclipse

NASA Astrophysics Data System (ADS)

Chen, Yajie; Tian, Hui; Su, Yingna; Qu, Zhongquan; Deng, Linhua; Jibben, Patricia R.; Yang, Zihao; Zhang, Jingwen; Samanta, Tanmoy; He, Jiansen; Wang, Linghua; Zhu, Yingjie; Zhong, Yue; Liang, Yu

2018-03-01

We present an investigation of a coronal cavity observed above the western limb in the coronal red line Fe X 6374 Å using a telescope of Peking University and in the green line Fe XIV 5303 Å using a telescope of Yunnan Observatories, Chinese Academy of Sciences, during the total solar eclipse on 2017 August 21. A series of magnetic field models is constructed based on the magnetograms taken by the Helioseismic and Magnetic Imager on board the Solar Dynamics Observatory (SDO) one week before the eclipse. The model field lines are then compared with coronal structures seen in images taken by the Atmospheric Imaging Assembly on board SDO and in our coronal red line images. The best-fit model consists of a flux rope with a twist angle of 3.1π, which is consistent with the most probable value of the total twist angle of interplanetary flux ropes observed at 1 au. Linear polarization of the Fe XIII 10747 Å line calculated from this model shows a “lagomorphic” signature that is also observed by the Coronal Multichannel Polarimeter of the High Altitude Observatory. We also find a ring-shaped structure in the line-of-sight velocity of Fe XIII 10747 Å, which implies hot plasma flows along a helical magnetic field structure, in the cavity. These results suggest that the magnetic structure of the cavity is a highly twisted flux rope, which may erupt eventually. The temperature structure of the cavity has also been investigated using the intensity ratio of Fe XIII 10747 Å and Fe X 6374 Å.

THERMAL STRUCTURE OF CORONAL LOOPS AS SEEN WITH NORIKURA CORONAGRAPH

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prasad, S. Krishna; Singh, Jagdev; Ichimoto, K., E-mail: krishna@iiap.res.in

2013-03-10

The thermal structure of a coronal loop, both along and across the loop, is vital in determining the exact plasma heating mechanism. High-resolution spectroscopic observations of the off-limb corona were made using the 25 cm Norikura coronagraph, located at Norikura, Japan. Observations on a number of days were made simultaneously in four forbidden iron emission lines, namely, the [Fe XI] 7892 A line, the [Fe XIII] 10747 A and 10798 A lines, and the [Fe XIV] 5303 A line and on some days made only in the [Fe XI] 7892 A and [Fe X] 6374 A lines. Using temperature sensitivemore » emission line ratios [Fe XIV] 5303 A/[Fe XIII] 10747 A and [Fe XI] 7892 A/[Fe X] 6374 A, we compute the electron temperatures along 18 different loop structures observed on different days. We find a significant negative temperature gradient in all of the structures observed in Fe XIV and Fe XIII and a positive temperature gradient in the structures observed in Fe XI and Fe X. Combining these results with the previous investigations by Singh and his collaborators, we infer that the loop tops, in general, appear hotter when observed in colder lines and colder when observed in relatively hotter lines as compared to their coronal foot points. We suggest that this contrasting trend observed in the temperature variation along the loop structures can be explained by a gradual interaction of different temperature plasma. The exact mechanism responsible for this interaction must be investigated further and has the potential to constrain loop heating models.« less

Double-stabilized neurotensin analogues as potential radiopharmaceuticals for NTR-positive tumors.

PubMed

García-Garayoa, Elisa; Maes, Veronique; Bläuenstein, Peter; Blanc, Alain; Hohn, Alexander; Tourwé, Dirk; Schubiger, P August

2006-05-01

Overexpression of neurotensin (NT) receptors in exocrine pancreatic cancer and other neuroendocrine cancers make them interesting targets for tumor imaging and therapy. Modifications at the cleavage bonds 8-9 and 11-12 led to the synthesis of NT-XII, NT-XIII and NT-XVIII, three new stabilized analogues. (NalphaHis)Ac was coupled to the N-terminus for labeling with [(99m)Tc]-tricarbonyl. Stability was tested in vitro in human plasma and HT-29 cells. Binding to NT1 receptors and internalization/efflux were analyzed in intact HT-29 cells. Biodistribution studies were performed in nude mice bearing HT-29 xenografts. All analogues were very stable in human plasma, with half-lives of 20-21 days. Degradation in HT-29 cells was more rapid (t(1/2) of 6.5, 5 and 2.5 h for NT-XII, NT-XIII and NT-XVIII, respectively). They also showed high affinity and specificity for NT1 receptors. Bound activity was rapidly internalized at 37 degrees C. The pattern of externalization was different. NT-XII was released more slowly than NT-XIII and NT-XVIII (half of the activity still inside the cells after 24 h). Bigger differences were found in the biodistribution studies. NT-XII showed the highest tumor uptake as well as the best tumor to nontumor ratios. The modifications introduced in NT(8-13) increased plasma stability, maintaining unaffected the in vitro binding properties. The best biodistribution corresponded to NT-XII, which shows to be a good candidate for NT1 receptors overexpressing tumors. First clinical trials are ongoing.

Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39

PubMed Central

Blombach, Fabian; Salvadori, Enrico; Fouqueau, Thomas; Yan, Jun; Reimann, Julia; Sheppard, Carol; Smollett, Katherine L; Albers, Sonja V; Kay, Christopher WM; Thalassinos, Konstantinos; Werner, Finn

2015-01-01

Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and β subunits. Here we have identified and characterised the function of the TFIIEβ homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39. Both archaeal TFEβ and hRPC39 harbour a cubane 4Fe-4S cluster, which is crucial for heterodimerization of TFEα/β and its engagement with the RNAP clamp. TFEα/β stabilises the preinitiation complex, enhances DNA melting, and stimulates abortive and productive transcription. These activities are strictly dependent on the β subunit and the promoter sequence. Our results suggest that archaeal TFEα/β is likely to represent the evolutionary ancestor of TFIIE-like factors in extant eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.08378.001 PMID:26067235

The Assembly Pathway of Mitochondrial Respiratory Chain Complex I.

PubMed

Guerrero-Castillo, Sergio; Baertling, Fabian; Kownatzki, Daniel; Wessels, Hans J; Arnold, Susanne; Brandt, Ulrich; Nijtmans, Leo

2017-01-10

Mitochondrial complex I is the largest integral membrane enzyme of the respiratory chain and consists of 44 different subunits encoded in the mitochondrial and nuclear genome. Its biosynthesis is a highly complicated and multifaceted process involving at least 14 additional assembly factors. How these subunits assemble into a functional complex I and where the assembly factors come into play is largely unknown. Here, we applied a dynamic complexome profiling approach to elucidate the assembly of human mitochondrial complex I and its further incorporation into respiratory chain supercomplexes. We delineate the stepwise incorporation of all but one subunit into a series of distinct assembly intermediates and their association with known and putative assembly factors, which had not been implicated in this process before. The resulting detailed and comprehensive model of complex I assembly is fully consistent with recent structural data and the remarkable modular architecture of this multiprotein complex. Copyright © 2017 Elsevier Inc. All rights reserved.

Assembly of Q{beta} viral RNA polymerase with host translational elongation factors EF-Tu and -Ts.

PubMed

Takeshita, Daijiro; Tomita, Kozo

2010-09-07

Replication and transcription of viral RNA genomes rely on host-donated proteins. Qbeta virus infects Escherichia coli and replicates and transcribes its own genomic RNA by Qbeta replicase. Qbeta replicase requires the virus-encoded RNA-dependent RNA polymerase (beta-subunit), and the host-donated translational elongation factors EF-Tu and -Ts, as active core subunits for its RNA polymerization activity. Here, we present the crystal structure of the core Qbeta replicase, comprising the beta-subunit, EF-Tu and -Ts. The beta-subunit has a right-handed structure, and the EF-Tu:Ts binary complex maintains the structure of the catalytic core crevasse of the beta-subunit through hydrophobic interactions, between the finger and thumb domains of the beta-subunit and domain-2 of EF-Tu and the coiled-coil motif of EF-Ts, respectively. These hydrophobic interactions are required for the expression and assembly of the Qbeta replicase complex. Thus, EF-Tu and -Ts have chaperone-like functions in the maintenance of the structure of the active Qbeta replicase. Modeling of the template RNA and the growing RNA in the catalytic site of the Qbeta replicase structure also suggests that structural changes of the RNAs and EF-Tu:Ts should accompany processive RNA polymerization and that EF-Tu:Ts in the Qbeta replicase could function to modulate the RNA folding and structure.

The Mediator complex of Caenorhabditis elegans: insights into the developmental and physiological roles of a conserved transcriptional coregulator.

PubMed

Grants, Jennifer M; Goh, Grace Y S; Taubert, Stefan

2015-02-27

The Mediator multiprotein complex ('Mediator') is an important transcriptional coregulator that is evolutionarily conserved throughout eukaryotes. Although some Mediator subunits are essential for the transcription of all protein-coding genes, others influence the expression of only subsets of genes and participate selectively in cellular signaling pathways. Here, we review the current knowledge of Mediator subunit function in the nematode Caenorhabditis elegans, a metazoan in which established and emerging genetic technologies facilitate the study of developmental and physiological regulation in vivo. In this nematode, unbiased genetic screens have revealed critical roles for Mediator components in core developmental pathways such as epidermal growth factor (EGF) and Wnt/β-catenin signaling. More recently, important roles for C. elegans Mediator subunits have emerged in the regulation of lipid metabolism and of systemic stress responses, engaging conserved transcription factors such as nuclear hormone receptors (NHRs). We emphasize instances where similar functions for individual Mediator subunits exist in mammals, highlighting parallels between Mediator subunit action in nematode development and in human cancer biology. We also discuss a parallel between the association of the Mediator subunit MED12 with several human disorders and the role of its C. elegans ortholog mdt-12 as a regulatory hub that interacts with numerous signaling pathways. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Analysis of colonization factor antigen I, an adhesin of enterotoxigenic Escherichia coli O78:H11: fimbrial morphology and location of the receptor-binding site.

PubMed Central

Bühler, T; Hoschützky, H; Jann, K

1991-01-01

Colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli was dissociated into one type of subunit (15 kDa). The dissociation was achieved either by heating CFA/I in sodium dodecyl sulfate at 100 degrees C or by heating it for 20 min in water. Heating in water to 100 degrees C yielded only in the 15-kDa subunit, but heating to 85 degree C yielded small amounts of oligomers in addition. The monomeric subunits obtained after heating in water are stable, as demonstrated by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis without heating prior to the electrophoretic run. These subunits inhibited CFA/I-induced hemagglutination, indicating that they had maintained their receptor-binding properties. When the hybridoma technique was used, two types of monoclonal anti-CFA/I antibodies were obtained. Antibodies obtained by immunization with the purified subunits were more reactive with subunits than with fimbriae, as shown by enzyme-linked immunosorbent assay. These antibodies strongly inhibited CFA/I-induced hemagglutination. When examined by immunoelectron microscopy, these antibodies seemed to label the fimbrial tips. A similar labeling pattern was obtained with gold particles modified with the receptor ganglioside GM2. Antibodies obtained by immunization with fimbriae reacted in enzyme-linked immunosorbent assays equally well with fimbriae and subunits. They inhibited CFA/I-induced hemagglutination only slightly. Immunoelectron microscopy revealed that these antibodies labeled the fimbriae densely and regularly over their entire lengths. In a coagglutination experiment with Staphylococcus aureus and monoclonal antibodies, the subunits retained their receptor-binding properties. From these results, we conclude that CFA/I fimbriae consist entirely of one type of adhesive subunit, of which only the one at the tip is accessible to the receptor. Images PMID:1682253

Design and Analysis of an Electron Gun/Booster and Free Electron Laser Optical Theory

DTIC Science & Technology

2010-09-01

42 23. Simplified cathode assembly model . . . . . . . . . . . . . . . . . . . . 45 24. Rossendorf and BNL RF chokes...225 123. Cross-correlation maps . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 124. BNL SDL optical field...amplitude . . . . . . . . . . . . . . . . . . . . . 230 125. BNL SDL Gain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 xiii THIS

8 CFR 214.4 - Denial of certification, denial of recertification or withdrawal of SEVP certification.

Code of Federal Regulations, 2011 CFR

2011-01-01

...). (xii) Failure to operate as a bona fide institution of learning. (xiii) Failure to employ adequate...) Failure to maintain the physical plant, curriculum, and teaching staff in the manner represented in the...

77 FR 67368 - Medicare and Medicaid Programs; Quarterly Listing of Program Issuances-July through September 2012

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-09

...) Facilities. XIII Medicare-Approved Lung JoAnna Baldwin, MS. (410) 786-7205 Volume Reduction Surgery Facilities. XIV Medicare-Approved Bariatric Kate Tillman, RN, (410) 786-9252 Surgery Facilities. MAS. XV...

Identifying and Analyzing Federal Government Market Opportunities for OpalSoft

DTIC Science & Technology

2004-09-01

OPPORTUNITIES, AND THREATS ( SWOT ) ANALYSIS......................................................................................13 1. Strengths...xiii LIST OF TABLES Table 1. SWOT Analysis...of industries to include Fortune 500 companies. Current key clients are Amkor, Symantec, Unisys Corporation, Apple Computers, Palm, Inc., and

Cross-linking of initiation factor IF3 to Escherichia coli 30S ribosomal subunit by trans-diamminedichloroplatinum(II): characterization of two cross-linking sites in 16S rRNA; a possible way of functioning for IF3.

PubMed Central

Ehresmann, C; Moine, H; Mougel, M; Dondon, J; Grunberg-Manago, M; Ebel, J P; Ehresmann, B

1986-01-01

The initiation factor IF3 is platinated with trans-diamminedichloroplatinum(II) and cross-linked to Escherichia coli 30S ribosomal subunit. Two cross-linking sites are unambiguously identified on the 16S rRNA: a major one, in the region 819-859 in the central domain, and a minor one, in the region 1506-1529 in the 3'-terminal domain. Specific features of these sequences together with their particular location within the 30S subunit lead us to postulate a role for IF3, that conciliates topographical and functional observations made so far. Images PMID:2425339

Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

NASA Astrophysics Data System (ADS)

Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

1995-02-01

The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

DIRECT MODULATION OF THE PROTEIN KINASE A CATALYTIC SUBUNIT α BY GROWTH FACTOR RECEPTOR TYROSINE KINASES

PubMed Central

Caldwell, George B.; Howe, Alan K.; Nickl, Christian K.; Dostmann, Wolfgang R.; Ballif, Bryan A.; Deming, Paula B.

2011-01-01

The cyclic-AMP-dependent protein kinase A (PKA) regulates processes such as cell proliferation and migration following activation of growth factor receptor tyrosine kinases (RTKs), yet the signaling mechanisms that link PKA with growth factor receptors remain largely undefined. Here we report that RTKs can directly modulate the function of the catalytic subunit of PKA (PKA-C) through post-translational modification. In vitro kinase assays revealed that both the epidermal growth factor and platelet derived growth factor receptors (EGFR and PDGFR, respectively) tyrosine phosphorylate PKA-C. Mass spectrometry identified tyrosine 330 (Y330) as a receptor-mediated phosphorylation site and mutation of Y330 to phenylalanine (Y330F) all but abolished the RTK-mediated phosphorylation of PKA-C in vitro. Y330 resides within a conserved region at the C-terminal tail of PKA-C that allosterically regulates enzymatic activity. Therefore, the effect of phosphorylation at Y330 on the activity of PKA-C was investigated. The Km for a peptide substrate was markedly decreased when PKA-C subunits were tyrosine phosphorylated by the receptors as compared to un-phosphorylated controls. Importantly, tyrosine-phosphorylated PKA-C subunits were detected in cells stimulated with EGF, PDGF and FGF2 and in fibroblasts undergoing PDGF-mediated chemotaxis. These results demonstrate a direct, functional interaction between RTKs and PKA-C and identify tyrosine phosphorylation as a novel mechansim for regulating PKA activity. PMID:21866565

The δ Subunit of RNA Polymerase Guides Promoter Selectivity and Virulence in Staphylococcus aureus

PubMed Central

Weiss, Andy; Ibarra, J. Antonio; Paoletti, Jessica; Carroll, Ronan K.

2014-01-01

In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the δ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the β and/or β′ subunits. To identify the impact of the δ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage ϕSA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the δ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection. PMID:24491578

The δ subunit of RNA polymerase guides promoter selectivity and virulence in Staphylococcus aureus.

PubMed

Weiss, Andy; Ibarra, J Antonio; Paoletti, Jessica; Carroll, Ronan K; Shaw, Lindsey N

2014-04-01

In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the δ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the β and/or β' subunits. To identify the impact of the δ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage SA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the δ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection.

A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants.

PubMed

Feiz, Leila; Williams-Carrier, Rosalind; Belcher, Susan; Montano, Monica; Barkan, Alice; Stern, David B

2014-12-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin-Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin-4α-carbinolamine dehydratases (PCD)-like protein is the causative mutation in a seedling-lethal, Rubisco-deficient mutant named Rubisco accumulation factor 2 (raf2-1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high-molecular weight complex, the formation of which requires a specific chaperonin 60-kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross-linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co-immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co-immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Kv channel subunits that contribute to voltage-gated K+ current in renal vascular smooth muscle.

PubMed

Fergus, Daniel J; Martens, Jeffrey R; England, Sarah K

2003-03-01

The rat renal arterial vasculature displays differences in K(+) channel current phenotypes along its length. Small arcuate to cortical radial arteries express a delayed rectifier phenotype, while the predominant Kv current in larger arcuate and interlobar arteries is composed of both transient and sustained components. We sought to determine whether Kvalpha subunits in the rat renal interlobar and arcuate arteries form heterotetramers, which may account for the unique currents, and whether modulatory Kvbeta subunits are present in renal vascular smooth muscle cells. RT-PCR indicated the presence of several different Kvalpha subunit isoform transcripts. Co-immunoprecipitation with immunoblotting and immunohistochemical evidence suggests that a portion of the K(+) current phenotype is a heteromultimer containing delayed-rectifier Kv1.2 and A-type Kv1.4 channel subunits. RT-PCR and immunoblot analyses also demonstrated the presence of both Kvbeta1.2 and Kvbeta1.3 in renal arteries. These results suggest that heteromultimeric formation of Kvalpha subunits and the presence of modulatory Kvbeta subunits are important factors in mediating Kv currents in the renal microvasculature and suggest a potentially critical role for these channel subunits in blood pressure regulation.

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export

PubMed Central

Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

2016-01-01

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms. PMID:26872259

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

PubMed

Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

2016-01-01

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

The A12.2 Subunit Is an Intrinsic Destabilizer of the RNA Polymerase I Elongation Complex.

PubMed

Appling, Francis D; Scull, Catherine E; Lucius, Aaron L; Schneider, David A

2018-06-05

Despite sharing a highly conserved core architecture with their prokaryotic counterparts, eukaryotic multisubunit RNA polymerases (Pols) have undergone structural divergence and biological specialization. Interesting examples of structural divergence are the A12.2 and C11 subunits of Pols I and III, respectively. Whereas all known cellular Pols possess cognate protein factors that stimulate cleavage of the nascent RNA, Pols I and III have incorporated their cleavage factors as bona fide subunits. Although it is not yet clear why these polymerases have incorporated their cleavage factors as subunits, a picture is emerging that identifies roles for these subunits beyond providing nucleolytic activity. Specifically, it appears that both A12.2 and C11 are required for efficient termination of transcription by Pols I and III. Given that termination involves destabilization of the elongation complex (EC), we tested whether A12.2 influences stability of the Pol I EC. Using, to our knowledge, a novel assay to measure EC dissociation kinetics, we have determined that A12.2 is an intrinsic destabilizer of the Pol I EC. In addition, the salt concentration dependence of Pol I EC dissociation kinetics suggests that A12.2 alters electrostatic interactions within the EC. Importantly, these data present a mechanistic basis for the requirement of A12.2 in Pol I termination. Combined with recent work demonstrating the direct involvement of A12.2 in Pol I nucleotide incorporation, this study further supports the concept that A12.2 cannot be viewed solely as a cleavage factor. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Super elongation complex contains a TFIIF-related subcomplex

PubMed Central

Knutson, Bruce A.; Smith, Marissa L.; Walker-Kopp, Nancy; Xu, Xia

2016-01-01

ABSTRACT Super elongation complex (SEC) belongs to a family of RNA polymerase II (Pol II) elongation factors that has similar properties as TFIIF, a general transcription factor that increases the transcription elongation rate by reducing pausing. Although SEC has TFIIF-like functional properties, it apparently lacks sequence and structural homology. Using HHpred, we find that SEC contains an evolutionarily related TFIIF-like subcomplex. We show that the SEC subunit ELL interacts with the Pol II Rbp2 subunit, as expected for a TFIIF-like factor. These findings suggest a new model for how SEC functions as a Pol II elongation factor and how it suppresses Pol II pausing. PMID:27223670

Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition.

PubMed

Doherty, Geoff P; Fogg, Mark J; Wilkinson, Anthony J; Lewis, Peter J

2010-12-01

Bacterial RNA polymerases (RNAPs) contain several small auxiliary subunits known to co-purify with the core α, β and β' subunits. The ω subunit is conserved between Gram-positive and Gram-negative bacteria, while the δ subunit is conserved within, but restricted to, Gram-positive bacteria. Although various functions have been assigned to these subunits via in vitro assays, very little is known about their in vivo roles. In this work we constructed a pair of vectors to investigate the subcellular localization of the δ and ω subunits in Bacillus subtilis with respect to the core RNAP. We found these subunits to be closely associated with RNAP involved in transcribing both mRNA and rRNA operons. Quantification of these subunits revealed δ to be present at equimolar levels with RNAP and ω to be present at around half the level of core RNAP. For comparison, the localization and quantification of RNAP β' and ω subunits in Escherichia coli was also investigated. Similar to B. subtilis, β' and ω closely associated with the nucleoid and formed subnucleoid regions of high green fluorescent protein intensity, but, unlike ω in B. subtilis, ω levels in E. coli were close to parity with those of β'. These results indicate that δ is likely to be an integral RNAP subunit in Gram-positives, whereas ω levels differ substantially between Gram-positives and -negatives. The ω subunit may be required for RNAP assembly and subsequently be turned over at different rates or it may play roles in Gram-negative bacteria that are performed by other factors in Gram-positives.

26 CFR 1.897-5T - Corporate distributions (temporary).

Code of Federal Regulations, 2014 CFR

2014-04-01

... provided in section 897 and the regulations thereunder, except as provided by Articles XIII (9) and XXX (5... June 6, 1988. With regard to Article XXX (5) of the Income Tax Treaty with Canada, see, Rev. Rul. 85-76...

26 CFR 1.897-5T - Corporate distributions (temporary).

Code of Federal Regulations, 2011 CFR

2011-04-01

... provided in section 897 and the regulations thereunder, except as provided by Articles XIII (9) and XXX (5... June 6, 1988. With regard to Article XXX (5) of the Income Tax Treaty with Canada, see, Rev. Rul. 85-76...

26 CFR 1.897-5T - Corporate distributions (temporary).

Code of Federal Regulations, 2012 CFR

2012-04-01

... provided in section 897 and the regulations thereunder, except as provided by Articles XIII (9) and XXX (5... June 6, 1988. With regard to Article XXX (5) of the Income Tax Treaty with Canada, see, Rev. Rul. 85-76...

26 CFR 1.897-5T - Corporate distributions (temporary).

Code of Federal Regulations, 2013 CFR

2013-04-01

... provided in section 897 and the regulations thereunder, except as provided by Articles XIII (9) and XXX (5... June 6, 1988. With regard to Article XXX (5) of the Income Tax Treaty with Canada, see, Rev. Rul. 85-76...

42 CFR 88.1 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-10-01

... sites in the respiratory system and intrathoracic organs. (xi) Mesothelioma. (xii) Malignant neoplasms of the peripheral nerves and autonomic nervous system, and other connective and soft tissue. (xiii... disorder means a chronic or recurrent disorder of the musculoskeletal system caused by heavy lifting or...

33 CFR 385.26 - Project Implementation Reports.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Implementation Report is a document that provides information on plan formulation and evaluation, engineering and..., environmental and/or economic benefits, engineering and design, costs, environmental impacts, real estate..., optimization and justification, cost-effectiveness, and engineering feasibility of the project; (xiii) Include...

26 CFR 1.897-5T - Corporate distributions (temporary).

Code of Federal Regulations, 2010 CFR

2010-04-01

... provided in section 897 and the regulations thereunder, except as provided by Articles XIII (9) and XXX (5... June 6, 1988. With regard to Article XXX (5) of the Income Tax Treaty with Canada, see, Rev. Rul. 85-76...

42 CFR 441.464 - State assurances.

Code of Federal Regulations, 2014 CFR

2014-10-01

... services, supports, and resources. (xii) Development of risk management agreements. (xiii) Development of... Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... protect the health and welfare of individuals furnished services under the program and to assure the...

42 CFR 441.464 - State assurances.

Code of Federal Regulations, 2012 CFR

2012-10-01

... services, supports, and resources. (xii) Development of risk management agreements. (xiii) Development of... Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... protect the health and welfare of individuals furnished services under the program and to assure the...

42 CFR 441.464 - State assurances.

Code of Federal Regulations, 2013 CFR

2013-10-01

... services, supports, and resources. (xii) Development of risk management agreements. (xiii) Development of... Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... protect the health and welfare of individuals furnished services under the program and to assure the...

Discrimination of Pathogenic Versus Non-Pathogenic Yersinia Pestis and Escherichia Coli Using Proteomics Mass Spectrometry

DTIC Science & Technology

2010-05-01

protein 1b (lb;c) thiol peroxidase attachment invasion locus protein trigger factor 50S ribosomal protein L9 urease (urea amidohydrolase) beta...subunit attachment invasion locus protein urease (urea amidohydrolase) beta subunit attachment invasion locus protein hypothetical protein y2159

A hypothesis: factor VII governs clot formation, tissue repair and apoptosis.

PubMed

Coleman, Lewis S

2007-01-01

A hypothesis: thrombin is a "Universal Enzyme of Energy Transduction" that employs ATP energy in flowing blood to activate biochemical reactions and cell effects in both hemostasis and tissue repair. All cells possess PAR-1 (thrombin) receptors and are affected by thrombin elevations, and thrombin effects on individual cell types are determined by their unique complement of PAR-1 receptors. Disruption of the vascular endothelium (VE) activates a tissue repair mechanism (TRM) consisting of the VE, tissue factor (TF), and circulating Factors VII, IX and X that governs localized thrombin elevations to activate clot formation and cellular effects that repair tissue damage. The culmination of the repair process occurs with the restoration of the VE followed by declines in thrombin production that causes Apoptosis ("programmed cell death") in wound-healing fibroblasts, which functions as a mechanism to draw wound edges together. The location and magnitude of TRM activity governs the location and magnitude of Factor VIII activity and clot formation, but the large size of Factor VIII prevents it from penetrating the clot formed by its activity, so that its effects are self-limiting. Factors VII, IX and X function primarily as tissue repair enzymes, while Factor VIII and Factor XIII are the only serine protease enzymes in the "Coagulation Cascade" that are exclusively associated with hemostasis.

Genetic factors in fetal growth restriction and miscarriage.

PubMed

Yamada, Hideto; Sata, Fumihiro; Saijo, Yasuaki; Kishi, Reiko; Minakami, Hisanori

2005-06-01

Recently, several investigations concerning disadvantageous genetic factors in human reproduction have progressed. Inherited thrombophilia, such as factor V Leiden, prothrombin, and methylenetetrahydrofolate reductase mutations; gene polymorphisms of detoxification enzyme (CYP1A1); growth factors (insulin-like growth factor-I); and hormones such as angiotensinogen and CYP17 are involved in the pathogenesis of fetal growth restriction. The inherited thrombophilia, gene polymorphisms of coagulation and anticoagulation factor such as thrombomodulin, endothelial protein C receptor, plasminogen activator inhibitor 1, and factor XIII; human lymphocyte antigen (HLA-G); detoxification enzymes (glutathione- S-transferase M1); cytokines such as interleukin (IL) -1 and IL-6; hormones (CYP17); vasodilators (nitric oxide synthase 3); and vitamins (transcobalamin) are involved in the pathogenesis of sporadic and recurrent miscarriage. It is likely that a gene polymorphism or mutation susceptible to reproductive failure has a beneficial effect on the process of human reproduction with or without the environmental interaction. The factor V Leiden mutation has genetic advantages that are believed to be an improved implantation rate in in vitro fertilization and a reduction of maternal intrapartum blood loss. It has also been demonstrated that the CYP17 A2 allele has bidirectional effects on human reproduction, including increases in susceptibility to recurrent miscarriage and fetal growth enhancement.

40 CFR 70.2 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-07-01

...: (i) Coal cleaning plants (with thermal dryers); (ii) Kraft pulp mills; (iii) Portland cement plants... plants; (xii) Phosphate rock processing plants; (xiii) Coke oven batteries; (xiv) Sulfur recovery plants...) totaling more than 250 million British thermal units per hour heat input; (xxii) Petroleum storage and...

40 CFR 71.2 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-07-01

...: (i) Coal cleaning plants (with thermal dryers); (ii) Kraft pulp mills; (iii) Portland cement plants... plants; (xii) Phosphate rock processing plants; (xiii) Coke oven batteries; (xiv) Sulfur recovery plants...) totaling more than 250 million British thermal units per hour heat input; (xxii) Petroleum storage and...

Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor

PubMed Central

Anandhakumar, Jayamani; Moustafa, Yara W.; Chowdhary, Surabhi; Kainth, Amoldeep S.

2016-01-01

Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. Although it has been generally assumed that in Saccharomyces cerevisiae, Mediator is a stable trimodular complex, its structural state in vivo remains unclear. Using the “anchor away” (AA) technique to conditionally deplete select subunits within Mediator and its reversibly associated Cdk8 kinase module (CKM), we provide evidence that Mediator's tail module is highly dynamic and that a subcomplex consisting of Med2, Med3, and Med15 can be independently recruited to the regulatory regions of heat shock factor 1 (Hsf1)-activated genes. Fluorescence microscopy of a scaffold subunit (Med14)-anchored strain confirmed parallel cytoplasmic sequestration of core subunits located outside the tail triad. In addition, and contrary to current models, we provide evidence that Hsf1 can recruit the CKM independently of core Mediator and that core Mediator has a role in regulating postinitiation events. Collectively, our results suggest that yeast Mediator is not monolithic but potentially has a dynamic complexity heretofore unappreciated. Multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 tail triad, and the four-subunit CKM, can be independently recruited by activated Hsf1 to its target genes in AA strains. PMID:27185874

Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

PubMed Central

Naveau, Marie; Lazennec-Schurdevin, Christine; Panvert, Michel; Dubiez, Etienne; Mechulam, Yves; Schmitt, Emmanuelle

2013-01-01

Heterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2–GDPNP–tRNA complex. PMID:23193270

Two sides of the same coin: TFIIH complexes in transcription and DNA repair.

PubMed

Zhovmer, Alexander; Oksenych, Valentyn; Coin, Frédéric

2010-04-13

TFIIH is organized into a seven-subunit core associated with a three-subunit Cdk-activating kinase (CAK) module. TFIIH has roles in both transcription initiation and DNA repair. During the last 15 years, several studies have been conducted to identify the composition of the TFIIH complex involved in DNA repair. Recently, a new technique combining chromatin immunoprecipitation and western blotting resolved the hidden nature of the TFIIH complex participating in DNA repair. Following the recruitment of TFIIH to the damaged site, the CAK module is released from the core TFIIH, and the core subsequently associates with DNA repair factors. The release of the CAK is specifically driven by the recruitment of the DNA repair factor XPA and is required to promote the incision/excision of the damaged DNA. Once the DNA lesions have been repaired, the CAK module returns to the core TFIIH on the chromatin, together with the release of the repair factors. These data highlight the dynamic composition of a fundamental cellular factor that adapts its subunit composition to the cell needs.

Defective ribosome assembly in Shwachman-Diamond syndrome.

PubMed

Wong, Chi C; Traynor, David; Basse, Nicolas; Kay, Robert R; Warren, Alan J

2011-10-20

Shwachman-Diamond syndrome (SDS), a recessive leukemia predisposition disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, skeletal abnormalities and poor growth, is caused by mutations in the highly conserved SBDS gene. Here, we test the hypothesis that defective ribosome biogenesis underlies the pathogenesis of SDS. We create conditional mutants in the essential SBDS ortholog of the ancient eukaryote Dictyostelium discoideum using temperature-sensitive, self-splicing inteins, showing that mutant cells fail to grow at the restrictive temperature because ribosomal subunit joining is markedly impaired. Remarkably, wild type human SBDS complements the growth and ribosome assembly defects in mutant Dictyostelium cells, but disease-associated human SBDS variants are defective. SBDS directly interacts with the GTPase elongation factor-like 1 (EFL1) on nascent 60S subunits in vivo and together they catalyze eviction of the ribosome antiassociation factor eukaryotic initiation factor 6 (eIF6), a prerequisite for the translational activation of ribosomes. Importantly, lymphoblasts from SDS patients harbor a striking defect in ribosomal subunit joining whose magnitude is inversely proportional to the level of SBDS protein. These findings in Dictyostelium and SDS patient cells provide compelling support for the hypothesis that SDS is a ribosomopathy caused by corruption of an essential cytoplasmic step in 60S subunit maturation.

Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III.

PubMed

Matsutani, Sachiko

2004-08-09

In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs). The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and alpha-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants than to fungi.

Toll-Like Receptor 2 Mediates Cellular Activation by the B Subunits of Type II Heat-Labile Enterotoxins

PubMed Central

Hajishengallis, George; Tapping, Richard I.; Martin, Michael H.; Nawar, Hesham; Lyle, Elizabeth A.; Russell, Michael W.; Connell, Terry D.

2005-01-01

The type II heat-labile enterotoxins (LT-IIa and LT-IIb) of Escherichia coli have an AB5 subunit structure similar to that of cholera toxin (CT) and other type I enterotoxins, despite significant differences in the amino acid sequences of their B subunits and different ganglioside receptor specificities. LT-II holotoxins and their nontoxic B subunits display unique properties as immunological adjuvants distinct from those of CT and its B subunits. In contrast to type II holotoxins, the corresponding pentameric B subunits, LT-IIaB and LT-IIbB, stimulated cytokine release in both human and mouse cells dependent upon Toll-like receptor 2 (TLR2). Induction of interleukin-1β (IL-1β), IL-6, IL-8, or tumor necrosis factor alpha in human THP-1 cells by LT-IIaB or LT-IIbB was inhibited by anti-TLR2 but not by anti-TLR4 antibody. Furthermore, transient expression of TLR1 and TLR2 in human embryonic kidney 293 cells resulted in activation of a nuclear factor-κB-dependent luciferase gene in response to LT-IIaB or LT-IIbB. Moreover, peritoneal macrophages from TLR2-deficient mice failed to respond to LT-IIaB or LT-IIbB, in contrast to wild-type or TLR4-deficient cells. These results demonstrate that besides their established binding to gangliosides, the B subunits of type II enterotoxins also interact with TLR2. Although a ganglioside-nonbinding mutant (T34I) of LT-IIaB effectively induced cytokine release, a phenotypically similar point mutation (T13I) in LT-IIbB abrogated cytokine induction, suggesting a variable requirement for gangliosides as coreceptors in TLR2 agonist activity. TLR2-dependent activation of mononuclear cells by type II enterotoxin B subunits appears to be a novel mechanism whereby these molecules may exert their immunomodulatory and adjuvant activities. PMID:15731031

Cloning of murine RNA polymerase I-specific TAF factors: conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1.

PubMed

Heix, J; Zomerdijk, J C; Ravanpay, A; Tjian, R; Grummt, I

1997-03-04

Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs). Unlike class II- and III-specific TBP-TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter. As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAFIs) and compared the amino acid sequences of the murine TAFIs with their human counterparts. The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAFIs, demonstrating that differences in the primary structure of human and mouse TAFIs do not dramatically alter the network of protein-protein contacts responsible for assembly of the multimeric complex. Thus, primate vs. rodent promoter selectivity mediated by the TBP-TAFI complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription.

Small things considered: the small accessory subunits of RNA polymerase in Gram-positive bacteria

PubMed Central

Weiss, Andy; Shaw, Lindsey N.

2015-01-01

The DNA-dependent RNA polymerase core enzyme in Gram-positive bacteria consists of seven subunits. Whilst four of them (α2ββ′) are essential, three smaller subunits, δ, ε and ω (∼9–21.5 kDa), are considered accessory. Both δ and ω have been viewed as integral components of RNAP for several decades; however, ε has only recently been described. Functionally these three small subunits carry out a variety of tasks, imparting important, supportive effects on the transcriptional process of Gram-positive bacteria. While ω is thought to have a wide range of roles, reaching from maintaining structural integrity of RNAP to σ factor recruitment, the only suggested function for ε thus far is in protecting cells from phage infection. The third subunit, δ, has been shown to have distinct influences in maintaining transcriptional specificity, and thus has a key role in cellular fitness. Collectively, all three accessory subunits, although dispensable under laboratory conditions, are often thought to be crucial for proper RNAP function. Herein we provide an overview of the available literature on each subunit, summarizing landmark findings that have deepened our understanding of these proteins and their function, and outline future challenges in understanding the role of these small subunits in the transcriptional process. PMID:25878038

Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation

PubMed Central

Joo, Yoo Jin; Ficarro, Scott B.; Soares, Luis M.; Chun, Yujin; Marto, Jarrod A.; Buratowski, Stephen

2017-01-01

TFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-binding protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-associated factor (TAF) subunits recognize downstream promoter elements, act as coactivators, and interact with nucleosomes. In yeast nuclear extracts, transcription induces stable TAF binding to downstream promoter DNA, promoting subsequent activator-independent transcription reinitiation. In vivo, promoter responses to TAF mutations correlate with the level of downstream, rather than overall, Taf1 cross-linking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations. PMID:29203645

The eukaryotic RNA exosome: same scaffold but variable catalytic subunits.

PubMed

Lykke-Andersen, Søren; Tomecki, Rafal; Jensen, Torben Heick; Dziembowski, Andrzej

2011-01-01

The RNA exosome is a versatile ribonucleolytic protein complex that participates in a multitude of cellular RNA processing and degradation events. It consists of an invariable nine-subunit core that associates with a variety of enzymatically active subunits and co-factors. These contribute to or even provide the catalytic activity and substrate specificity of the complex. The S. cerevisiae exosome has been intensively studied since its discovery in 1997 and thus serves as the archetype of eukaryotic exosomes. Notably, its catalytic potential, derived exclusively from associated subunits, differs between the nuclear and cytoplasmic versions of the complex. The same holds true for other eukaryotes, however, recent discoveries from various laboratories including our own have revealed that there are variations on this theme. Here, we review the latest findings concerning catalytic subunits of eukaryotic exosomes, and we discuss the apparent need for differential composition and subcellular distribution of exosome variants.

Disseminated intravascular coagulation: pathophysiology and principles of management.

PubMed

Marwaha, R K; Mitra, S; Marwaha, N

1998-03-01

DIC is a thrombohemorrhagic syndrome which occurs in association with well-defined clinical disorders such as septicemia, acute leukemia, snake envenomation, hypoxic states, etc. These disease conditions trigger the coagulation cascade in vivo resulting in formation of microthrombi, activation of fibrinolysis and a bleeding tendency. The important and most frequently observed laboratory abberrations include reduced platelet counts, low levels of fibrinogen, factors V and XIII with increased FDP's. Therapy primarily consists of recognizing the cause of DIC, removing the triggering process and administering anticoagulant therapy in specific situations. Component replacement is required if patients continue to bleed inspite of instituting the above mentioned measures. Rarely, drugs which inhibit fibrinolysis may be indicated. Early recognition and prompt institution of appropriate remedial measures coupled with adequate laboratory monitoring help in reducing morbidity and mortality due to DIC.

Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor

PubMed Central

Simonetti, Angelita; Marzi, Stefano; Billas, Isabelle M. L.; Tsai, Albert; Fabbretti, Attilio; Myasnikov, Alexander G.; Roblin, Pierre; Vaiana, Andrea C.; Hazemann, Isabelle; Eiler, Daniel; Steitz, Thomas A.; Puglisi, Joseph D.; Gualerzi, Claudio O.; Klaholz, Bruno P.

2013-01-01

Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl(fMet)-tRNAfMet positioning and efficient transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process. PMID:24029017

46 CFR 199.180 - Training and drills.

Code of Federal Regulations, 2014 CFR

2014-10-01

... survival craft engine and accessories; (xiii) The recovery of survival craft and rescue boats, including... and operating the lifeboat engine; and (vii) Operating davits used for launching the liferafts. (2) Abandon-ship drills should also include conducting a mock search and rescue of passengers or special...

46 CFR 199.180 - Training and drills.

Code of Federal Regulations, 2012 CFR

2012-10-01

... survival craft engine and accessories; (xiii) The recovery of survival craft and rescue boats, including... and operating the lifeboat engine; and (vii) Operating davits used for launching the liferafts. (2) Abandon-ship drills should also include conducting a mock search and rescue of passengers or special...

46 CFR 199.180 - Training and drills.

Code of Federal Regulations, 2013 CFR

2013-10-01

... survival craft engine and accessories; (xiii) The recovery of survival craft and rescue boats, including... and operating the lifeboat engine; and (vii) Operating davits used for launching the liferafts. (2) Abandon-ship drills should also include conducting a mock search and rescue of passengers or special...

78 FR 16302 - Sunshine Act Meeting; Regular Board of Directors Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-14

..., Washington, DC 20005. STATUS: Open. CONTACT PERSON FOR MORE INFORMATION: Erica Hall, Assistant Corporate... Discussion IV. Financial Report V. DC Office Move VI. NCST Board VII. Homeownership Challenges..., NFMC & EHLP Reports XII. Executive Session XIII. Adjournment Erica Hall, Assistant Corporate Secretary...

The Business Case for Systems Engineering Study: Detailed Response Data

DTIC Science & Technology

2012-11-01

of Carnegie Mellon University. DM -0000794 CMU/SEI-2012-SR-011 | i Table of Contents Acknowledgments ix Executive Summary xi Abstract xiii...providing an SRD25 upfront with crisp requirements. Customer/acquirer consistently talks about SE but never practices SE. Customer practices SE

24 CFR 576.400 - Area-wide systems coordination requirements.

Code of Federal Regulations, 2012 CFR

2012-04-01

...): (8) Head Start (45 CFR chapter XIII, subchapter B); (9) Mental Health and Substance Abuse Block..., if any, on the homelessness prevention or rapid re-housing assistance that each program participant... HOUSING AND URBAN DEVELOPMENT COMMUNITY FACILITIES EMERGENCY SOLUTIONS GRANTS PROGRAM Program Requirements...

24 CFR 576.400 - Area-wide systems coordination requirements.

Code of Federal Regulations, 2014 CFR

2014-04-01

...): (8) Head Start (45 CFR chapter XIII, subchapter B); (9) Mental Health and Substance Abuse Block..., if any, on the homelessness prevention or rapid re-housing assistance that each program participant... HOUSING AND URBAN DEVELOPMENT COMMUNITY FACILITIES EMERGENCY SOLUTIONS GRANTS PROGRAM Program Requirements...

24 CFR 576.400 - Area-wide systems coordination requirements.

Code of Federal Regulations, 2013 CFR

2013-04-01

...): (8) Head Start (45 CFR chapter XIII, subchapter B); (9) Mental Health and Substance Abuse Block..., if any, on the homelessness prevention or rapid re-housing assistance that each program participant... HOUSING AND URBAN DEVELOPMENT COMMUNITY FACILITIES EMERGENCY SOLUTIONS GRANTS PROGRAM Program Requirements...

Highway Safety Information System guidebook for the Maine state data files. Volume 1 : SAS file formats

DOT National Transportation Integrated Search

2012-05-05

As part of the Federal Highway Administration (FHWA) Traffic Analysis Toolbox (Volume XIII), this guide was designed to help corridor stakeholders implement the ICM AMS methodology successfully and effectively. It provides a step-by-step approach to ...

40 CFR 60.53c - Operator training and qualification requirements.

Code of Federal Regulations, 2011 CFR

2011-07-01

... and types of emissions; (ii) Basic combustion principles, including products of combustion; (iii) Operation of the type of incinerator to be used by the operator, including proper startup, waste charging... procedures; (xii) Pre-startup inspections; and (xiii) Recordkeeping requirements. (2) An examination designed...

40 CFR 60.53c - Operator training and qualification requirements.

Code of Federal Regulations, 2012 CFR

2012-07-01

... and types of emissions; (ii) Basic combustion principles, including products of combustion; (iii) Operation of the type of incinerator to be used by the operator, including proper startup, waste charging... procedures; (xii) Pre-startup inspections; and (xiii) Recordkeeping requirements. (2) An examination designed...

46 CFR 199.180 - Training and drills.

Code of Federal Regulations, 2010 CFR

2010-10-01

... survival craft engine and accessories; (xiii) The recovery of survival craft and rescue boats, including... and operating the lifeboat engine; and (vii) Operating davits used for launching the liferafts. (2) Abandon-ship drills should also include conducting a mock search and rescue of passengers or special...

46 CFR 199.180 - Training and drills.

Code of Federal Regulations, 2011 CFR

2011-10-01

... survival craft engine and accessories; (xiii) The recovery of survival craft and rescue boats, including... and operating the lifeboat engine; and (vii) Operating davits used for launching the liferafts. (2) Abandon-ship drills should also include conducting a mock search and rescue of passengers or special...

Sylphellapuccoon gen. n., sp. n. and two additional new species of aquatic oligochaetes (Lumbriculidae, Clitellata) from poorly-known lotic habitats in North Carolina (USA).

PubMed

Rodriguez, Pilar; Fend, Steven V; Lenat, David R

2014-01-01

Three new species of Lumbriculidae were collected from floodplain seeps and small streams in southeastern North America. Some of these habitats are naturally acidic. Sylphellapuccoon gen. n., sp. n. has prosoporous male ducts in X-XI, and spermathecae in XII-XIII. Muscular, spherical atrial ampullae and acuminate penial sheaths distinguish this monotypic new genus from other lumbriculid genera having similar arrangements of reproductive organs. Cookidriluspocosinus sp. n. resembles its two subterranean, Palearctic congeners in the arrangement of reproductive organs, but is easily distinguished by the position of the spermathecal pores in front of the chaetae in X-XIII. Stylodriluscoreyi sp. n. differs from congeners having simple-pointed chaetae and elongate atria primarily by the structure of the male duct and the large clusters of prostate cells. Streams and wetlands of Southeastern USA have a remarkably high diversity of endemic lumbriculids, and these poorly-known invertebrates should be considered in conservation efforts.

Tabulation of comet observations.

NASA Astrophysics Data System (ADS)

1985-04-01

Concerning comets: 1961 VIII Seki, 1962 III Seki-Lines, 1963 I Ikeya, 1963 III Alcock, 1964 VIII Ikeya, 1965 VIII Ikeya-Seki, 1966 V Kilston, 1967 II Rudnicki, 1968 I Ikeya-Seki, 1968 VI Honda, 1969 IX Tago-Sato-Kosaka, 1970 II Bennett, 1971 V Toba, 1973 XII Kohoutek, 1974 II P/Schwassmann-Wachmann 1, 1974 III Bradfield, 1975 IX Kobayashi-Berger-Milon, 1975 X Suzuki-Saigusa-Mori, 1975 XII Mori-Sato-Fujikawa, 1976 VI West, 1976 XI P/d'Arrest, 1979 X Bradfield, 1980 XI P/Encke, 1980 XIII P/Tuttle, 1980 XV Bradfield, 1981 II Panther, 1982i P/Halley, 1983 XIII P/Kopff, 1983n P/Crommelin, 1983v P/Hartley-IRAS, 1983w P/Clark, 1984c P/Neujmin, 1984f Shoemaker, 1984g P/Wolf-Harrington, 1984h P/Faye, 1984i Austin, 1984j P/Takamizawa, 1984k P/Arend-Rigaux, 1984m P/Schaumasse, 1984p Tsuchinshan 1, 1984q P/Shoemaker 1, 1984s Shoemaker, 1984t Levy-Rudenko.

Sylphella puccoon gen. n., sp. n. and two additional new species of aquatic oligochaetes (Lumbriculidae, Clitellata) from poorly-known lotic habitats in North Carolina (USA)

USGS Publications Warehouse

Rodriguez, Pilar; Fend, Steven V.; Lenat, David R.

2014-01-01

Three new species of Lumbriculidae were collected from floodplain seeps and small streams in southeastern North America. Some of these habitats are naturally acidic. Sylphella puccoon gen. n., sp. n. has prosoporous male ducts in X-XI, and spermathecae in XII-XIII. Muscular, spherical atrial ampullae and acuminate penial sheaths distinguish this monotypic new genus from other lumbriculid genera having similar arrangements of reproductive organs. Cookidrilus pocosinus sp. n. resembles its two subterranean, Palearctic congeners in the arrangement of reproductive organs, but is easily distinguished by the position of the spermathecal pores in front of the chaetae in X-XIII. Stylodrilus coreyi sp. n. differs from congeners having simple-pointed chaetae and elongate atria primarily by the structure of the male duct and the large clusters of prostate cells. Streams and wetlands of Southeastern USA have a remarkably high diversity of endemic lumbriculids, and these poorly-known invertebrates should be considered in conservation efforts.

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression.

PubMed

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Kandimalla, Ramesh J L; Bal, Amanjit; Gill, Kiran Dip

2013-12-01

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10mg/kgb.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) and Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits-NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. © 2013.

A Synthetic Fibrin-Crosslinking Polymer for Modulating Clot Properties and Inducing Hemostasis

PubMed Central

Chan, Leslie W.-G.; Wang, Xu; Wei, Hua; Pozzo, Lilo D.; White, Nathan J.; Pun, Suzie H.

2015-01-01

Clotting factor replacement is the standard management of acute bleeding in congenital and acquired bleeding disorders. We present a synthetic approach to hemostasis using an engineered hemostatic polymer (PolySTAT) that circulates innocuously in the blood, identifies sites of vascular injury, and promotes clot formation to stop bleeding. PolySTAT induces hemostasis by crosslinking the fibrin matrix within clots, mimicking the function of the transglutaminase Factor XIII. Furthermore, synthetic PolySTAT binds specifically to fibrin monomers and is uniformly integrated into fibrin fibers during fibrin polymerization, resulting in a fortified, hybrid polymer network with enhanced resistance to enzymatic degradation. In vivo hemostatic activity was confirmed in a rat model of trauma and fluid resuscitation in which intravenous administration of PolySTAT improved survival by reducing blood loss and resuscitation fluid requirements. PolySTAT-induced fibrin crosslinking is a novel approach to hemostasis utilizing synthetic polymers for non-invasive modulation of clot architecture with potentially wide-ranging therapeutic applications. PMID:25739763

In situ cell manipulation through enzymatic hydrogel photopatterning

NASA Astrophysics Data System (ADS)

Mosiewicz, Katarzyna A.; Kolb, Laura; van der Vlies, André J.; Martino, Mikaël M.; Lienemann, Philipp S.; Hubbell, Jeffrey A.; Ehrbar, Martin; Lutolf, Matthias P.

2013-11-01

The physicochemical properties of hydrogels can be manipulated in both space and time through the controlled application of a light beam. However, methods for hydrogel photopatterning either fail to maintain the bioactivity of fragile proteins and are thus limited to short peptides, or have been used in hydrogels that often do not support three-dimensional (3D) cell growth. Here, we show that the 3D invasion of primary human mesenchymal stem cells can be spatiotemporally controlled by micropatterning the hydrogel with desired extracellular matrix (ECM) proteins and growth factors. A peptide substrate of activated transglutaminase factor XIII (FXIIIa)—a key ECM crosslinking enzyme—is rendered photosensitive by masking its active site with a photolabile cage group. Covalent incorporation of the caged FXIIIa substrate into poly(ethylene glycol) hydrogels and subsequent laser-scanning lithography affords highly localized biomolecule tethering. This approach for the 3D manipulation of cells within gels should open up avenues for the study and manipulation of cell signalling.

G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity.

PubMed

Stott, Jennifer B; Povstyan, Oleksandr V; Carr, Georgina; Barrese, Vincenzo; Greenwood, Iain A

2015-05-19

Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein βγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K(+) currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gβγ subunits (2-250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein-coupled receptors. Gallein, an inhibitor of Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein-coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone.

G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity

PubMed Central

Stott, Jennifer B.; Povstyan, Oleksandr V.; Carr, Georgina; Barrese, Vincenzo; Greenwood, Iain A.

2015-01-01

Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein βγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K+ currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gβγ subunits (2–250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein–coupled receptors. Gallein, an inhibitor of Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein–coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone. PMID:25941381

Cyclin A recruits p33cdk2 to the cellular transcription factor DRTF1.

PubMed

Bandara, L R; Adamczewski, J P; Zamanian, M; Poon, R Y; Hunt, T; Thangue, N B

1992-01-01

Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they control important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p107. We now show, using biologically active fusion proteins, that cyclin A can direct the binding of the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p107, as well as activate its histone H1 kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin A-p33cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an activating and targeting subunit of p33cdk2. The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progression and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.

Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen

PubMed Central

Gründel, Anne; Friedrich, Kathleen; Pfeiffer, Melanie; Jacobs, Enno; Dumke, Roger

2015-01-01

The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhesins and release of cell-damaging factors, surface-displayed glycolytic enzymes may facilitate the further interaction between host and microbe. In this study, we described detection of the four subunits of pyruvate dehydrogenase complex (PDHA-D) among the cytosolic and membrane-associated proteins of M. pneumoniae. Subunits of PDH were cloned, expressed and purified to produce specific polyclonal guinea pig antisera. Using colony blotting, fractionation of total proteins and immunofluorescence experiments, the surface localization of PDHA-C was demonstrated. All recombinant PDH subunits are able to bind to HeLa cells and human plasminogen. These interactions can be specifically blocked by the corresponding polyclonal antisera. In addition, an influence of ionic interactions on PDHC-binding to plasminogen as well as of lysine residues on the association of PDHA-D with plasminogen was confirmed. The PDHB subunit was shown to activate plasminogen and the PDHB-plasminogen complex induces degradation of human fibrinogen. Hence, our data indicate that the surface-associated PDH subunits might play a role in the pathogenesis of M. pneumoniae infections by interaction with human plasminogen. PMID:25978044

Mediator kinase module and human tumorigenesis.

PubMed

Clark, Alison D; Oldenbroek, Marieke; Boyer, Thomas G

2015-01-01

Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit "kinase" module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways.

Mediator kinase module and human tumorigenesis

PubMed Central

Clark, Alison D.; Oldenbroek, Marieke; Boyer, Thomas G.

2016-01-01

Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit “kinase” module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways. PMID:26182352

Ferrite Loaded Coils for Improved Wireless Power Transfer Efficiency

DTIC Science & Technology

2015-09-01

visible in yellow, from [4]. ..........................................................................................4  Figure 4.  The Odyssey AUV ...47  Figure 35.  System mockup of AUV in docking/charging station...42  xii THIS PAGE INTENTIONALLY LEFT BLANK xiii LIST OF ACRONYMS AND ABBREVIATIONS AUV Autonomous Underwater Vehicle CST

38 CFR 26.6 - Environmental documents.

Code of Federal Regulations, 2011 CFR

2011-07-01

... significant impact on, official local or regional zoning or comprehensive land use plans; and, (xiii...) Environmental Impact Statements. The head of each VA element shall include a detailed written statement “in... Regulations, 40 CFR part 1502. An environmental impact statement shall be prepared in accordance with the...

42 CFR 417.166 - Waiver of assurances.

Code of Federal Regulations, 2010 CFR

2010-10-01

... (CONTINUED) MEDICARE PROGRAM HEALTH MAINTENANCE ORGANIZATIONS, COMPETITIVE MEDICAL PLANS, AND HEALTH CARE... law; or (2) The HMO shows good cause, consistent with the purposes of title XIII of the PHS Act. (b... the reorganization can only be approved with the waiver of the assurances. (ii) State laws governing...

12 CFR 27.3 - Recordkeeping requirements.

Code of Federal Regulations, 2010 CFR

2010-01-01

... employed by the current employer of the applicant(s). For self-employed persons, the number of continuous years self-employed. (xiii) Gross total monthly income of each applicant, comprising the sum of normal... or disability income and income from part-time employment. For self-employed persons, include the...

25 CFR 36.40 - Standard XIII-Library/media program.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR EDUCATION MINIMUM ACADEMIC STANDARDS FOR THE BASIC EDUCATION OF INDIAN CHILDREN AND NATIONAL CRITERIA FOR DORMITORY SITUATIONS Instructional Support... inclusive of materials located in the classrooms shall be maintained. This category includes some of each of...

27 CFR 9.108 - Ozark Mountain.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Creek; (xii) Then northeastward along Rock Creek to Petit Jean Creek; (xiii) Then generally northeastward and eastward along Petit Jean Creek until it becomes the Petit Jean River (on the Russellville map); (xiv) Then generally eastward along the Petit Jean River, flowing through Blue Mountain Lake, until the...

27 CFR 9.108 - Ozark Mountain.

Code of Federal Regulations, 2012 CFR

2012-04-01

... Creek; (xii) Then northeastward along Rock Creek to Petit Jean Creek; (xiii) Then generally northeastward and eastward along Petit Jean Creek until it becomes the Petit Jean River (on the Russellville map); (xiv) Then generally eastward along the Petit Jean River, flowing through Blue Mountain Lake, until the...

27 CFR 9.108 - Ozark Mountain.

Code of Federal Regulations, 2013 CFR

2013-04-01

... Creek; (xii) Then northeastward along Rock Creek to Petit Jean Creek; (xiii) Then generally northeastward and eastward along Petit Jean Creek until it becomes the Petit Jean River (on the Russellville map); (xiv) Then generally eastward along the Petit Jean River, flowing through Blue Mountain Lake, until the...

27 CFR 9.108 - Ozark Mountain.

Code of Federal Regulations, 2014 CFR

2014-04-01

... Creek; (xii) Then northeastward along Rock Creek to Petit Jean Creek; (xiii) Then generally northeastward and eastward along Petit Jean Creek until it becomes the Petit Jean River (on the Russellville map); (xiv) Then generally eastward along the Petit Jean River, flowing through Blue Mountain Lake, until the...

Incentive Pay for Remotely Piloted Aircraft Career Fields

DTIC Science & Technology

2012-01-01

Fields C.1. Mathematical Symbols for Non-Stochastic Values and Shock Terms...78 C.2. Mathematical Symbols for Taste and Compensation . . . . . . . . . . . 79 xiii Summary Background and...manning requirement, even with the current incentive pays and reenlistment bonuses. 2 The mathematical foundations, data, and estimation methods for the

40 CFR 62.14595 - What are the operator training and qualification requirements?

Code of Federal Regulations, 2010 CFR

2010-07-01

... charging, and shutdown procedures. (iv) Combustion controls and monitoring. (v) Operation of air pollution... the incinerator and air pollution control devices. (vii) Actions to correct malfunctions or conditions... requirements. (xiii) Methods to continuously monitor CISWI unit and air pollution control device operating...

40 CFR 62.14595 - What are the operator training and qualification requirements?

Code of Federal Regulations, 2011 CFR

2011-07-01

... charging, and shutdown procedures. (iv) Combustion controls and monitoring. (v) Operation of air pollution... the incinerator and air pollution control devices. (vii) Actions to correct malfunctions or conditions... requirements. (xiii) Methods to continuously monitor CISWI unit and air pollution control device operating...

75 FR 42000 - Requirements for Fingerprint-Based Criminal History Records Checks for Individuals Seeking...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-20

... Compatibility VIII. Plain Language IX. Voluntary Consensus Standards X. Finding of No Significant Environmental Impact: Availability XI. Paperwork Reduction Act Statement XII. Regulatory Analysis: Availability XIII... received seven comment letters from interested parties: Four from RTR licensees, one from the Nuclear...

76 FR 6199 - Enhanced Weapons, Firearms Background Checks, and Security Event Notifications

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-03

... XI. Voluntary Consensus Standards XII. Finding of No Significant Environmental Impact XIII. Paperwork... potential advantages to NRC licensees and certificate holders to enhance security. The first advantage is... advantage is that security personnel of certain licensees or certificate holders will be permitted to...

40 CFR Appendix Xiii to Part 266 - Mercury Bearing Wastes That May Be Processed in Exempt Mercury Recovery Units

Code of Federal Regulations, 2011 CFR

2011-07-01

... are exempt mercury-bearing materials with less than 500 ppm of 40 CFR Part 261, appendix VIII organic... tank sludge 13. Mercury cell process solids 14. Recoverable levels of mercury contained in soil [59 FR...

40 CFR Appendix Xiii to Part 266 - Mercury Bearing Wastes That May Be Processed in Exempt Mercury Recovery Units

Code of Federal Regulations, 2012 CFR

2012-07-01

... are exempt mercury-bearing materials with less than 500 ppm of 40 CFR Part 261, appendix VIII organic... tank sludge 13. Mercury cell process solids 14. Recoverable levels of mercury contained in soil [59 FR...

40 CFR Appendix Xiii to Part 266 - Mercury Bearing Wastes That May Be Processed in Exempt Mercury Recovery Units

Code of Federal Regulations, 2014 CFR

2014-07-01

... are exempt mercury-bearing materials with less than 500 ppm of 40 CFR Part 261, appendix VIII organic... tank sludge 13. Mercury cell process solids 14. Recoverable levels of mercury contained in soil [59 FR...

40 CFR Appendix Xiii to Part 266 - Mercury Bearing Wastes That May Be Processed in Exempt Mercury Recovery Units

Code of Federal Regulations, 2010 CFR

2010-07-01

... are exempt mercury-bearing materials with less than 500 ppm of 40 CFR Part 261, appendix VIII organic... tank sludge 13. Mercury cell process solids 14. Recoverable levels of mercury contained in soil [59 FR...

40 CFR Appendix Xiii to Part 266 - Mercury Bearing Wastes That May Be Processed in Exempt Mercury Recovery Units

Code of Federal Regulations, 2013 CFR

2013-07-01

... are exempt mercury-bearing materials with less than 500 ppm of 40 CFR Part 261, appendix VIII organic... tank sludge 13. Mercury cell process solids 14. Recoverable levels of mercury contained in soil [59 FR...

7. Photocopied December 1977, form F.B. Tower, Illustrations of the ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

7. Photocopied December 1977, form F.B. Tower, Illustrations of the Croton Aqueduct, New York: Wiley and Putnam, 1843. CROTON AQUEDUCT AT SING SING, PLATE XIII, PAGE 101. - Old Croton Aqueduct, Sing Sing Kill Bridge, Spanning Aqueduct Street & Broadway, Ossining, Westchester County, NY

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.

PubMed

Fuchs, Gabriele; Petrov, Alexey N; Marceau, Caleb D; Popov, Lauren M; Chen, Jin; O'Leary, Seán E; Wang, Richard; Carette, Jan E; Sarnow, Peter; Puglisi, Joseph D

2015-01-13

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

Tabulation of comet observations.

NASA Astrophysics Data System (ADS)

1993-10-01

Concerning comets: 1955 III Mrkos, 1955 IV Bakharev-Macfarlane-Krienke, 1955 V Honda, 1956 III Mrkos, 1956 IV P/Olbers, 1957 III Arend-Roland, 1957 V Mrkos, 1958 III Burnham, 1959 VIII P/Giacobini-Zinner, 1960 II Burnham, 1973 XII Kohoutek, 1974 III Bradfield, 1975 IX Kobayashi-Berger-Milon, 1975 X Suzuki-Saigusa-Mori, 1975 XI Bradfield, 1975 XII Mori-Sato-Fujikawa, 1976 IV Bradfield, 1976 VI West, 1979 VII Bradfield, 1980 X P/Stephan-Oerma, 1980 XII Meier, 1980 XIII P/Tuttle, 1981 II Panther, 1981 IV P/Borrelly, 1981 XIX P/Swift-Gehrels, 1982 I Bowell, 1982 IV P/Grigg-Skjellerup, 1982 VI Austin, 1982 VII P/d'Arrest, 1982 VIII P/Churyumov-Gerasimenko, 1983 V Sugano-Saigusa-Fujikawa, 1983 VII IRAS-Araki-Alcock, 1983 X P/Tempel 2, 1983 XI P/Tempel 1, 1983 XIII P/Kopff, 1983 XIV P/IRAS, 1983 XV Shoemaker, 1984 III P/Hartley-IRAS, 1984 IV P/Crommelin, 1984 XI P/Faye, 1984 XIII Austin, 1984 XIV P/Wild 2, 1984 XVI P/Shoemaker 1, 1984 XXIII Levy-Rudenko, 1985 I P/Tsuchinshan 1, 1985 XIII P/Giacobini-Zinner, 1985 XV P/Giclas, 1985 XVI P/Ciffréo, 1985 XVII Hartley-Good, 1985 XVIII P/Shoemaker 3, 1985 XIX Thiele, 1986 I P/Boethin, 1986 III P/Halley, 1986 VIII P/Machholz, 1986 XVII Levy, 1986 XVIII Terasako, 1987 II Sorrells, 1987 VII Wilson, 1987 XIX P/Schwassmann-Wachmann 2, 1987 XXI Levy, 1987 XXIII Rudenko, 1987 XXIV P/Brooks 2, 1987 XXVII P/Kohoutek, 1987 XXIX Bradfield, 1988 IV Furuyama, 1988 XIV P/Tempel 2, 1989 III Shoemaker, 1989 XV P/Schwassmann-Wachmann 1, 1989 XIX Okazaki-Levy-Rudenko, 1990 V Austin, 1990 XVII Tsuchiya-Kiuchi, 1990 XX Levy, 1990 XXI P/Encke, 1990 XXVI Arai, 1991 I P/Metcalf-Brewington, 1991 XV P/Hartley 2, 1991 XVII P/Arend-Rigaux, 1991a1 Shoemaker-Levy, 1991g1 Zanotta-Brewington, 1992c P/Howell, 1992d Tanaka-Machholz, 1992e P/Singer Brewster, 1992f P/Shoemaker-Levy 8, 1992h Spacewatch, 1992j P/Ashbrook-Jackson, 1992t P/Swift-Tuttle, 1992u P/Väisälä 1, 1992w P/Slaughter-Burnham, 1992x P/Schaumasse, 1992y Shoemaker, 1993a Mueller, 1993d Mueller, 1993e P/Shoemaker-Levy 9, 1993f P/Forbes, 1993i P/Holmes, 1993j P/Neujmin 3, 1993k P/Shajn-Schaldach, 1993l P/Helin-Lawrence, 1993m P/Hartley 3, 1993n P/Whipple, 1993ο P/West-Kohoutek-Ikemura, 1993p Mueller, P/Smirnova-Chernykh.

Regular approximate factorization of a class of matrix-function with an unstable set of partial indices

PubMed Central

Rogosin, S.

2018-01-01

From the classic work of Gohberg & Krein (1958 Uspekhi Mat. Nauk. XIII, 3–72. (Russian).), it is well known that the set of partial indices of a non-singular matrix function may change depending on the properties of the original matrix. More precisely, it was shown that if the difference between the largest and the smallest partial indices is larger than unity then, in any neighbourhood of the original matrix function, there exists another matrix function possessing a different set of partial indices. As a result, the factorization of matrix functions, being an extremely difficult process itself even in the case of the canonical factorization, remains unresolvable or even questionable in the case of a non-stable set of partial indices. Such a situation, in turn, has became an unavoidable obstacle to the application of the factorization technique. This paper sets out to answer a less ambitious question than that of effective factorizing matrix functions with non-stable sets of partial indices, and instead focuses on determining the conditions which, when having known factorization of the limiting matrix function, allow to construct another family of matrix functions with the same origin that preserves the non-stable partial indices and is close to the original set of the matrix functions. PMID:29434502

Regular approximate factorization of a class of matrix-function with an unstable set of partial indices.

PubMed

Mishuris, G; Rogosin, S

2018-01-01

From the classic work of Gohberg & Krein (1958 Uspekhi Mat. Nauk. XIII , 3-72. (Russian).), it is well known that the set of partial indices of a non-singular matrix function may change depending on the properties of the original matrix. More precisely, it was shown that if the difference between the largest and the smallest partial indices is larger than unity then, in any neighbourhood of the original matrix function, there exists another matrix function possessing a different set of partial indices. As a result, the factorization of matrix functions, being an extremely difficult process itself even in the case of the canonical factorization, remains unresolvable or even questionable in the case of a non-stable set of partial indices. Such a situation, in turn, has became an unavoidable obstacle to the application of the factorization technique. This paper sets out to answer a less ambitious question than that of effective factorizing matrix functions with non-stable sets of partial indices, and instead focuses on determining the conditions which, when having known factorization of the limiting matrix function, allow to construct another family of matrix functions with the same origin that preserves the non-stable partial indices and is close to the original set of the matrix functions.

Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor.

PubMed

Anandhakumar, Jayamani; Moustafa, Yara W; Chowdhary, Surabhi; Kainth, Amoldeep S; Gross, David S

2016-07-15

Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. Although it has been generally assumed that in Saccharomyces cerevisiae, Mediator is a stable trimodular complex, its structural state in vivo remains unclear. Using the "anchor away" (AA) technique to conditionally deplete select subunits within Mediator and its reversibly associated Cdk8 kinase module (CKM), we provide evidence that Mediator's tail module is highly dynamic and that a subcomplex consisting of Med2, Med3, and Med15 can be independently recruited to the regulatory regions of heat shock factor 1 (Hsf1)-activated genes. Fluorescence microscopy of a scaffold subunit (Med14)-anchored strain confirmed parallel cytoplasmic sequestration of core subunits located outside the tail triad. In addition, and contrary to current models, we provide evidence that Hsf1 can recruit the CKM independently of core Mediator and that core Mediator has a role in regulating postinitiation events. Collectively, our results suggest that yeast Mediator is not monolithic but potentially has a dynamic complexity heretofore unappreciated. Multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 tail triad, and the four-subunit CKM, can be independently recruited by activated Hsf1 to its target genes in AA strains. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Chemical factors that control lignin polymerization.

PubMed

Sangha, Amandeep K; Davison, Brian H; Standaert, Robert F; Davis, Mark F; Smith, Jeremy C; Parks, Jerry M

2014-01-09

Lignin is a complex, branched polymer that reinforces plant tissue. Understanding the factors that govern lignin structure is of central importance to the development of technologies for converting lignocellulosic biomass into fuels because lignin imparts resistance to chemical, enzymatic, and mechanical deconstruction. Lignin is formed by enzymatic oxidation of phenolic monomers (monolignols) of three main types, guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) subunits. It is known that increasing the relative abundance of H subunits results in lower molecular weight lignin polymers and hence more easily deconstructed biomass, but it is not known why. Here, we report an analysis of frontier molecular orbitals in mono-, di-, and trilignols, calculated using density functional theory, which points to a requirement of strong p-electron density on the reacting phenolic oxygen atom of the neutral precursor for enzymatic oxidation to occur. This model is consistent with a proton-coupled electron transfer (PCET) mechanism and for the first time explains why H subunits in certain linkages (β-β or β-5) react poorly and tend to "cap" the polymer. In general, β-5 linkages with either a G or H terminus are predicted to inhibit elongation. More broadly, the model correctly accounts for the reactivity of the phenolic groups in a diverse set of dilignols comprising H and G subunits. Thus, we provide a coherent framework for understanding the propensity toward growth or termination of different terminal subunits in lignin.

Crystal Structure of the 25 kDa Subunit of Human Cleavage Factor I{m}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coseno,M.; Martin, G.; Berger, C.

Cleavage factor Im is an essential component of the pre-messenger RNA 3'-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein-protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic {alpha}/{beta}/{alpha} fold and a conserved catalytic sequence or Nudix box. We present heremore » the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Angstroms, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3'-end processing.« less

Crystal structure of the 25 kDa subunit of human cleavage factor Im

PubMed Central

Coseno, Molly; Martin, Georges; Berger, Christopher; Gilmartin, Gregory; Keller, Walter; Doublié, Sylvie

2008-01-01

Cleavage factor Im is an essential component of the pre-messenger RNA 3′-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein–protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic α/β/α fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Å, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3′-end processing. PMID:18445629

7 CFR 1485.16 - Contribution rules.

Code of Federal Regulations, 2014 CFR

2014-01-01

... business cards that target a foreign audience; (viii) The cost of seasonal greeting cards; (ix) Fees for... conducted overseas; (xii) Credit card fees; (xiii) The cost of any independent evaluation or audit that is...) Membership fees in clubs and social organizations; and (xi) Any expenditure for an activity prior to CCC's...

7 CFR 1485.13 - Application process and strategic plan.

Code of Federal Regulations, 2012 CFR

2012-01-01

... business cards; (viii) The cost of seasonal greeting cards; (ix) Fees for office parking; (x) The cost of subscriptions to publications; (xi) The cost of activities conducted overseas; (xii) Credit card fees; (xiii... fees or similar sales expenditures; (x) Membership fees in clubs and social organizations; and (xi) Any...

7 CFR 1485.16 - Contribution rules.

Code of Federal Regulations, 2013 CFR

2013-01-01

... business cards that target a foreign audience; (viii) The cost of seasonal greeting cards; (ix) Fees for... conducted overseas; (xii) Credit card fees; (xiii) The cost of any independent evaluation or audit that is...) Membership fees in clubs and social organizations; and (xi) Any expenditure for an activity prior to CCC's...

[Bio-nanoscience and bio-nanotechnology: social and ethical aspects].

PubMed

Martín-Lomas, Manuel

2006-01-01

This article is an abstract of a conference with the same title presented at the XIII Jornadas sobre Derecho y Genoma Humano and is basically centred in a report for the Royal Society and the Royal Academy of Engineering entitled Nanoscience and Nanotechnology made publicly available July 2004.

46 CFR 56.01-10 - Plan approval.

Code of Federal Regulations, 2010 CFR

2010-10-01

... ballast piping. (vii) Tank cleaning piping. (viii) Condenser circulating water piping. (ix) Vent, sound....) (xii) Cargo piping. (xiii) Hot water heating systems if the temperature is greater than 121 °C(250 °F... substantiate their compliance with the regulations of this subchapter; (3) A thermal stress analysis is not...

Higher Education: Handbook of Theory and Research. Volume XIII.

ERIC Educational Resources Information Center

Smart, John C., Ed.

The 10 papers in this handbook consider higher education theory and research. Following an opening essay, "Recollections and Reflections," by C. Robert Pace, which offers reflections on higher education as a field, on its evolution, and it future research needs, papers include: "Reflections on the Study of Effective College Teaching…

Single-cycle Optical Pulses and Isolated Attosecond Pulse Generation

DTIC Science & Technology

2012-02-29

picosecond green light from a frequency-doubled hybrid cryogenic Yb:YAG laser system,” 36 UFO /HFSW 2009 (Arcachon, France, Aug. 31-Sept. 4, 2009...High Fields Short Wavelength,” ( UFO VII – HFSW XIII), Arcachon, France, August 31 – September 4, 2009 (invited). 25) Kyung-Han Hong, Juliet Gopinath

45 CFR 307.11 - Functional requirements for computerized support enforcement systems in operation by October 1...

Code of Federal Regulations, 2011 CFR

2011-10-01

... violence or child abuse); (xi) Indication of an order; (xii) Locate request type (optional); (xiii) Locate... to Public Welfare OFFICE OF CHILD SUPPORT ENFORCEMENT (CHILD SUPPORT ENFORCEMENT PROGRAM), ADMINISTRATION FOR CHILDREN AND FAMILIES, DEPARTMENT OF HEALTH AND HUMAN SERVICES COMPUTERIZED SUPPORT...

45 CFR 307.11 - Functional requirements for computerized support enforcement systems in operation by October 1...

Code of Federal Regulations, 2013 CFR

2013-10-01

... violence or child abuse); (xi) Indication of an order; (xii) Locate request type (optional); (xiii) Locate... to Public Welfare OFFICE OF CHILD SUPPORT ENFORCEMENT (CHILD SUPPORT ENFORCEMENT PROGRAM), ADMINISTRATION FOR CHILDREN AND FAMILIES, DEPARTMENT OF HEALTH AND HUMAN SERVICES COMPUTERIZED SUPPORT...

45 CFR 307.11 - Functional requirements for computerized support enforcement systems in operation by October 1...

Code of Federal Regulations, 2012 CFR

2012-10-01

... violence or child abuse); (xi) Indication of an order; (xii) Locate request type (optional); (xiii) Locate... to Public Welfare OFFICE OF CHILD SUPPORT ENFORCEMENT (CHILD SUPPORT ENFORCEMENT PROGRAM), ADMINISTRATION FOR CHILDREN AND FAMILIES, DEPARTMENT OF HEALTH AND HUMAN SERVICES COMPUTERIZED SUPPORT...

32 CFR 505.7 - Disclosure of personal information to other agencies and third parties.

Code of Federal Regulations, 2010 CFR

2010-07-01

... for federal employment); (xii) Social Security Number (SSN); and (xiii) The information that would... released if their positions or duties require frequent interaction with the public. (3) Disclosure of.... (g) Social rosters. (1) Before including personal information such as a spouse's name, home addresses...

76 FR 17870 - Medicare and Medicaid Programs; Quarterly Listing of Program Issuances-October Through December 2010

Federal Register 2010, 2011, 2012, 2013, 2014

2011-03-31

... Registry (NOPR) Sites. Addendum XIII: Medicare-approved Ventricular Assist Device (Destination Therapy... Therapy) Facilities (October Through December 2010) On October 1, 2003, we issued our decision memorandum on ventricular assist devices (VADs) for the clinical indication of destination therapy. We...

40 CFR 63.1207 - What are the performance testing requirements?

Code of Federal Regulations, 2011 CFR

2011-07-01

... incinerators, cement kilns, and lightweight aggregate kilns, you must commence the initial comprehensive... performance test operating conditions, as provided by paragraph (g)(1)(iii) of this section; (xiii) For cement... preheater or preheater/precalciner cement kilns with dual stacks, if you elect to use the emissions...

78 FR 70352 - Notice of Government in the Sunshine Regular Board of Directors Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-25

... NEIGHBORHOOD REINVESTMENT CORPORATION Notice of Government in the Sunshine Regular Board of.... Approval of Minutes III. Management Internal Operations Review IV. Non-Network Grant Policy V. MHA... Board XIII. Adjournment Jeffrey T. Bryson, General Counsel/Secretary. [FR Doc. 2013-28325 Filed 11-21-13...

38 CFR 3.815 - Monetary allowance under 38 U.S.C. chapter 18 for an individual with disability from covered...

Code of Federal Regulations, 2012 CFR

2012-07-01

..., the following: (i) Down syndrome and other Trisomies; (ii) Fragile X syndrome; (iii) Klinefelter's... atresia; (vi) Hallerman-Streiff syndrome; (vii) Hip dysplasia; (viii) Hirschprung's disease (congenital...) Neural tube defects (including spina bifida, encephalocele, and anencephaly); (xiii) Poland syndrome...

38 CFR 3.815 - Monetary allowance under 38 U.S.C. chapter 18 for an individual with disability from covered...

Code of Federal Regulations, 2013 CFR

2013-07-01

..., the following: (i) Down syndrome and other Trisomies; (ii) Fragile X syndrome; (iii) Klinefelter's... atresia; (vi) Hallerman-Streiff syndrome; (vii) Hip dysplasia; (viii) Hirschprung's disease (congenital...) Neural tube defects (including spina bifida, encephalocele, and anencephaly); (xiii) Poland syndrome...

38 CFR 3.815 - Monetary allowance under 38 U.S.C. chapter 18 for an individual with disability from covered...

Code of Federal Regulations, 2014 CFR

2014-07-01

..., the following: (i) Down syndrome and other Trisomies; (ii) Fragile X syndrome; (iii) Klinefelter's... atresia; (vi) Hallerman-Streiff syndrome; (vii) Hip dysplasia; (viii) Hirschprung's disease (congenital...) Neural tube defects (including spina bifida, encephalocele, and anencephaly); (xiii) Poland syndrome...

7 CFR 25.1 - Applicability and scope.

Code of Federal Regulations, 2014 CFR

2014-01-01

... Reconciliation Act of 1993, title XIII, subchapter C, part I (Round I), the Taxpayer Relief Act of 1997, title IX, subtitle F (Round II), the Agriculture, Rural Development, Food and Drug Administration, and Related Agencies Appropriations Act, 1999 (Public Law 105-277) (Round IIS), and the Community Renewal Tax Relief...

7 CFR 25.1 - Applicability and scope.

Code of Federal Regulations, 2012 CFR

2012-01-01

... Reconciliation Act of 1993, title XIII, subchapter C, part I (Round I), the Taxpayer Relief Act of 1997, title IX, subtitle F (Round II), the Agriculture, Rural Development, Food and Drug Administration, and Related Agencies Appropriations Act, 1999 (Public Law 105-277) (Round IIS), and the Community Renewal Tax Relief...

7 CFR 25.1 - Applicability and scope.

Code of Federal Regulations, 2013 CFR

2013-01-01

... Reconciliation Act of 1993, title XIII, subchapter C, part I (Round I), the Taxpayer Relief Act of 1997, title IX, subtitle F (Round II), the Agriculture, Rural Development, Food and Drug Administration, and Related Agencies Appropriations Act, 1999 (Public Law 105-277) (Round IIS), and the Community Renewal Tax Relief...

32 CFR 865.114 - Decisional document.

Code of Federal Regulations, 2010 CFR

2010-07-01

... punishment). (xii) Conviction by court-martial. (xiii) Prior military service and type of discharge received. (3) A list of the type of documents submitted by or on behalf of the applicant (including a written... documentary evidence), if any. (4) A statement whether the applicant testified, and a list of the type of...

15 CFR 30.3 - Electronic Export Information filer requirements, parties to export transactions, and...

Code of Federal Regulations, 2013 CFR

2013-01-01

.../units of measure. (ix) Value. (x) Export Control Classification Number (ECCN) or sufficient technical information to determine the ECCN. (xi) All licensing information necessary to file the EEI for commodities... export. (xi) Foreign port of unloading. (xii) Shipping weight. (xiii) ECCN. (xiv) License or license...

15 CFR 30.3 - Electronic Export Information filer requirements, parties to export transactions, and...

Code of Federal Regulations, 2010 CFR

2010-01-01

.../units of measure. (ix) Value. (x) Export Control Classification Number (ECCN) or sufficient technical information to determine the ECCN. (xi) All licensing information necessary to file the EEI for commodities... export. (xi) Foreign port of unloading. (xii) Shipping weight. (xiii) ECCN. (xiv) License or license...

15 CFR 30.3 - Electronic Export Information filer requirements, parties to export transactions, and...

Code of Federal Regulations, 2012 CFR

2012-01-01

.../units of measure. (ix) Value. (x) Export Control Classification Number (ECCN) or sufficient technical information to determine the ECCN. (xi) All licensing information necessary to file the EEI for commodities... export. (xi) Foreign port of unloading. (xii) Shipping weight. (xiii) ECCN. (xiv) License or license...

15 CFR 30.3 - Electronic Export Information filer requirements, parties to export transactions, and...

Code of Federal Regulations, 2014 CFR

2014-01-01

.../units of measure. (ix) Value. (x) Export Control Classification Number (ECCN) or sufficient technical information to determine the ECCN. (xi) All licensing information necessary to file the EEI for commodities... export. (xi) Foreign port of unloading. (xii) Shipping weight. (xiii) ECCN. (xiv) License or license...

15 CFR 30.3 - Electronic Export Information filer requirements, parties to export transactions, and...

Code of Federal Regulations, 2011 CFR

2011-01-01

.../units of measure. (ix) Value. (x) Export Control Classification Number (ECCN) or sufficient technical information to determine the ECCN. (xi) All licensing information necessary to file the EEI for commodities... export. (xi) Foreign port of unloading. (xii) Shipping weight. (xiii) ECCN. (xiv) License or license...

Photogrammetric camera calibration

USGS Publications Warehouse

Tayman, W.P.; Ziemann, H.

1984-01-01

Section 2 (Calibration) of the document "Recommended Procedures for Calibrating Photogrammetric Cameras and Related Optical Tests" from the International Archives of Photogrammetry, Vol. XIII, Part 4, is reviewed in the light of recent practical work, and suggestions for changes are made. These suggestions are intended as a basis for a further discussion. ?? 1984.

38 CFR 17.81 - Contracts for residential treatment services for veterans with alcohol or drug dependence or...

Code of Federal Regulations, 2014 CFR

2014-07-01

... contract. (6) Demonstrate an existing capability to furnish the following: (i) A supervised alcohol and... social service. (x) Individual counseling as appropriate. (xi) Opportunities for learning/development of...-free life style). (xiii) Opportunities for learning, testing, and internalizing knowledge of illness...

38 CFR 17.81 - Contracts for residential treatment services for veterans with alcohol or drug dependence or...

Code of Federal Regulations, 2013 CFR

2013-07-01

... contract. (6) Demonstrate an existing capability to furnish the following: (i) A supervised alcohol and... social service. (x) Individual counseling as appropriate. (xi) Opportunities for learning/development of...-free life style). (xiii) Opportunities for learning, testing, and internalizing knowledge of illness...

75 FR 55274 - Change of Address for Region 5 State and Local Agencies; Technical Correction

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-10

... Health District, Air Pollution Control, 33 Mill Street, Painesville, OH 44077. (xiv) Mahoning and...; Director, Canton City Health Department, Air Pollution Control Division, 420 Market Avenue North, Canton.... (xiii) Geauga and Lake Counties; Lake County General Health District, Air Pollution Control, 33 Mill...

Emergence of new sub-genotypes of virulent Newcastle disease virus with panzootic features

USDA-ARS?s Scientific Manuscript database

Newcastle disease virus (NDV) strains with epizootic characteristics from three new sub-genotypes of genotypes VII and XIII are rapidly spreading through Asia and the Middle East causing outbreaks of Newcastle disease (ND) that are producing significant illness and mortality in vaccinated poultry, s...

Accurate Computation of Electric Field Enhancement Factors for Metallic Nanoparticles Using the Discrete Dipole Approximation

PubMed Central

2010-01-01

We model the response of nanoscale Ag prolate spheroids to an external uniform static electric field using simulations based on the discrete dipole approximation, in which the spheroid is represented as a collection of polarizable subunits. We compare the results of simulations that employ subunit polarizabilities derived from the Clausius–Mossotti relation with those of simulations that employ polarizabilities that include a local environmental correction for subunits near the spheroid’s surface [Rahmani et al. Opt Lett 27: 2118 (2002)]. The simulations that employ corrected polarizabilities give predictions in very good agreement with exact results obtained by solving Laplace’s equation. In contrast, simulations that employ uncorrected Clausius–Mossotti polarizabilities substantially underestimate the extent of the electric field “hot spot” near the spheroid’s sharp tip, and give predictions for the field enhancement factor near the tip that are 30 to 50% too small. PMID:20672062

In vivo effects on intron retention and exon skipping by the U2AF large subunit and SF1/BBP in the nematode Caenorhabditis elegans

PubMed Central

Ma, Long; Tan, Zhiping; Teng, Yanling; Hoersch, Sebastian; Horvitz, H. Robert

2011-01-01

The in vivo analysis of the roles of splicing factors in regulating alternative splicing in animals remains a challenge. Using a microarray-based screen, we identified a Caenorhabditis elegans gene, tos-1, that exhibited three of the four major types of alternative splicing: intron retention, exon skipping, and, in the presence of U2AF large subunit mutations, the use of alternative 3′ splice sites. Mutations in the splicing factors U2AF large subunit and SF1/BBP altered the splicing of tos-1. 3′ splice sites of the retained intron or before the skipped exon regulate the splicing pattern of tos-1. Our study provides in vivo evidence that intron retention and exon skipping can be regulated largely by the identities of 3′ splice sites. PMID:22033331

Archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2B alpha-beta-delta subunit families

NASA Technical Reports Server (NTRS)

Kyrpides, N. C.; Woese, C. R.

1998-01-01

As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits alpha, beta, and delta, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.

New insights into the structure-function relationships and therapeutic applications of cholera-like enterotoxins.

PubMed

Hirst, Timothy R; Fraser, Sylvia; Soriani, Marco; Aman, A Tholib; de, Haan Lolke; Hearn, Arron; Merritt, Ethan

2002-02-01

Cholera toxin and E. coli heat-labile enterotoxin are structurally homologous proteins comprised of an enzymatically active A-subunit and five B-subunits that bind with high affinity to GM1-ganglioside receptors found on the surface of mammalian cells. The B-subunits have long been thought of simply as trafficking vehicles that trigger entry and subsequent delivery of the 'toxic' A-subunit into cells. Indeed, such is the capacity of the B-subunits to enter cells, that they have been developed as generic carriers for attachment and delivery of a variety of peptides into mammalian cells. However, the B-subunits also appear to possess discrete 'signalling functions', that induce both transcription factor and cell activation. These are thought to be directly responsible for the potent immunomodulatory properties of the B-subunits, and have resulted in their use as adjuvants and as agents to suppress inflammatory immune disorders. The relationship between the signalling properties of the B-subunits and their capacity to act as trafficking vehicles has remained unclear. In an effort to understand the structural requirements for these two functions, a set of mutant B-subunits, with amino acid substitutions at position His-57, have been generated and studied. Importantly, such mutant B-subunits retain an ability to bind with high affinity to GM1 and to traffic into cells, but have entirely lost their capacity to activate immune cell populations. Thus, while binding via GM1 appears to be sufficient to trigger cellular uptake it is not sufficient to activate signal transduction. The His-57 region is therefore speculated to be actively engaged in triggering signalling events, possibly via cognate interaction with other cell surface molecules.

Highly acidic C-terminal domain of pp32 is required for the interaction with histone chaperone, TAF-Ibeta.

PubMed

Lee, In-Seon; Oh, Sang-Min; Kim, Sung-Mi; Lee, Dong-Seok; Seo, Sang-Beom

2006-12-01

We have previously reported that INHAT (inhibitor of acetyltransferases) complex subunits, TAF (template activating factor)-Ialpha, TAF-Ibeta and pp32 can inhibit histone acetylation and HAT (histone acetyltransferase)-dependent transcription by binding to histones. Evidences are accumulating that INHAT complex subunits have important regulatory roles in various cellular activities such as replication, transcription, and apoptosis etc. However, how these subunits interact each other remains largely unknown. Using immunoprecipitation (IP) and protein-protein interaction assays with TAF-Ibeta and pp32 deletion mutant proteins, we identify INHAT complex subunits, TAF-Ibeta and pp32 interaction requires highly acidic C-terminal domain of pp32. We also show that the interaction between the INHAT complex subunits is stronger in the presence of histones. In this study, we report that the synergistic inhibition of HAT-mediated transcription by TAF-Ibeta and pp32 is dependent on the highly acidic C-terminal domain of pp32.

Some assembly required: Contributions of Tom Stevens' lab to the V-ATPase field.

PubMed

Graham, Laurie A; Finnigan, Gregory C; Kane, Patricia M

2018-06-01

Tom Stevens' lab has explored the subunit composition and assembly of the yeast V-ATPase for more than 30 years. Early studies helped establish yeast as the predominant model system for study of V-ATPase proton pumps and led to the discovery of protein splicing of the V-ATPase catalytic subunit. The Vma - phenotype, characteristic of loss-of-V-ATPase activity in yeast was key in determining the enzyme's subunit composition via yeast genetics. V-ATPase subunit composition proved to be highly conserved among eukaryotes. Genetic screens for new vma mutants led to identification of a set of dedicated V-ATPase assembly factors and helped unravel the complex pathways for V-ATPase assembly. In later years, exploration of the evolutionary history of several V-ATPase subunits provided new information about the enzyme's structure and function. This review highlights V-ATPase work in the Stevens' lab between 1987 and 2017. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Purification and properties of the heterogeneous subunits of elongation factor EF-1 from Guerin epithelioma cells.

PubMed

Marcinkiewicz, C; Gajko, A; Gałasiński, W

1991-01-01

Elongation factor EF-1 from Guerin epithelioma was separated into two subunit forms EF-1A and EF-1B by chromatography in the presence of 25% glycerol, successively on CM-Sephadex and DEAE-Sephadex. It was shown that EF-1A is a thermolabile, single polypeptide which catalyses the binding of aminoacyl-tRNA to ribosomes, similarly as eukaryotic EF-1 alpha or prokaryotic EF-Tu. EF-1B was characterized as a complex composed of at least two polypeptides. One of them is EF-1A, the other EF-1C, which stimulates EF-1A activity and protects this elongation factor from thermal inactivation.

The challenges of classical swine fever control: modified live and E2 subunit vaccines.

PubMed

Huang, Yu-Liang; Deng, Ming-Chung; Wang, Fun-In; Huang, Chin-Cheng; Chang, Chia-Yi

2014-01-22

Classical swine fever (CSF) is an economically important, highly contagious disease of swine worldwide. CSF is caused by classical swine fever virus (CSFV), and domestic pigs and wild boars are its only natural hosts. The two main strategies used to control CSF epidemic are systematic prophylactic vaccination and a non-vaccination stamping-out policy. This review compares the protective efficacy of the routinely used modified live vaccine (MLV) and E2 subunit vaccines and summarizes the factors that influence the efficacy of the vaccines and the challenges that both vaccines face to CSF control. Although MLV provide earlier and more complete protection than E2 subunit vaccines, it has the drawback of not allowing differentiation between infected and vaccinated animals (DIVA). The marker vaccine of E2 protein with companion discriminatory test to detect antibodies against E(rns) allows DIVA and is a promising strategy for future control and eradication of CSF. Maternal derived antibody (MDA) is the critical factor in impairing the efficacy of both MLV and E2 subunit vaccines, so the well-designed vaccination programs of sows and piglets should be considered together. Because of the antigen variation among various genotypes of CSFV, antibodies raised by either MLV or subunit vaccine neutralize genotypically homologous strains better than heterologous ones. However, although this is not a major concern for MLV as the induced immune responses can protect pigs against the challenge of various genotypes of CSFVs, it is critical for E2 subunit vaccines. It is thus necessary to evaluate whether the E2 subunit vaccine can completely protect against the current prevalent strains in the field. An ideal new generation of vaccine should be able to maintain the high protective efficiency of MLV and overcome the problem of antigenic variations while allowing for DIVA. Copyright © 2013 Elsevier B.V. All rights reserved.

Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery

PubMed Central

Lanfranco, Maria Fe; Gárate, Fernanda; Engdahl, Ashton J.; Maillard, Rodrigo A.

2017-01-01

Many allosteric proteins form homo-oligomeric complexes to regulate a biological function. In homo-oligomers, subunits establish communication pathways that are modulated by external stimuli like ligand binding. A challenge for dissecting the communication mechanisms in homo-oligomers is identifying intermediate liganded states, which are typically transiently populated. However, their identities provide the most mechanistic information on how ligand-induced signals propagate from bound to empty subunits. Here, we dissected the directionality and magnitude of subunit communication in a reengineered single-chain version of the homodimeric transcription factor cAMP receptor protein. By combining wild-type and mutant subunits in various asymmetric configurations, we revealed a linear relationship between the magnitude of cooperative effects and the number of mutant subunits. We found that a single mutation is sufficient to change the global allosteric behavior of the dimer even when one subunit was wild type. Dimers harboring two mutations with opposite cooperative effects had different allosteric properties depending on the arrangement of the mutations. When the two mutations were placed in the same subunit, the resulting cooperativity was neutral. In contrast, when placed in different subunits, the observed cooperativity was dominated by the mutation with strongest effects over cAMP affinity relative to wild type. These results highlight the distinct roles of intrasubunit interactions and intersubunit communication in allostery. Finally, dimers bound to either one or two cAMP molecules had similar DNA affinities, indicating that both asymmetric and symmetric liganded states activate DNA interactions. These studies have revealed the multiple communication pathways that homo-oligomers employ to transduce signals. PMID:28188293

Isolated juvenile xanthogranuloma in the bone marrow: report of a case and review of the literature.

PubMed

Kesserwan, Chimen; Boué, Daniel R; Kahwash, Samir B

2007-01-01

We report a case of juvenile xanthogranuloma limited to involvement of the bone marrow in a 6-week-old male infant. Evaluation of the bone marrow was a part of the workup for peripheral blood cytopenia. Examination showed hypercellular marrow with paratrabecular clusters of lipidized histiocytes positive for CD68, CD4, and factor XIII(a) and negative for S100 and CD1a. Clinical and radiological workup showed no associated skin lesions or osseous or visceral involvement. The patient was started on chemotherapy with clinical improvement and gradual decreased bone marrow involvement. The child is alive and well at 16 months of age. This case represents, to the best of our knowledge, the 1st documented case of juvenile xanthogranuloma with isolated bone marrow involvement sparing skin and viscera.

Decreased store operated Ca2+ entry in dendritic cells isolated from mice expressing PKB/SGK-resistant GSK3.

PubMed

Schmid, Evi; Yan, Jing; Nurbaeva, Meerim K; Russo, Antonella; Yang, Wenting; Faggio, Caterina; Shumilina, Ekaterina; Lang, Florian

2014-01-01

Dendritic cells (DCs), key players of immunity, are regulated by glycogen synthase kinase GSK3. GSK3 activity is suppressed by PKB/Akt and SGK isoforms, which are in turn stimulated by the PI3K pathway. Exposure to bacterial lipopolysaccharides increases cytosolic Ca(2+)-concentration ([Ca(2+)]i), an effect augmented in DCs isolated from mutant mice expressing PKB/SGK-resistant GSK3α,β (gsk3(KI) ). Factors affecting [Ca(2+)]i include Ca(2+)-release from intracellular stores (CRIS), store-operated Ca(2+)-entry (SOCE) through STIM1/STIM2-regulated Orai1, K(+)-dependent Na(+)/Ca(2+)-exchangers (NCKX), K(+)-independent Na(+)/Ca(2+)-exchangers (NCX) and calbindin-D28k. The present study explored whether PKB/SGK-dependent GSK3α, β-activity impacts on CRIS, SOCE, NCKX, NCX or calbindin. DCs were isolated from gsk3(KI) mice and respective wild-type mice (gsk3(WT) ), [Ca(2+)]i estimated from Fura2 fluorescence, Orai1, STIM1, STIM2 as well as calbindin-D28k protein abundance determined by Western blotting and mRNA levels quantified by real time PCR. As a result, thapsigargin-induced CRIS and SOCE were significantly blunted by GSK3-inhibitors SB216763 (1-10 µM, 30 min) or GSK-XIII (10 µM, 30 min) but were significantly lower in gsk3(WT) than in gsk3(KI) DCs. Orai1, STIM1 and STIM2 protein abundance was significantly lower and calbindin-D28k abundance significantly higher in gsk3(KI) than in gsk3(WT) DCs. Activity of NCKX and NCX was significantly higher in gsk3(KI) than in gsk3(WT) DCs and was significantly increased by SB216763 (1 µM, 30 min) or GSK-XIII (10 µM, 30 min). Treatment of gsk3(WT) DCs with SB216763 (1 µM, 4-24 h) or GSK-XIII (10 µM, 4-24 h) did not significantly modify the protein abundance of Orai1, STIM1 and STIM2. The present observations point to a dual role of GSK3 in the regulation of Ca(2+) in DCs. Acute inhibition of GSK3 blunted the increase of [Ca(2+)]i following CRIS and SOCE and stimulated NCKX/NCX activity. However, expression of PKB/SGK-resistant GSK3α, β downregulated the increase of [Ca(2+)]i following CRIS and SOCE, an effect at least partially due to downregulation of Orai1, STIM1 and STIM2 expression as well as upregulation of Na(+)/Ca(2+)-exchanger activity and calbindin D28k expression.

Decreased Store Operated Ca2+ Entry in Dendritic Cells Isolated from Mice Expressing PKB/SGK-Resistant GSK3

PubMed Central

Nurbaeva, Meerim K.; Russo, Antonella; Yang, Wenting; Faggio, Caterina; Shumilina, Ekaterina; Lang, Florian

2014-01-01

Dendritic cells (DCs), key players of immunity, are regulated by glycogen synthase kinase GSK3. GSK3 activity is suppressed by PKB/Akt and SGK isoforms, which are in turn stimulated by the PI3K pathway. Exposure to bacterial lipopolysaccharides increases cytosolic Ca2+-concentration ([Ca2+]i), an effect augmented in DCs isolated from mutant mice expressing PKB/SGK-resistant GSK3α,β (gsk3KI). Factors affecting [Ca2+]i include Ca2+-release from intracellular stores (CRIS), store-operated Ca2+-entry (SOCE) through STIM1/STIM2-regulated Orai1, K+-dependent Na+/Ca2+-exchangers (NCKX), K+-independent Na+/Ca2+-exchangers (NCX) and calbindin-D28k. The present study explored whether PKB/SGK-dependent GSK3α, β-activity impacts on CRIS, SOCE, NCKX, NCX or calbindin. DCs were isolated from gsk3KI mice and respective wild-type mice (gsk3WT), [Ca2+]i estimated from Fura2 fluorescence, Orai1, STIM1, STIM2 as well as calbindin-D28k protein abundance determined by Western blotting and mRNA levels quantified by real time PCR. As a result, thapsigargin-induced CRIS and SOCE were significantly blunted by GSK3-inhibitors SB216763 (1–10 µM, 30 min) or GSK-XIII (10 µM, 30 min) but were significantly lower in gsk3WT than in gsk3KIDCs. Orai1, STIM1 and STIM2 protein abundance was significantly lower and calbindin-D28k abundance significantly higher in gsk3KI than in gsk3WTDCs. Activity of NCKX and NCX was significantly higher in gsk3KI than in gsk3WTDCs and was significantly increased by SB216763 (1 µM, 30 min) or GSK-XIII (10 µM, 30 min). Treatment of gsk3WT DCs with SB216763 (1 µM, 4–24 h) or GSK-XIII (10 µM, 4–24 h) did not significantly modify the protein abundance of Orai1, STIM1 and STIM2. The present observations point to a dual role of GSK3 in the regulation of Ca2+ in DCs. Acute inhibition of GSK3 blunted the increase of [Ca2+]i following CRIS and SOCE and stimulated NCKX/NCX activity. However, expression of PKB/SGK-resistant GSK3α, β downregulated the increase of [Ca2+]i following CRIS and SOCE, an effect at least partially due to downregulation of Orai1, STIM1 and STIM2 expression as well as upregulation of Na+/Ca2+-exchanger activity and calbindin D28k expression. PMID:24523925

Cell- and subunit-specific mechanisms of CNG channel ciliary trafficking and localization in C. elegans

PubMed Central

Wojtyniak, Martin; Brear, Andrea G.; O'Halloran, Damien M.; Sengupta, Piali

2013-01-01

Summary Primary cilia are ubiquitous sensory organelles that concentrate transmembrane signaling proteins essential for sensing environmental cues. Mislocalization of crucial ciliary signaling proteins, such as the tetrameric cyclic nucleotide-gated (CNG) channels, can lead to cellular dysfunction and disease. Although several cis- and trans-acting factors required for ciliary protein trafficking and localization have been identified, whether these mechanisms act in a protein- and cell-specific manner is largely unknown. Here, we show that CNG channel subunits can be localized to discrete ciliary compartments in individual sensory neurons in C. elegans, suggesting that channel composition is heterogeneous across the cilium. We demonstrate that ciliary localization of CNG channel subunits is interdependent on different channel subunits in specific cells, and identify sequences required for efficient ciliary targeting and localization of the TAX-2 CNGB and TAX-4 CNGA subunits. Using a candidate gene approach, we show that Inversin, transition zone proteins, intraflagellar transport motors and a MYND-domain protein are required to traffic and/or localize CNG channel subunits in both a cell- and channel subunit-specific manner. We further find that TAX-2 and TAX-4 are relatively immobile in specific sensory cilia subcompartments, suggesting that these proteins undergo minimal turnover in these domains in mature cilia. Our results uncover unexpected diversity in the mechanisms that traffic and localize CNG channel subunits to cilia both within and across cell types, highlighting the essential contribution of this process to cellular functions. PMID:23886944

Cell- and subunit-specific mechanisms of CNG channel ciliary trafficking and localization in C. elegans.

PubMed

Wojtyniak, Martin; Brear, Andrea G; O'Halloran, Damien M; Sengupta, Piali

2013-10-01

Primary cilia are ubiquitous sensory organelles that concentrate transmembrane signaling proteins essential for sensing environmental cues. Mislocalization of crucial ciliary signaling proteins, such as the tetrameric cyclic nucleotide-gated (CNG) channels, can lead to cellular dysfunction and disease. Although several cis- and trans-acting factors required for ciliary protein trafficking and localization have been identified, whether these mechanisms act in a protein- and cell-specific manner is largely unknown. Here, we show that CNG channel subunits can be localized to discrete ciliary compartments in individual sensory neurons in C. elegans, suggesting that channel composition is heterogeneous across the cilium. We demonstrate that ciliary localization of CNG channel subunits is interdependent on different channel subunits in specific cells, and identify sequences required for efficient ciliary targeting and localization of the TAX-2 CNGB and TAX-4 CNGA subunits. Using a candidate gene approach, we show that Inversin, transition zone proteins, intraflagellar transport motors and a MYND-domain protein are required to traffic and/or localize CNG channel subunits in both a cell- and channel subunit-specific manner. We further find that TAX-2 and TAX-4 are relatively immobile in specific sensory cilia subcompartments, suggesting that these proteins undergo minimal turnover in these domains in mature cilia. Our results uncover unexpected diversity in the mechanisms that traffic and localize CNG channel subunits to cilia both within and across cell types, highlighting the essential contribution of this process to cellular functions.

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

PubMed Central

McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

1992-01-01

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli.

PubMed

Smith, Corey L; Matheson, Timothy D; Trombly, Daniel J; Sun, Xiaoming; Campeau, Eric; Han, Xuemei; Yates, John R; Kaufman, Paul D

2014-09-15

Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element-containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus. © 2014 Smith et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Quantitative RT-PCR for inhibin/activin subunits: measurements of rat hypothalamic and ovarian inhibin/activin subunit mRNAs during the estrous cycle.

PubMed

Murata, T; Takizawa, T; Funaba, M; Fujimura, H; Murata, E; Takahashi, M; Torii, K

1997-02-01

Inhibins (alpha-beta(A) and alpha-beta(B)) and activins (beta(A)-beta(A), beta(A)-beta(B) and beta(B)-beta(B)) were originally isolated from ovarian follicular fluids as FSH secretion modifiers. Inhibin/activin subunits, alpha, beta(A) and beta(B), are widely distributed in several tissues, including gonads and brain, and inhibins and activins have been reported to be involved in ovarian or hypothalamic functions. In this study, we established and employed a competitive RT-PCR assay system for rat inhibin/activin subunits by capillary electrophoresis to determine rat hypothalamic and ovarian inhibin/activin subunit mRNA levels during the estrous cycle. Linearity of standards for alpha, beta(A), and beta(B) subunit assays were between 0.01-0.3 amol, 0.003-0.09 amol and 0.002-0.02 amol of each fragment DNA as a standard, respectively. Hypothalamic beta(A) subunit mRNA during the estrous morning (1000 h) tended to be increased compared with that of the proestrous evening (1700 h), although they were not significantly different. Ovarian alpha subunit mRNA levels tended to be increased during the proestrous morning (1000 h) and were significantly increased in the proestrous evening (1700 h), compared with diestrus and estrus (P < 0.05). Ovarian beta(A) subunit mRNA was also significantly higher in the proestrous evening, compared with diestrus and estrus (P < 0.05), but in the case of beta(B) subunit mRNA there was no difference among diestrus, proestrus and estrus. We thus established a sensitive competitive RT-PCR system for the measurement of inhibin/activin alpha, beta(A) and beta(B) subunits, and this assay system would be helpful for the study of inhibin/activin action in brain and other tissues where these factors are expressed at low levels.

Waiving a requirement of clause 6(a) of rule XIII with respect to consideration of certain resolutions reported from the Committee on Rules.

THOMAS, 111th Congress

Rep. Cardoza, Dennis A. [D-CA-18

2010-06-30

House - 07/14/2010 Pursuant to the provisions of H. Res. 1509, H. Res. 1496 is laid on the table. (All Actions) Tracker: This bill has the status IntroducedHere are the steps for Status of Legislation:

11 CFR 300.2 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 6, 2002, the receipt of such funds prior to November 6, 2002 shall have no bearing on determining... if we can count on you for $10,000.” (xiii) “Your contribution to this campaign would mean a great... entire Democratic ticket in November.” (vi) A Federal officeholder says: “Our Senator has done a great...

11 CFR 300.2 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 6, 2002, the receipt of such funds prior to November 6, 2002 shall have no bearing on determining... if we can count on you for $10,000.” (xiii) “Your contribution to this campaign would mean a great... entire Democratic ticket in November.” (vi) A Federal officeholder says: “Our Senator has done a great...

11 CFR 300.2 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 6, 2002, the receipt of such funds prior to November 6, 2002 shall have no bearing on determining... if we can count on you for $10,000.” (xiii) “Your contribution to this campaign would mean a great... entire Democratic ticket in November.” (vi) A Federal officeholder says: “Our Senator has done a great...

11 CFR 300.2 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 6, 2002, the receipt of such funds prior to November 6, 2002 shall have no bearing on determining... if we can count on you for $10,000.” (xiii) “Your contribution to this campaign would mean a great... entire Democratic ticket in November.” (vi) A Federal officeholder says: “Our Senator has done a great...

Testing of New Bridge Rail and Transition Designs Vol. XIII Appendix L 32-in (813-mm) Thrie-Beam Transition

DOT National Transportation Integrated Search

1997-06-01

This report presents the results of a State Planning and Research (SP&R) pooled-fund study to develop safer bridge rail and transition designsThis pooled-fund study was sponsored by the Federal Highway Administration, 23 States, and the District of C...

38 CFR 36.4340 - Underwriting standards, processing procedures, lender responsibility, and lender certification.

Code of Federal Regulations, 2013 CFR

2013-07-01

... equity in refinancing loans; (vii) Little or no increase in shelter expense; (viii) Military benefits...; (xii) Tax credits of a continuing nature, such as tax credits for child care; and (xiii) Tax benefits... supplied in the Consumer Expenditure Survey (CES) published by the Department of Labor's Bureau of Labor...

38 CFR 36.4340 - Underwriting standards, processing procedures, lender responsibility, and lender certification.

Code of Federal Regulations, 2014 CFR

2014-07-01

... streamlined refinance loan would not increase the principal balance outstanding on the prior existing... refinancing loans; (vii) Little or no increase in shelter expense; (viii) Military benefits; (ix) Satisfactory... continuing nature, such as tax credits for child care; and (xiii) Tax benefits of home ownership. (6) The...

38 CFR 36.4340 - Underwriting standards, processing procedures, lender responsibility, and lender certification.

Code of Federal Regulations, 2011 CFR

2011-07-01

... equity in refinancing loans; (vii) Little or no increase in shelter expense; (viii) Military benefits...; (xii) Tax credits of a continuing nature, such as tax credits for child care; and (xiii) Tax benefits... supplied in the Consumer Expenditure Survey (CES) published by the Department of Labor's Bureau of Labor...

38 CFR 36.4340 - Underwriting standards, processing procedures, lender responsibility, and lender certification.

Code of Federal Regulations, 2012 CFR

2012-07-01

... equity in refinancing loans; (vii) Little or no increase in shelter expense; (viii) Military benefits...; (xii) Tax credits of a continuing nature, such as tax credits for child care; and (xiii) Tax benefits... supplied in the Consumer Expenditure Survey (CES) published by the Department of Labor's Bureau of Labor...

Distance Learning Plan for the Defense Finance and Accounting Service (DFAS): A Study for the DBMU

DTIC Science & Technology

1994-09-01

according to the standard (H.261) motion video compression algorithm.24 n Schaphorst, Richard, notes presented at TELECON XIII, San Jose , California, 10...include automatic microphone mixing systems with one microphone for every two student seats, a large screen interactive computer display and the Socrates

Bangladesh v. India: A Positive Step Forward in Public Order of the Seas

DTIC Science & Technology

2017-09-05

between India and Bangladesh, “regarding the delimitation of the maritime boundary between them in the territorial sea, the exclusive economic zone...Diplomatic Relations ................................................................................................... 31 Political and Economic ...xiii Glossary CLCS Commission on the Limits of the Continental Shelf EEZ Exclusive Economic Zone ICJ International Court of

The History of Disability Services in Higher Education

ERIC Educational Resources Information Center

Madaus, Joseph W.

2011-01-01

In 2002, Brinckerhoff, McGuire, and Shaw observed that the field of postsecondary education and disability services had "moved through its adolescence and was embarking on adulthood" (xiii). Indeed, the field had undergone rapid expansion nationwide in the prior 30 years and grew into a full-fledged profession within higher education (Jarrow…

Chemical and Photographic Evaluation of Rigid Explosive Transfer Lines.

DTIC Science & Technology

1984-05-01

and Kaplan , L. A., J. Org. Chem., Vol. 31, 1966, p. 857. 4. Dacons, J. C., Adolph, H. G., and Kpmlet, M. J., Heat Resistant Explosives, XIII...Sand Canyon Road % Saugus, CA 91351 Jet Propulsion Laboratory Attn: W. Gin 125-124 1 Rockwell International 4800 Oak Grove Drive Attn: L. Corvin ( MA2

Love, Charity, & Pope Leo XIII: A Leadership Paradigm for Catholic Education

ERIC Educational Resources Information Center

Davis, Henry J.

2015-01-01

The treatment of workers is an ongoing social issue affecting society. No organization is immune to questionable employee practices, including Catholic educational institutions. For Catholic leadership to fully embody its intended justice-based role, it must first be aware of the social teachings put forth by the Roman Catholic Church. In this…

Air Quality Side Event Proposal November 2016 GEO XIII Plenary in St. Petersburg, Russia

EPA Science Inventory

The Group on Earth Observations (GEO), which EPA has participated in since 2003, has put out a call for Side Events for its thirteenth annual international Plenary Meeting which is in St. Petersburg, Russia this year during November, 2016. EPA has put on Side Events on Air Quali...

Military Review. Volume 80, Number 4, July-August 2000

DTIC Science & Technology

2000-08-01

Politics, Book 7, written in 350 B.C., trans. Benjamin Jowett, xiii, 1331 b24b39. 3.Stephen Covey, The 7 Habits of Highly Effective People : Restoring the...daily activities of subordinates. Stephen Covey, The 7 Habits of Highly Effective People : Restoring the Character Ethic, uses the notion of alignment to

75 FR 44144 - Washington: Final Authorization of State Hazardous Waste Management Program Revisions

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-28

....19. 400(3)(c)(xiii)(A) Correction--the 265.300 Subpart word N--Landfills, carbonaceous related... recycling Subpart G exception from related; closure and 264.140, financial 265.140 Subpart responsibility H...) Correction--chang 264.143. e ``resource reclamation units'' to ``recycling units''. 620(4)(d)(iv...

The Mississippi River Campaign 1862-1863: The Impact of Climate and Pathogens on Operational Art at the Port Hudson Siege

DTIC Science & Technology

2017-04-13

48 1 Introduction Sun Tzu , for one, advised commanders to, “camp on hard ground, the army...51 Bibliography ...Society Papers Volume XIII January to December 1885 (Richmond, VA: The Society, 1891), 329; Bell, 31. 248 Bell, 30. 52 Bibliography Army Doctrine

Apollo XIII Spacecraft - Splashdown - South Pacific Ocean

NASA Image and Video Library

1970-04-17

S70-35652 (17 April 1970) --- The Apollo 13 spacecraft heads toward a splashdown in the South Pacific Ocean. The Apollo 13 Command Module splashed down in the South Pacific at 12:07:44 p.m., April 17, 1970. Note the capsule and its parachutes just visible against a gap in the dark clouds.

[Growth of the neurocranium].

PubMed

Hodacová, Z; Skalská, H

1998-01-01

The present study demonstrates the results of evaluation of growth changes of the neurocranium in a set of 98 human skulls of immature individuals aged 6-18/20/ years from the XIII.-XVIII. century. Craniometric values were matched with the corresponding values measured on the skulls of the same age groups from the IX. century and IX.-XII. centuries.

Development of an NPS Middle Ultraviolet Spectrograph (Mustang) Electronic Interface

DTIC Science & Technology

1991-12-01

connecting coaxial shield ................................ 140 xiii Figure 7-12 Encode Command Signal (top) and Video Data Signal (bottom) after connecting...coaxial shield ................................................ 142 Figure 7-13 Data Ready Signal (top) and Video Data Signal (bottom) after...connecting coaxial shield ....................................................... 142 Figure 7-14 Word Clock (top) and Gated Enable Signal Rising Edge (bottom

A novel Sarcocystis neurona genotype XIII is associated with severe encephalitis in an unexpectedly broad range of marine mammals from the northeastern Pacific Ocean.

PubMed

Barbosa, Lorraine; Johnson, Christine K; Lambourn, Dyanna M; Gibson, Amanda K; Haman, Katherine H; Huggins, Jessica L; Sweeny, Amy R; Sundar, Natarajan; Raverty, Stephen A; Grigg, Michael E

2015-08-01

Sarcocystis neurona is an important cause of protozoal encephalitis among marine mammals in the northeastern Pacific Ocean. To characterise the genetic type of S. neurona in this region, samples from 227 stranded marine mammals, most with clinical or pathological evidence of protozoal disease, were tested for the presence of coccidian parasites using a nested PCR assay. The frequency of S. neurona infection was 60% (136/227) among pinnipeds and cetaceans, including seven marine mammal species not previously known to be susceptible to infection by this parasite. Eight S. neurona fetal infections identified this coccidian parasite as capable of being transmitted transplacentally. Thirty-seven S. neurona-positive samples were multilocus sequence genotyped using three genetic markers: SnSAG1-5-6, SnSAG3 and SnSAG4. A novel genotype, referred to as Type XIII within the S. neurona population genetic structure, has emerged recently in the northeastern Pacific Ocean and is significantly associated with an increased severity of protozoal encephalitis and mortality among multiple stranded marine mammal species. Published by Elsevier Ltd.

Energy levels, lifetimes, and transition rates for the selenium isoelectronic sequence Pd XIII-Te XIX, Xe XXI-Nd XXVII, W XLI

NASA Astrophysics Data System (ADS)

Wang, K.; Yang, X.; Chen, Z. B.; Si, R.; Chen, C. Y.; Yan, J.; Zhao, X. H.; Dang, W.

2017-09-01

Energy levels, wavelengths, lifetimes, oscillator strengths, and electric dipole (E1), magnetic dipole (M1), electric quadrupole (E2), magnetic quadrupole (M2) transition rates among the 46 fine structure levels belonging to the ([ Ar ] 3d10) 4s2 4p4, ([ Ar ] 3d10) 4s2 4p3 4 d, and ([ Ar ] 3d10) 4 s 4p5 configurations for the selenium isoelectronic sequence Pd XIII-Te XIX, Xe XXI-Nd XXVII, W XLI are reported. These data are determined in the multi-configuration Dirac-Fock (MCDF) approach, in which relativistic effects, main electron correlations within the n = 7 complex, Breit interaction (BI), and quantum electrodynamic (QED) corrections are included. The many-body perturbation theory (MBPT) method is also employed as an independent calculation to confirm the present accuracy, taking W XLI as an example. Comparisons and analysis are made between the present results and available experimental and theoretical ones, and good agreements are obtained. These accurate data are expected to be useful in nuclear fusion research and astrophysical applications.

Intrinsic thermodynamics of ethoxzolamide inhibitor binding to human carbonic anhydrase XIII

PubMed Central

2012-01-01

Background Human carbonic anhydrases (CAs) play crucial role in various physiological processes including carbon dioxide and hydrocarbon transport, acid homeostasis, biosynthetic reactions, and various pathological processes, especially tumor progression. Therefore, CAs are interesting targets for pharmaceutical research. The structure-activity relationships (SAR) of designed inhibitors require detailed thermodynamic and structural characterization of the binding reaction. Unfortunately, most publications list only the observed thermodynamic parameters that are significantly different from the intrinsic parameters. However, only intrinsic parameters could be used in the rational design and SAR of the novel compounds. Results Intrinsic binding parameters for several inhibitors, including ethoxzolamide, trifluoromethanesulfonamide, and acetazolamide, binding to recombinant human CA XIII isozyme were determined. The parameters were the intrinsic Gibbs free energy, enthalpy, entropy, and the heat capacity. They were determined by titration calorimetry and thermal shift assay in a wide pH and temperature range to dissect all linked protonation reaction contributions. Conclusions Precise determination of the inhibitor binding thermodynamics enabled correct intrinsic affinity and enthalpy ranking of the compounds and provided the means for SAR analysis of other rationally designed CA inhibitors. PMID:22676044

A novel Sarcocystis neurona genotype XIII is associated with severe encephalitis in an unexpectedly broad range of marine mammals from the northeastern Pacific Ocean

PubMed Central

Barbosa, Lorraine; Johnson, Christine K.; Lambourn, Dyanna M.; Gibson, Amanda K.; Haman, Katherine H.; Huggins, Jessica L.; Sweeny, Amy R.; Sundar, Natarajan; Raverty, Stephen A.; Grigg, Michael E.

2015-01-01

Sarcocystis neurona is an important cause of protozoal encephalitis among marine mammals in the northeastern Pacific Ocean. To characterize the genetic type of S. neurona in this region, samples from 227 stranded marine mammals, most with clinical or pathological evidence of protozoal disease, were tested for the presence of coccidian parasites using a nested PCR assay. The frequency of S. neurona infection was 60% (136/227) among pinnipeds and cetaceans, including seven marine mammal species not previously known to be susceptible to infection by this parasite. Eight S. neurona fetal infections identified this coccidian parasite as capable of being transmitted transplacentally. Thirty-seven S. neurona-positive samples were multilocus sequence genotyped using three genetic markers: SnSAG1-5-6, SnSAG3 and SnSAG4. A novel genotype, referred to as Type XIII within the S. neurona population genetic structure, has emerged recently in the northeastern Pacific Ocean and is significantly associated with an increased severity of protozoal encephalitis and mortality among multiple stranded marine mammal species. PMID:25997588

The Cryogenic Near Infrared Spectropolarimeter for the Daniel K. Inouye Solar Telescope

NASA Astrophysics Data System (ADS)

Fehlmann, Andre; Giebink, Cindy; Kuhn, Jeffrey Richard; Mickey, D. L.; Scholl, Isabelle

2017-08-01

The Cryogenic Near Infrared Spectropolarimeter is one of the first light instruments for the Daniel K. Inouye Solar Telescope. This dual-beam instrument, which is currently characterized at the University of Hawaii’s Institute for Astronomy, is designed to sensitively measure the solar spectrum at wavelengths from 1 to 5 μm. The high dynamic range of the spectrograph and its context imager will provide sensitive data of the solar disk in the CO bands; unique observations of the low corona and unprecedented measurements of the coronal magnetic field. Observations near the limb and in the corona will greatly benefit from DKIST’s limb occulting system. The initial suite of filters includes selecting filters for the spectrograph at He I / Fe XIII 1080 nm, Si X 1430 nm, Si IX 3934 nm and CO 4651 nm as well as narrow band filters for the context imager at Fe XIII 1074.7 nm, He I 1083.0, Si X 1430.0 nm and J band 1250 nm. In this paper we will present an update on the ongoing instrument characterization and CryoNIRSP’s capabilities.

Regulation of Na(+)/K(+)-ATPase by nuclear respiratory factor 1: implication in the tight coupling of neuronal activity, energy generation, and energy consumption.

PubMed

Johar, Kaid; Priya, Anusha; Wong-Riley, Margaret T T

2012-11-23

NRF-1 regulates mediators of neuronal activity and energy generation. NRF-1 transcriptionally regulates Na(+)/K(+)-ATPase subunits α1 and β1. NRF-1 functionally regulates mediators of energy consumption in neurons. NRF-1 mediates the tight coupling of neuronal activity, energy generation, and energy consumption at the molecular level. Energy generation and energy consumption are tightly coupled to neuronal activity at the cellular level. Na(+)/K(+)-ATPase, a major energy-consuming enzyme, is well expressed in neurons rich in cytochrome c oxidase, an important enzyme of the energy-generating machinery, and glutamatergic receptors that are mediators of neuronal activity. The present study sought to test our hypothesis that the coupling extends to the molecular level, whereby Na(+)/K(+)-ATPase subunits are regulated by the same transcription factor, nuclear respiratory factor 1 (NRF-1), found recently by our laboratory to regulate all cytochrome c oxidase subunit genes and some NMDA and AMPA receptor subunit genes. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation, promoter mutational analysis, and real-time quantitative PCR, NRF-1 was found to functionally bind to the promoters of Atp1a1 and Atp1b1 genes but not of the Atp1a3 gene in neurons. The transcripts of Atp1a1 and Atp1b1 subunit genes were up-regulated by KCl and down-regulated by tetrodotoxin. Atp1b1 is positively regulated by NRF-1, and silencing of NRF-1 with small interference RNA blocked the up-regulation of Atp1b1 induced by KCl, whereas overexpression of NRF-1 rescued these transcripts from being suppressed by tetrodotoxin. On the other hand, Atp1a1 is negatively regulated by NRF-1. The binding sites of NRF-1 on Atp1a1 and Atp1b1 are conserved among mice, rats, and humans. Thus, NRF-1 regulates key Na(+)/K(+)-ATPase subunits and plays an important role in mediating the tight coupling between energy consumption, energy generation, and neuronal activity at the molecular level.

Deregulation of DNA-dependent protein kinase catalytic subunit contributes to human hepatocarcinogenesis development and has a putative prognostic value.

PubMed

Evert, M; Frau, M; Tomasi, M L; Latte, G; Simile, M M; Seddaiu, M A; Zimmermann, A; Ladu, S; Staniscia, T; Brozzetti, S; Solinas, G; Dombrowski, F; Feo, F; Pascale, R M; Calvisi, D F

2013-11-12

The DNA-repair gene DNA-dependent kinase catalytic subunit (DNA-PKcs) favours or inhibits carcinogenesis, depending on the cancer type. Its role in human hepatocellular carcinoma (HCC) is unknown. DNA-dependent protein kinase catalytic subunit, H2A histone family member X (H2AFX) and heat shock transcription factor-1 (HSF1) levels were assessed by immunohistochemistry and/or immunoblotting and qRT-PCR in a collection of human HCC. Rates of proliferation, apoptosis, microvessel density and genomic instability were also determined. Heat shock factor-1 cDNA or DNA-PKcs-specific siRNA were used to explore the role of both genes in HCC. Activator protein 1 (AP-1) binding to DNA-PKcs promoter was evaluated by chromatin immunoprecipitation. Kaplan-Meier curves and multivariate Cox model were used to study the impact on clinical outcome. Total and phosphorylated DNA-PKcs and H2AFX were upregulated in HCC. Activated DNA-PKcs positively correlated with HCC proliferation, genomic instability and microvessel density, and negatively with apoptosis and patient's survival. Proliferation decline and massive apoptosis followed DNA-PKcs silencing in HCC cell lines. Total and phosphorylated HSF1 protein, mRNA and activity were upregulated in HCC. Mechanistically, we demonstrated that HSF1 induces DNA-PKcs upregulation through the activation of the MAPK/JNK/AP-1 axis. DNA-dependent protein kinase catalytic subunit transduces HSF1 effects in HCC cells, and might represent a novel target and prognostic factor in human HCC.

The XPB subunit of repair/transcription factor TFIIH directly interacts with SUG1, a subunit of the 26S proteasome and putative transcription factor.

PubMed

Weeda, G; Rossignol, M; Fraser, R A; Winkler, G S; Vermeulen, W; van 't Veer, L J; Ma, L; Hoeijmakers, J H; Egly, J M

1997-06-15

Mutations in the basal transcription initiation/DNA repair factor TFIIH are responsible for three human disorders: xeroderma pigmentosum (XP), cockayne syndrome (CS) and trichothiodystrophy (TTD). The non-repair features of CS and TTD are thought to be due to a partial inactivation of the transcription function of the complex. To search for proteins whose interaction with TFIIH subunits is disturbed by mutations in patients we used the yeast two-hybrid system and report the isolation of a novel XPB interacting protein, SUG1. The interaction was validated in vivo and in vitro in the following manner. (i) SUG1 interacts with XPB but not with the other core TFIIH subunits in the two-hybrid assay. (ii) Physical interaction is observed in a baculovirus co-expression system. (iii) In fibroblasts under non-overexpression conditions a portion of SUG1 is bound to the TFIIH holocomplex as deduced from co-purification, immunopurification and nickel-chelate affinity chromatography using functional tagged TFIIH. Furthermore, overexpression of SUG1 in normal fibroblasts induced arrest of transcription and a chromatin collapse in vivo. Interestingly, the interaction was diminished with a mutant form of XPB, thus providing a potential link with the clinical features of XP-B patients. Since SUG1 is an integral component of the 26S proteasome and may be part of the mediator, our findings disclose a SUG1-dependent link between TFIIH and the cellular machinery involved in protein modelling/degradation.

Translation Initiation Factor eIF4B Interacts with a Picornavirus Internal Ribosome Entry Site in both 48S and 80S Initiation Complexes Independently of Initiator AUG Location

PubMed Central

Ochs, Kerstin; Rust, René C.; Niepmann, Michael

1999-01-01

Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation. PMID:10438840

The Evolution of COP9 Signalosome in Unicellular and Multicellular Organisms.

PubMed

Barth, Emanuel; Hübler, Ron; Baniahmad, Aria; Marz, Manja

2016-05-02

The COP9 signalosome (CSN) is a highly conserved protein complex, recently being crystallized for human. In mammals and plants the COP9 complex consists of nine subunits, CSN 1-8 and CSNAP. The CSN regulates the activity of culling ring E3 ubiquitin and plays central roles in pleiotropy, cell cycle, and defense of pathogens. Despite the interesting and essential functions, a thorough analysis of the CSN subunits in evolutionary comparative perspective is missing. Here we compared 61 eukaryotic genomes including plants, animals, and yeasts genomes and show that the most conserved subunits of eukaryotes among the nine subunits are CSN2 and CSN5. This may indicate a strong evolutionary selection for these two subunits. Despite the strong conservation of the protein sequence, the genomic structures of the intron/exon boundaries indicate no conservation at genomic level. This suggests that the gene structure is exposed to a much less selection compared with the protein sequence. We also show the conservation of important active domains, such as PCI (proteasome lid-CSN-initiation factor) and MPN (MPR1/PAD1 amino-terminal). We identified novel exons and alternative splicing variants for all CSN subunits. This indicates another level of complexity of the CSN. Notably, most COP9-subunits were identified in all multicellular and unicellular eukaryotic organisms analyzed, but not in prokaryotes or archaeas. Thus, genes encoding CSN subunits present in all analyzed eukaryotes indicate the invention of the signalosome at the root of eukaryotes. The identification of alternative splice variants indicates possible "mini-complexes" or COP9 complexes with independent subunits containing potentially novel and not yet identified functions. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The Evolution of COP9 Signalosome in Unicellular and Multicellular Organisms

PubMed Central

Barth, Emanuel; Hübler, Ron; Baniahmad, Aria; Marz, Manja

2016-01-01

The COP9 signalosome (CSN) is a highly conserved protein complex, recently being crystallized for human. In mammals and plants the COP9 complex consists of nine subunits, CSN 1–8 and CSNAP. The CSN regulates the activity of culling ring E3 ubiquitin and plays central roles in pleiotropy, cell cycle, and defense of pathogens. Despite the interesting and essential functions, a thorough analysis of the CSN subunits in evolutionary comparative perspective is missing. Here we compared 61 eukaryotic genomes including plants, animals, and yeasts genomes and show that the most conserved subunits of eukaryotes among the nine subunits are CSN2 and CSN5. This may indicate a strong evolutionary selection for these two subunits. Despite the strong conservation of the protein sequence, the genomic structures of the intron/exon boundaries indicate no conservation at genomic level. This suggests that the gene structure is exposed to a much less selection compared with the protein sequence. We also show the conservation of important active domains, such as PCI (proteasome lid-CSN-initiation factor) and MPN (MPR1/PAD1 amino-terminal). We identified novel exons and alternative splicing variants for all CSN subunits. This indicates another level of complexity of the CSN. Notably, most COP9-subunits were identified in all multicellular and unicellular eukaryotic organisms analyzed, but not in prokaryotes or archaeas. Thus, genes encoding CSN subunits present in all analyzed eukaryotes indicate the invention of the signalosome at the root of eukaryotes. The identification of alternative splice variants indicates possible “mini-complexes” or COP9 complexes with independent subunits containing potentially novel and not yet identified functions. PMID:27044515

Role of Mex67-Mtr2 in the Nuclear Export of 40S Pre-Ribosomes

PubMed Central

Occhipinti, Laura; Kemmler, Stefan; Panse, Vikram G.

2012-01-01

Nuclear export of mRNAs and pre-ribosomal subunits (pre40S and pre60S) is fundamental to all eukaryotes. While genetic approaches in budding yeast have identified bona fide export factors for mRNAs and pre60S subunits, little is known regarding nuclear export of pre40S subunits. The yeast heterodimeric transport receptor Mex67-Mtr2 (TAP-p15 in humans) binds mRNAs and pre60S subunits in the nucleus and facilitates their passage through the nuclear pore complex (NPC) into the cytoplasm by interacting with Phe-Gly (FG)-rich nucleoporins that line its transport channel. By exploiting a combination of genetic, cell-biological, and biochemical approaches, we uncovered an unanticipated role of Mex67-Mtr2 in the nuclear export of 40S pre-ribosomes. We show that recruitment of Mex67-Mtr2 to pre40S subunits requires loops emanating from its NTF2-like domains and that the C-terminal FG-rich nucleoporin interacting UBA-like domain within Mex67 contributes to the transport of pre40S subunits to the cytoplasm. Remarkably, the same loops also recruit Mex67-Mtr2 to pre60S subunits and to the Nup84 complex, the respective interactions crucial for nuclear export of pre60S subunits and mRNAs. Thus Mex67-Mtr2 is a unique transport receptor that employs a common interaction surface to participate in the nuclear export of both pre-ribosomal subunits and mRNAs. Mex67-Mtr2 could engage a regulatory crosstalk among the three major export pathways for optimal cellular growth and proliferation. PMID:22956913

Tabulation of comet observations.

NASA Astrophysics Data System (ADS)

1993-01-01

Concerning comets: 1973 XII Kohoutek, 1975 IX Kobayashi-Berger-Milon, 1976 VI West, 1976 XI P/d'Arrest, 1977 XIV Kohler, 1979 X Bradfield, 1980 X P/Stephan-Oterma, 1980 XV Bradfield, 1981 II Panther, 1982 VI Austin, 1983 V Sugano-Saigusa-Fujikawa, 1983 VII IRAS-Araki-Alcock, 1983 XIII P/Kopff, 1984 XIII Austin, 1984 XXIII Levy-Rudenko, 1985 XIII P/Giacobini-Zinner, 1985 XVII Hartley-Good, 1985 XIX Thiele, 1986 I P/Boethin, 1986 III P/Halley, 1986 XVIII Terasako, 1987 II Sorrells, 1987 III Nishikawa-Takamizawa-Tago, 1987 X P/Grigg-Skjellerup, 1987 XXIII Rudenko, 1987 XXIX Bradfield, 1987 XXXII McNaught, 1987 XXXIII P/Borrelly, 1988 IV Furuyama, 1988 V Liller, 1988 XIV P/Tempel 2, 1988 XV Machholz, 1988 XX Yanaka, 1988 XXIV Yanaka, 1989 X P/Brorsen-Metcalf, 1989 XV P/Schwassmann-Wachmann 1, 1989 XIX Okazaki-Levy-Rudenko, 1989 XXI Helin-Roman-Alu, 1989 XXII Aarseth-Brewington, 1990 III Černis-Kiuchi-Nakamura, 1990 VI Skorichenko-George, 1990 VIII P/Schwassmann-Wachmann 3, 1990 IX P/Peters-Hartley, 1990 X P/Wild 4, 1990 XIV P/Honda Mrkos-Pajdušáková, 1990 XVII Tsuchiya-Kiuchi, 1990 XXI P/Encke, 1990 XXVI Arai, 1991 XI P/Levy, 1991 XV P/Hartley 2, 1991 XVI P/Wirtanen, 1991 XVII P/Arend-Rigaux, 1991 XXI P/Faye, 1991 XXIII P/Shoemaker 1, 1991 XXIV Shoemaker-Levy, 1991l Helin-Lawrence, 1991ο P/Chernykh, 1991r Helin-Alu, 1991a1 Shoemaker-Levy, 1991g1 Zanotta-Brewington, 1991h1 Mueller, 1912d Tanaka-Machholz, 1992f P/Shoemaker-Levy 8, 1992k Machholz, 1992l P/Giclas, 1992p P/Brewington, 1992q Helin-Lawrence, 1992s P/Ciffréo, 1992t P/Swift-Tuttle, 1992u P/Väisälä, 1992x P/Schaumasse, 1992y Shoemaker, 1992a1 Ohshita, 1993a Mueller, P/Smirnova-Chernykh.

The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans.

PubMed

Grants, Jennifer M; Ying, Lisa T L; Yoda, Akinori; You, Charlotte C; Okano, Hideyuki; Sawa, Hitoshi; Taubert, Stefan

2016-02-01

Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals. Copyright © 2016 by the Genetics Society of America.

Expression of the pituitary transcription factor Ptx-1, but not that of the trans-activating factor prop-1, is reduced in human corticotroph adenomas and is associated with decreased alpha-subunit secretion.

PubMed

Skelly, R H; Korbonits, M; Grossman, A; Besser, G M; Monson, J P; Geddes, J F; Burrin, J M

2000-07-01

We have studied the expression of the pituitary transcription factors Ptx-1 and Prop-1 in a series of 34 pituitary adenomas fully characterized for in vitro hormone secretion and histological staining. In studies involving mammalian cell lines, the pituitary transcription factor Ptx-1 has been shown to be a pituitary hormone panactivator, whereas more recent studies have shown that it plays an important role in alpha-subunit gene expression. Its expression has not been examined previously in human pituitary adenomas characterized by in vitro hormone secretory profiles. Of the 34 pituitary adenomas studied, Ptx-1 expression was reduced by more than 50% compared to that of the housekeeping gene human glyceraldehyde-3-phosphate dehydrogenase in the 6 corticotroph adenomas, which also had significantly reduced alpha-subunit production (all 6 tumors secreting < or =0.5 ng/24 h). Mutations of the pituitary transcription factor Prop-1, which is responsible for the syndrome of Ames dwarfism in mice, are being increasingly recognized as a cause of combined pituitary hormone deficiency in humans, although ACTH deficiency has been described only once. Prop-1 expression was detected in all 34 pituitary adenomas, including 6 corticotroph adenomas and 5 gonadotroph adenomas. The expression of Prop-1 has not been described previously in these cell phenotypes.

Cloning of murine RNA polymerase I-specific TAF factors: Conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1

PubMed Central

Heix, Jutta; Zomerdijk, Joost C. B. M.; Ravanpay, Ali; Tjian, Robert; Grummt, Ingrid

1997-01-01

Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs). Unlike class II- and III-specific TBP–TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter. As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAFIs) and compared the amino acid sequences of the murine TAFIs with their human counterparts. The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAFIs, demonstrating that differences in the primary structure of human and mouse TAFIs do not dramatically alter the network of protein–protein contacts responsible for assembly of the multimeric complex. Thus, primate vs. rodent promoter selectivity mediated by the TBP–TAFI complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription. PMID:9050847

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parvinen, M.; Soeder, O.M.; Mali, P.

Levels of rat testicular interleukin-1-like factor (tIL-1) have been shown to correlate with DNA synthetic activity during the cycle of the rat seminiferous epithelium, suggesting its role as a spermatogonial or meiotic growth factor. To explore this further, a new in vitro model system was developed. Rat seminiferous tubule segments from stages I, V, VIIa, and VIII-IX of the cycle were isolated by transillumination-assisted microdissection, cultured in chemically defined serum-free medium supplemented with human recombinant IL-1 {alpha}, and labeled with (3H)thymidine. During incubation, spontaneous progression of spermatogenesis was noted. Inactive stage VIIa tubule segments differentiated to stage VIII and initiatedmore » DNA synthesis, and concomitantly started to secrete IL-1-like factor. DNA synthesis of stages VIII-IX ceased through differentiation of spermatocytes to leptotene-zygotene (stages XII-XIII of the cycle). IL-1 {alpha} stimulated DNA synthesis significantly in spermatogonia of stage I. Meiotic DNA synthesis at stage VIIa was stimulated (48 h/34 C) and maintained at stages VIII-IX (48 h/34 C). IL-1 {alpha} seems to act as a regulator of spermatogenic DNA synthesis in both mitotic and meiotic phases. It has mainly stimulating and maintaining effects, but it may also be inhibitory under certain conditions.« less

Alternatives to allogeneic platelet transfusion.

PubMed

Desborough, Michael J R; Smethurst, Peter A; Estcourt, Lise J; Stanworth, Simon J

2016-11-01

Allogeneic platelet transfusions are widely used for the prevention and treatment of bleeding in thrombocytopenia. Recent evidence suggests platelet transfusions have limited efficacy and are associated with uncertain immunomodulatory risks and concerns about viral or bacterial transmission. Alternatives to transfusion are a well-recognised tenet of Patient Blood Management, but there has been less focus on different strategies to reduce bleeding risk by comparison to platelet transfusion. Direct alternatives to platelet transfusion include agents to stimulate endogenous platelet production (thrombopoietin mimetics), optimising platelet adhesion to endothelium by treating anaemia or increasing von Willebrand factor levels (desmopressin), increasing formation of cross-linked fibrinogen (activated recombinant factor VII, fibrinogen concentrate or recombinant factor XIII), decreasing fibrinolysis (tranexamic acid or epsilon aminocaproic acid) or using artificial or modified platelets (cryopreserved platelets, lyophilised platelets, haemostatic particles, liposomes, engineered nanoparticles or infusible platelet membranes). The evidence base to support the use of these alternatives is variable, but an area of active research. Much of the current randomised controlled trial focus is on evaluation of the use of thrombopoietin mimetics and anti-fibrinolytics. It is also recognised that one alternative strategy to platelet transfusion is choosing not to transfuse at all. © 2016 John Wiley & Sons Ltd.

A functional network involved in the recycling of nucleocytoplasmic pre-60S factors

PubMed Central

Lebreton, Alice; Saveanu, Cosmin; Decourty, Laurence; Rain, Jean-Christophe; Jacquier, Alain; Fromont-Racine, Micheline

2006-01-01

Eukaryotic pre-ribosomes go through cytoplasmic maturation steps before entering translation. The nucleocytoplasmic proteins participating in these late stages of maturation are reimported to the nucleus. In this study, we describe a functional network focused on Rei1/Ybr267w, a strictly cytoplasmic pre-60S factor indirectly involved in nuclear 27S pre-ribosomal RNA processing. In the absence of Rei1, the nuclear import of at least three other pre-60S factors is impaired. The accumulation in the cytoplasm of a small complex formed by the association of Arx1 with a novel factor, Alb1/Yjl122w, inhibits the release of the putative antiassociation factor Tif6 from the premature large ribosomal subunits and its recycling to the nucleus. We propose a model in which Rei1 is a key factor for the coordinated dissociation and recycling of the last pre-60S factors before newly synthesized large ribosomal subunits enter translation. PMID:16651379

Identification of a domain within human TAF(I)48, a subunit of Selectivity Factor 1, that interacts with helix 2 of TBP.

PubMed

Xu, Shuping; Hori, Roderick T

2004-09-01

RNA polymerase I transcription in human cells requires Selectivity Factor 1, a multisubunit complex composed of the TATA-box-binding protein (TBP) and three TBP-associated factors (TAFs) called TAF(I)48, TAF(I)63 and TAF(I)110. Each of the Selectivity Factor 1 subunits binds directly to the other three components, but these interactions have not been characterized. This study is the initial identification and analysis of a TBP-binding domain within a Selectivity Factor 1 TAF. The interaction between human TBP and human TAF(I)48 was initially examined using the yeast two-hybrid assay, and a TBP-binding domain was identified in the carboxyl-terminus of human (h)TAF(I)48. Consistent with this result, the hTAF(I)48 carboxyl-terminus was able to bind directly to TBP in protein-protein interaction assays. When mutations were introduced into the hTAF(I)48 carboxyl-terminus, we identified changes in uncharged and positive residues that affect its interaction with TBP. By examining TBP mutants, residues within and adjacent to helix 2 of TBP, previously demonstrated to interact with subunits of other TBP-containing complexes [Transcription Factor IID (TFIID) and TFIIIB] were also found to diminish its affinity for the carboxyl-terminus of hTAF(I)48. The regions of hTAF(I)48 and TBP that interact are compared to those identified within other complexes containing TBP.

Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation

PubMed Central

Chakraborty, Anirban; Mazumder, Abhishek; Lin, Miaoxin; Hasemeyer, Adam; Xu, Qumiao; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

2015-01-01

Summary A three-step procedure comprising (i) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (ii) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (iii) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a crosslinking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP. PMID:25665560

Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

PubMed Central

Baldauf, Keegan J.; Royal, Joshua M.; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

2015-01-01

Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction. PMID:25802972

Functional Role of N- and C-Terminal Amino Acids in the Structural Subunits of Colonization Factor CS6 Expressed by Enterotoxigenic Escherichia coli

PubMed Central

Debnath, Anusuya; Sabui, Subrata; Wajima, Takeaki; Hamabata, Takashi; Banerjee, Rajat

2016-01-01

ABSTRACT CS6 is a common colonization factor expressed by enterotoxigenic Escherichia coli. It is a two-subunit protein consisting of CssA and CssB in an equal stoichiometry, assembled via the chaperone-usher pathway into an afimbrial, oligomeric assembly on the bacterial cell surface. A recent structural study has predicted the involvement of the N- and C-terminal regions of the CS6 subunits in its assembly. Here, we identified the functionally important residues in the N- and C-terminal regions of the CssA and CssB subunits during CS6 assembly by alanine scanning mutagenesis. Bacteria expressing mutant proteins were tested for binding with Caco-2 cells, and the results were analyzed with respect to the surface expression of mutant CS6. In this assay, many mutant proteins were not expressed on the surface while some showed reduced expression. It appeared that some, but not all, of the residues in both the N and C termini of CssA and CssB played an important role in the intermolecular interactions between these two structural subunits, as well as chaperone protein CssC. Our results demonstrated that T20, K25, F27, S36, Y143, and V147 were important for the stability of CssA, probably through interaction of CssC. We also found that I22, V29, and I33 of CssA and G154, Y156, L160, V162, F164, and Y165 of CssB were responsible for CssA-CssB intermolecular interactions. In addition, some of the hydrophobic residues in the C terminus of CssA and the N terminus of CssB were involved in the stabilization of higher-order complex formation. Overall, the results presented here might help in understanding the pathway used to assemble CS6 and predict its structure. IMPORTANCE Unlike most other colonization factors, CS6 is nonfimbrial, and in a sense, its subunit composition and assembly are also unique. Here we report that both the N- and C-terminal amino acid residues of CssA and CssB play a critical role in the intermolecular interactions between them and assembly proteins. We found mainly that alternate hydrophobic residues present in these motifs are essential for the interaction between the structural subunits, as well as the chaperone and usher assembly proteins. Our results indicate the involvement of the side chains of identified amino acids in CS6 assembly. This study adds a step toward understanding the interactions between structural subunits of CS6 and assembly proteins during CS6 biogenesis. PMID:26929298

The function of the Mediator complex in plant immunity.

PubMed

An, Chuanfu; Mou, Zhonglin

2013-03-01

Upon pathogen infection, plants undergo dramatic transcriptome reprogramming to shift from normal growth and development to immune response. During this rapid process, the multiprotein Mediator complex has been recognized as an important player to fine-tune gene-specific and pathway-specific transcriptional reprogramming by acting as an adaptor/coregulator between sequence-specific transcription factor and RNA polymerase II (RNAPII). Here, we review current understanding of the role of five functionally characterized Mediator subunits (MED8, MED15, MED16, MED21 and MED25) in plant immunity. All these Mediator subunits positively regulate resistance against leaf-infecting biotrophic bacteria or necrotrophic fungi. While MED21 appears to regulate defense against fungal pathogens via relaying signals from upstream regulators and chromatin modification to RNAPII, the other four Mediator subunits locate at different positions of the defense network to convey phytohormone signal(s). Fully understanding the role of Mediator in plant immunity needs to characterize more Mediator subunits in both Arabidopsis and other plant species. Identification of interacting proteins of Mediator subunits will further help to reveal their specific regulatory mechanisms in plant immunity.

Large subunit of the ribonucleotide reductase gene is a virulent factor and plays a critical role in Marek's disease virus pathogenesis

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) encodes a ribonucleotide reductase (RR) gene consisting of two subunits UL39 (RR1) and UL40 (RR2). Both RR1 and RR2 form an active holoenzyme and are necessary for enzyme activity. This gene was indentified by monoclonal antibody T81 in a gt11 MDV expression library and f...

Ribulose-1,5-bis-phosphate carboxylase/oxygenase accumulation factor1 is required for holoenzyme assembly in maize.

PubMed

Feiz, Leila; Williams-Carrier, Rosalind; Wostrikoff, Katia; Belcher, Susan; Barkan, Alice; Stern, David B

2012-08-01

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process.

The cryo-electron microscopy structure of human transcription factor IIH

DOE PAGES

Greber, Basil J.; Nguyen, Thi Hoang Duong; Fang, Jie; ...

2017-09-13

We report human transcription factor IIH (TFIIH) is part of the general transcriptional machinery required by RNA polymerase II for the initiation of eukaryotic gene transcription. Composed of ten subunits that add up to a molecular mass of about 500 kDa, TFIIH is also essential for nucleotide excision repair. The seven-subunit TFIIH core complex formed by XPB, XPD, p62, p52, p44, p34, and p8 is competent for DNA repair, while the CDK-activating kinase subcomplex, which includes the kinase activity of CDK7 as well as the cyclin H and MAT1 subunits, is additionally required for transcription initiation. Mutations in the TFIIHmore » subunits XPB, XPD, and p8 lead to severe premature ageing and cancer propensity in the genetic diseases xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy, highlighting the importance of TFIIH for cellular physiology. Here we present the cryo-electron microscopy structure of human TFIIH at 4.4 Å resolution. The structure reveals the molecular architecture of the TFIIH core complex, the detailed structures of its constituent XPB and XPD ATPases, and how the core and kinase subcomplexes of TFIIH are connected. Also, our structure provides insight into the conformational dynamics of TFIIH and the regulation of its activity.« less

Regulation of Memory T Cells by Interleukin-23.

PubMed

Li, Yanchun; Wang, Hongbo; Lu, Honghua; Hua, Shucheng

2016-01-01

Interleukin-23 (IL-23), a member of the IL-12 family of cytokines, is a heterodimeric cytokine. It is composed of subunits p40 (shared with IL-12) and p19 (an IL-12 p35-related subunit) and is secreted by several types of immune cells, such as natural killer cells and dendritic cells. The IL-23 receptor is composed of the subunit IL-12Rβ1 and the IL-23-specific subunit IL-23R. The binding of IL-23 to its specific cell surface receptor regulates a number of functions, including proliferation and differentiation of cells and secretion of cell factors. Memory T cells are a subset of T cells that secrete numerous important cell factors, and they function in the immune response to infection and diseases like cancer, autoimmune disease and bronchial asthma. IL-23R is expressed on the surface of memory T cells, which suggests that it can specifically regulate memory T cell function. IL-23 has been widely used as a clinical indicator in immune-related diseases and shows potential for use in disease treatment. Here we review the current progress in the study of the role of IL-23 in the regulation of memory T cells. © 2016 S. Karger AG, Basel.

The cryo-electron microscopy structure of human transcription factor IIH

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greber, Basil J.; Nguyen, Thi Hoang Duong; Fang, Jie

We report human transcription factor IIH (TFIIH) is part of the general transcriptional machinery required by RNA polymerase II for the initiation of eukaryotic gene transcription. Composed of ten subunits that add up to a molecular mass of about 500 kDa, TFIIH is also essential for nucleotide excision repair. The seven-subunit TFIIH core complex formed by XPB, XPD, p62, p52, p44, p34, and p8 is competent for DNA repair, while the CDK-activating kinase subcomplex, which includes the kinase activity of CDK7 as well as the cyclin H and MAT1 subunits, is additionally required for transcription initiation. Mutations in the TFIIHmore » subunits XPB, XPD, and p8 lead to severe premature ageing and cancer propensity in the genetic diseases xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy, highlighting the importance of TFIIH for cellular physiology. Here we present the cryo-electron microscopy structure of human TFIIH at 4.4 Å resolution. The structure reveals the molecular architecture of the TFIIH core complex, the detailed structures of its constituent XPB and XPD ATPases, and how the core and kinase subcomplexes of TFIIH are connected. Also, our structure provides insight into the conformational dynamics of TFIIH and the regulation of its activity.« less

Virulence factor NSs of rift valley fever virus recruits the F-box protein FBXO3 to degrade subunit p62 of general transcription factor TFIIH.

PubMed

Kainulainen, Markus; Habjan, Matthias; Hubel, Philipp; Busch, Laura; Lau, Simone; Colinge, Jacques; Superti-Furga, Giulio; Pichlmair, Andreas; Weber, Friedemann

2014-03-01

The nonstructural protein NSs is the main virulence factor of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), a serious pathogen of livestock and humans in Africa. RVFV NSs blocks transcriptional upregulation of antiviral type I interferons (IFN) and destroys the general transcription factor TFIIH subunit p62 via the ubiquitin/proteasome pathway. Here, we identified a subunit of E3 ubiquitin ligases, F-box protein FBXO3, as a host cell interactor of NSs. Small interfering RNA (siRNA)-mediated depletion of FBXO3 rescued p62 protein levels in RVFV-infected cells and elevated IFN transcription by 1 order of magnitude. NSs interacts with the full-length FBXO3 protein as well as with a truncated isoform that lacks the C-terminal acidic and poly(R)-rich domains. These isoforms are present in both the nucleus and the cytoplasm. NSs exclusively removes the nuclear pool of full-length FBXO3, likely due to consumption during the degradation process. F-box proteins form the variable substrate recognition subunit of the so-called SCF ubiquitin ligases, which also contain the constant components Skp1, cullin 1 (or cullin 7), and Rbx1. siRNA knockdown of Skp1 also protected p62 from degradation, suggesting involvement in NSs action. However, knockdown of cullin 1, cullin 7, or Rbx1 could not rescue p62 degradation by NSs. Our data show that the enzymatic removal of p62 via the host cell factor FBXO3 is a major mechanism of IFN suppression by RVFV. Rift Valley fever virus is a serious emerging pathogen of animals and humans. Its main virulence factor, NSs, enables unhindered virus replication by suppressing the antiviral innate immune system. We identified the E3 ubiquitin ligase FBXO3 as a novel host cell interactor of NSs. NSs recruits FBXO3 to destroy the general host cell transcription factor TFIIH-p62, resulting in suppression of the transcriptional upregulation of innate immunity.

Virulence Factor NSs of Rift Valley Fever Virus Recruits the F-Box Protein FBXO3 To Degrade Subunit p62 of General Transcription Factor TFIIH

PubMed Central

Kainulainen, Markus; Habjan, Matthias; Hubel, Philipp; Busch, Laura; Lau, Simone; Colinge, Jacques; Superti-Furga, Giulio; Pichlmair, Andreas

2014-01-01

ABSTRACT The nonstructural protein NSs is the main virulence factor of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), a serious pathogen of livestock and humans in Africa. RVFV NSs blocks transcriptional upregulation of antiviral type I interferons (IFN) and destroys the general transcription factor TFIIH subunit p62 via the ubiquitin/proteasome pathway. Here, we identified a subunit of E3 ubiquitin ligases, F-box protein FBXO3, as a host cell interactor of NSs. Small interfering RNA (siRNA)-mediated depletion of FBXO3 rescued p62 protein levels in RVFV-infected cells and elevated IFN transcription by 1 order of magnitude. NSs interacts with the full-length FBXO3 protein as well as with a truncated isoform that lacks the C-terminal acidic and poly(R)-rich domains. These isoforms are present in both the nucleus and the cytoplasm. NSs exclusively removes the nuclear pool of full-length FBXO3, likely due to consumption during the degradation process. F-box proteins form the variable substrate recognition subunit of the so-called SCF ubiquitin ligases, which also contain the constant components Skp1, cullin 1 (or cullin 7), and Rbx1. siRNA knockdown of Skp1 also protected p62 from degradation, suggesting involvement in NSs action. However, knockdown of cullin 1, cullin 7, or Rbx1 could not rescue p62 degradation by NSs. Our data show that the enzymatic removal of p62 via the host cell factor FBXO3 is a major mechanism of IFN suppression by RVFV. IMPORTANCE Rift Valley fever virus is a serious emerging pathogen of animals and humans. Its main virulence factor, NSs, enables unhindered virus replication by suppressing the antiviral innate immune system. We identified the E3 ubiquitin ligase FBXO3 as a novel host cell interactor of NSs. NSs recruits FBXO3 to destroy the general host cell transcription factor TFIIH-p62, resulting in suppression of the transcriptional upregulation of innate immunity. PMID:24403578

Mapping of the Rsd Contact Site on the Sigma 70 Subunit of Escherichia coli RNA Polymerase

PubMed Central

Jishage, Miki; Dasgupta, Dipak; Ishihama, Akira

2001-01-01

Rsd (regulator of sigma D) is an anti-sigma factor for the Escherichia coli RNA polymerase ς70 subunit. The contact site of Rsd on ς70 was analyzed after mapping of the contact-dependent cleavage sites by Rsd-tethered iron-p-bromoacetamidobenzyl EDTA and by analysis of the complex formation between Ala-substituted ς70 and Rsd. Results indicate that the Rsd contact site is located downstream of the promoter −35 recognition helix-turn-helix motif within region 4, overlapping with the regions involved in interaction with both core enzyme and ς70 contact transcription factors. PMID:11292818

Mapping of the Rsd contact site on the sigma 70 subunit of Escherichia coli RNA polymerase.

PubMed

Jishage, M; Dasgupta, D; Ishihama, A

2001-05-01

Rsd (regulator of sigma D) is an anti-sigma factor for the Escherichia coli RNA polymerase sigma(70) subunit. The contact site of Rsd on sigma(70) was analyzed after mapping of the contact-dependent cleavage sites by Rsd-tethered iron-p-bromoacetamidobenzyl EDTA and by analysis of the complex formation between Ala-substituted sigma(70) and Rsd. Results indicate that the Rsd contact site is located downstream of the promoter -35 recognition helix-turn-helix motif within region 4, overlapping with the regions involved in interaction with both core enzyme and sigma(70) contact transcription factors.

Inactivation of chloroplast H(+)-ATPase by modification of Lys beta 359, Lys alpha 176 and Lys alpha 266.

PubMed

Horbach, M; Meyer, H E; Bickel-Sandkötter, S

1991-09-01

Treatment of isolated, latent chloroplast ATPase with pyridoxal-5-phosphate (pyridoxal-P) in presence of Mg2+ causes inhibition of dithiothreitol-activated plus heat-activated ATP hydrolysis. The amount of [3H]pyridoxal-P bound to chloroplast coupling factor 1 (CF1) was estimated to run up to 6 +/- 1 pyridoxal-P/enzyme, almost equally distributed between the alpha- and beta-subunits. Inactivation, however, is complete after binding of 1.5-2 pyridoxal-P/CF1, suggesting that two covalently modified lysines prevent the activation of the enzyme. ADP as well as ATP in presence of Mg2+ protects the enzyme against inactivation and concomittantly prevents incorporation of a part of the 3H-labeled pyridoxal-P into beta- and alpha-subunits. Phosphate prevents labeling of the alpha-subunit, but has only a minor effect on protection against inactivation. The data indicate a binding site at the interface between the alpha- and beta-subunits. Cleavage of the pyridoxal-P-labeled subunits with cyanogen bromide followed by sequence analysis of the labeled peptides led to the detection of Lys beta 359, Lys alpha 176 and Lys alpha 266, which are closely related to proposed nucleotide-binding regions of the alpha- and beta-subunits.

77 FR 76871 - Approval and Promulgation of Implementation Plans; State of Colorado; Regional Haze State...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-31

... nitrogen oxides. xiii. The initials NPS mean or refer to National Park Service. xiv. The initials PM 2.5..., nitrogen deposition, and mercury emissions and deposition. The State spent considerable time and conducted sequential and extended hearings to develop a plan which seeks to balance a number of variables beyond those...

The ELISE II Project: A Digital Image Library for Europe.

ERIC Educational Resources Information Center

Strunz, Bob; Waters, Mairead

This paper describes the progress made under the ELISE II electronic image library project from a technical standpoint. The ELISE II project is a European-wide initiative that aims to provide a comprehensive electronic image library service for Europe. It is funded under the European Commission, DG XIII-E, Telematics for Libraries Initiative. The…

Speckle Interferometry at the US Naval Observatory. XIII

DTIC Science & Technology

2007-10-01

18443+3940 ............................. STF 2382 AB 6.394 348.9 2.35 1 0.3 0.06 Mason et al. (2004a) 0.2 0.03 Novakovic & Todorovic (2005) 18443+3940...1952, Bull. Astron. Paris, 16, 263 Novakovic , B., & Todorovic, N. 2005, Circ. d’Inf. 157 Olevic, D. 2002, Circ. d’Inf. 147 Olevic, D., & Cvetkovic, Z

Russian Function Catalog and Rolebooks. Methods for Determining Language Objectives and Criteria, Volume XIII.

ERIC Educational Resources Information Center

Setzler, Hubert H., Jr.; And Others

A Russian Function Catalog and Instructor and Advisor Rolebooks for Russian are presented. The catalog and rolebooks are part of the communication/language objectives-based system (C/LOBS), which supports the front-end analysis efforts of the Defense Language Institute Foreign Language Center. The C/LOBS projects, which is described in 13 volumes…

AUTOMOTIVE DIESEL MAINTENANCE 2. UNIT XIII, BATTERY SERVICE AND TESTING PROCEDURES--PART II.

ERIC Educational Resources Information Center

Human Engineering Inst., Cleveland, OH.

THIS MODULE OF A 25-MODULE COURSE IS DESIGNED TO FAMILIARIZE THE TRAINEE WITH PROCEDURES FOR SERVICING LEAD-ACID STORAGE BATTERIES USED ON DIESEL POWERED EQUIPMENT. TOPICS ARE (1) ELECTROLYTE AND SPECIFIC GRAVITY, (2) BATTERY CHARGING, (3) STORAGE BATTERY TYPES AND DESIGN, (4) BATTERY CAPACITY RATINGS, (5) BATTERY INSTALLATION, SERVICING, AND…

Osteogenesis Imperfecta

PubMed Central

Sam, Justin Easow; Dharmalingam, Mala

2017-01-01

Osteogenesis imperfecta is a common heritable connective tissue disorder. Nearly ninety percent are due to Type I collagen mutations. Type I-IV are autosomal dominant, and Type VI–XIII are autosomal recessive. They are Graded 1-5 based on severity. Genomic testing is done by collagen analysis from fibroblasts. The mainstay of treatment is bisphosphonate therapy. The prognosis is variable. PMID:29285457

Energy Policy Act Transportation Rate Study: Final Report on Coal Transportation

EIA Publications

2000-01-01

This is the final in a series of reports prepared for the U.S. Congress by the Secretary of Energy on coal distribution and transportation rates as mandated by Title XIII, Section 1340, Establishment of Data Base and Study of Transportation Rates, of the Energy Policy Act of 1992 (P.L. 102-486).

Improving Soldier and Unit Effectiveness with the Stryker Brigade Combat Team Warfighters’ Forum

DTIC Science & Technology

2011-01-01

Figures S.1. Differences Attributable to the ICEA Handbook During a Training Rotation . . . . . . . . . . . xiii 2.1. Distribution of Respondents...of Differences Attributable to the Handbook During a Training Rotation ...in an SBCT that received the ICEA did significantly better on tactical tasks during combat training center rotations than platoons that did not

Public health. Gates Foundation on big funding spree.

PubMed

Hagmann, M

2000-08-11

The Bill and Melinda Gates Foundation dished up $200 million in grants for scientists in various fields late last month, including research into malaria and tuberculosis. The foundation kicked off its spending spree in mid-July at the XIII International AIDS Conference in Durban, South Africa, by announcing several AIDS/HIV-related grants totaling $90 million.

38 CFR 17.81 - Contracts for residential treatment services for veterans with alcohol or drug dependence or...

Code of Federal Regulations, 2010 CFR

2010-07-01

... the Department of Veterans Affairs, Office of Regulations Management (02D), Room 1154, 810 Vermont... management must agree that all the other residents in any building housing veterans will also have such...) Support for the individual desire for sobriety (alcohol/drug abuse-free life style). (xiii) Opportunities...

Carrascolendas: Evaluation of a Spanish/English Educational Television Series Within Region XIII. Final Report. Evaluation Component.

ERIC Educational Resources Information Center

Van Wart, Geraldine

This fourth year evaluation reports the effects and usage of "Carrascolendas," a children's television series in Spanish and English. Research was conducted in Texas schools and encompassed three phases: a field experiment to measure learning effects; attitudinal surveys among teachers, parents, and children; and a process evaluation of…

7 CFR 2.22 - Under Secretary for Marketing and Regulatory Programs.

Code of Federal Regulations, 2014 CFR

2014-01-01

... Consultative Group to Eliminate the Use of Child Labor and Forced Labor in Imported Agricultural Products. (2..., control or eradicate foot-and-mouth disease and other foreign animal diseases (21 U.S.C. 113a); (xiii) The... States in control and eradication of plant and animal diseases and pests; (xv) The Federal Noxious Weed...

7 CFR 2.22 - Under Secretary for Marketing and Regulatory Programs.

Code of Federal Regulations, 2013 CFR

2013-01-01

... Consultative Group to Eliminate the Use of Child Labor and Forced Labor in Imported Agricultural Products. (2..., control or eradicate foot-and-mouth disease and other foreign animal diseases (21 U.S.C. 113a); (xiii) The... States in control and eradication of plant and animal diseases and pests; (xv) The Federal Noxious Weed...

7 CFR 2.22 - Under Secretary for Marketing and Regulatory Programs.

Code of Federal Regulations, 2012 CFR

2012-01-01

... Consultative Group to Eliminate the Use of Child Labor and Forced Labor in Imported Agricultural Products. (2..., control or eradicate foot-and-mouth disease and other foreign animal diseases (21 U.S.C. 113a); (xiii) The... States in control and eradication of plant and animal diseases and pests; (xv) The Federal Noxious Weed...

7 CFR 2.22 - Under Secretary for Marketing and Regulatory Programs.

Code of Federal Regulations, 2011 CFR

2011-01-01

... Consultative Group to Eliminate the Use of Child Labor and Forced Labor in Imported Agricultural Products. (2..., control or eradicate foot-and-mouth disease and other foreign animal diseases (21 U.S.C. 113a); (xiii) The... States in control and eradication of plant and animal diseases and pests; (xv) The Federal Noxious Weed...

78 FR 15988 - Self-Regulatory Organizations; the NASDAQ Stock Market LLC; Notice of Filing and Immediate...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-13

.... ``dark venues'' or ``dark pools''). QCST orders, pursuant to Rule 4758(a)(1)(A)(xiii), check the System for available shares and simultaneously route to select dark venues and to certain low cost exchanges... Web site ( http://www.sec.gov/rules/sro.shtml ). Copies of the submission, all subsequent amendments...

Improving Operational Effectiveness of Tactical Long Endurance Unmanned Aerial Systems (TALEUAS) by Utilizing Solar Power

DTIC Science & Technology

2014-06-01

Speed xiii TEK Total Energy Compensated TSP traveling salesman problem UAV unmanned aerial vehicle UDP user datagram protocol UKF unscented...discretized map, and use the map to optimally solve the navigation task. The optimal navigation solution utilizes the well-known “ travelling salesman problem ...2 C. FORMULATION OF THE PROBLEM .................................................. 3 D

Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fan, Haitian; Hahm, Joseph; Diggs, Stephen

The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3', 5'-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes tomore » small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. Finally, this molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response.« less

Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli

DOE PAGES

Fan, Haitian; Hahm, Joseph; Diggs, Stephen; ...

2015-07-10

The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3', 5'-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes tomore » small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. Finally, this molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response.« less

Age-related decline of the cytochrome c oxidase subunit expression in the auditory cortex of the mimetic aging rat model associated with the common deletion.

PubMed

Zhong, Yi; Hu, Yujuan; Peng, Wei; Sun, Yu; Yang, Yang; Zhao, Xueyan; Huang, Xiang; Zhang, Honglian; Kong, Weijia

2012-12-01

The age-related deterioration in the central auditory system is well known to impair the abilities of sound localization and speech perception. However, the mechanisms involved in the age-related central auditory deficiency remain unclear. Previous studies have demonstrated that mitochondrial DNA (mtDNA) deletions accumulated with age in the auditory system. Also, a cytochrome c oxidase (CcO) deficiency has been proposed to be a causal factor in the age-related decline in mitochondrial respiratory activity. This study was designed to explore the changes of CcO activity and to investigate the possible relationship between the mtDNA common deletion (CD) and CcO activity as well as the mRNA expression of CcO subunits in the auditory cortex of D-galactose (D-gal)-induced mimetic aging rats at different ages. Moreover, we explored whether peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM) were involved in the changes of nuclear- and mitochondrial-encoded CcO subunits in the auditory cortex during aging. Our data demonstrated that d-gal-induced mimetic aging rats exhibited an accelerated accumulation of the CD and a gradual decline in the CcO activity in the auditory cortex during the aging process. The reduction in the CcO activity was correlated with the level of CD load in the auditory cortex. The mRNA expression of CcO subunit III was reduced significantly with age in the d-gal-induced mimetic aging rats. In contrast, the decline in the mRNA expression of subunits I and IV was relatively minor. Additionally, significant increases in the mRNA and protein levels of PGC-1α, NRF-1 and TFAM were observed in the auditory cortex of D-gal-induced mimetic aging rats with aging. These findings suggested that the accelerated accumulation of the CD in the auditory cortex may induce a substantial decline in CcO subunit III and lead to a significant decline in the CcO activity progressively with age despite compensatory increases of PGC-1α, NRF-1 and TFAM. Therefore, CcO may be a specific intramitochondrial site of age-related deterioration in the auditory cortex, and CcO subunit III might be a target in the development of presbycusis. Copyright © 2012 Elsevier B.V. All rights reserved.

CONCEPTUAL DESIGN OF A LUNAR REGOLITH CLUSTERED-REACTOR SYSTEM

DOE Office of Scientific and Technical Information (OSTI.GOV)

John Darrell Bess

2009-06-01

It is proposed that a fast-fission, heatpipe-cooled, lunar-surface power reactor system be divided into subcritical units that could be launched safely without the incorporation of additional spectral shift absorbers or other complex means of control. The reactor subunits are to be emplaced directly into the lunar regolith utilizing the regolith not just for shielding but as the reflector material to increase the neutron economy of the system. While a single subunit cannot achieve criticality by itself, coordinated placement of additional subunits will provide a critical reactor system for lunar surface power generation. A lunar regolith clustered-reactor system promotes reliability, safety,more » and ease of manufacture and testing at the cost of a slight increase in launch mass per rated power level and an overall reduction in neutron economy when compared to a single-reactor system. Additional subunits may be launched with future missions to increase the cluster size and power according to desired lunar base power demand and lifetime. The results address the potential uncertainties associated with the lunar regolith material and emplacement of the subunit systems. Physical distance between subunits within the clustered emplacement exhibits the most significant feedback regarding changes in overall system reactivity. Narrow, deep holes will be the most effective in reducing axial neutron leakage from the core. The variation in iron concentration in the lunar regolith can directly influence the overall system reactivity although its effects are less than the more dominant factors of subunit emplacement.« less

Off-pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli.

PubMed

Bao, Rui; Liu, Yang; Savarino, Stephen J; Xia, Di

2016-12-01

The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone-usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor-strand complementation (DSC) and cleft-mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus additionally assist in subunit refolding. Surprisingly, over-stabilization of the chaperone-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off-pathway self-polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Molecular Microbiology published by John Wiley & Sons Ltd.

The XPB subunit of repair/transcription factor TFIIH directly interacts with SUG1, a subunit of the 26S proteasome and putative transcription factor.

PubMed Central

Weeda, G; Rossignol, M; Fraser, R A; Winkler, G S; Vermeulen, W; van 't Veer, L J; Ma, L; Hoeijmakers, J H; Egly, J M

1997-01-01

Mutations in the basal transcription initiation/DNA repair factor TFIIH are responsible for three human disorders: xeroderma pigmentosum (XP), cockayne syndrome (CS) and trichothiodystrophy (TTD). The non-repair features of CS and TTD are thought to be due to a partial inactivation of the transcription function of the complex. To search for proteins whose interaction with TFIIH subunits is disturbed by mutations in patients we used the yeast two-hybrid system and report the isolation of a novel XPB interacting protein, SUG1. The interaction was validated in vivo and in vitro in the following manner. (i) SUG1 interacts with XPB but not with the other core TFIIH subunits in the two-hybrid assay. (ii) Physical interaction is observed in a baculovirus co-expression system. (iii) In fibroblasts under non-overexpression conditions a portion of SUG1 is bound to the TFIIH holocomplex as deduced from co-purification, immunopurification and nickel-chelate affinity chromatography using functional tagged TFIIH. Furthermore, overexpression of SUG1 in normal fibroblasts induced arrest of transcription and a chromatin collapse in vivo. Interestingly, the interaction was diminished with a mutant form of XPB, thus providing a potential link with the clinical features of XP-B patients. Since SUG1 is an integral component of the 26S proteasome and may be part of the mediator, our findings disclose a SUG1-dependent link between TFIIH and the cellular machinery involved in protein modelling/degradation. PMID:9173976

Transcription coactivator SAYP combines chromatin remodeler Brahma and transcription initiation factor TFIID into a single supercomplex

PubMed Central

Vorobyeva, Nadezhda E.; Soshnikova, Nataliya V.; Nikolenko, Julia V.; Kuzmina, Julia L.; Nabirochkina, Elena N.; Georgieva, Sofia G.; Shidlovskii, Yulii V.

2009-01-01

Transcription activation by RNA polymerase II is a complicated process driven by combined, precisely coordinated action of a wide array of coactivator complexes, which carry out chromatin-directed activities and nucleate the assembly of the preinitiation complex on the promoter. Using various techniques, we have shown the existence of a stable coactivator supercomplex consisting of the chromatin-remodeling factor Brahma (SWI/SNF) and the transcription initiation factor TFIID, named BTFly (Brahma and TFIID in one assembly). The coupling of Brahma and TFIID is mediated by the SAYP factor, whose evolutionarily conserved activation domain SAY can directly bind to both BAP170 subunit of Brahma and TAF5 subunit of TFIID. The integrity of BTFly is crucial for its ability to activate transcription. BTFly is distributed genome-wide and appears to be a means of effective transcription activation. PMID:19541607

Rapid changes in ovarian mRNA induced by brief photostimulation in Siberian hamsters (Phodopus sungorus)

PubMed Central

Shahed, Asha; McMichael, Carling F.; Young, Kelly A.

2017-01-01

This study sought to characterize the rapid intraovarian mRNA response of key folliculogenic factors that may contribute to the restoration of folliculogenesis during 2-10 days of photostimulation in Siberian hamsters. Adult hamsters were exposed to short photoperiod (8L:16D) for 14 weeks (SD). A subset were then transferred to long photoperiod (16L:8D) for 2(PT day-2), 4(PT day-4), or 10 days (PT day-10). Quantitative real-time PCR was used to measure intraovarian mRNA expression of: gonadotropin releasing hormone (GnRH), follicle stimulating hormone β-subunit (FSHβ-subunit), luteinizing hormone β-subunit (LHβ-subunit), FSH and LH receptors, estrogen receptorsα and β (Esr1 and Esr2), matrix metalloproteinase (MMP)-2 and -9, anti-Müllerian hormone (AMH), inhibin-α subunit, fibroblast growth factor-2 (FGF-2) and proliferating cell nuclear antigen (PCNA). Compared to SD, plasma FSH concentrations increased on PT day-4 and the number of antral follicles and corpora lutea increased on PT day-10. FSHR and inhibin-α mRNA expression also increased on PT day-4, whereas LHR and proliferation marker PCNA both increased on PT day-10 as compared to SD. Esr1 mRNA increased on PT day-2 and remained significantly increased as compared to SD, whereas Esr1 mRNA increased only on PT day-2, similar to FGF-2 and MMP-2 results. No differences were observed in mRNA expression in ovarian GnRH, FSHβ- and LHβ-subunits, AMH, and MMP-9 mRNA with 2-10 days of photostimulation. Rapid increases in intraovarian FSHR and inhibin-α mRNA and antral follicle/corpora lutea numbers suggest that the ovary is primed to react quickly to the FSH released in response to brief periods of photostimulation. PMID:26174001

The B55α Regulatory Subunit of Protein Phosphatase 2A Mediates Fibroblast Growth Factor-Induced p107 Dephosphorylation and Growth Arrest in Chondrocytes

PubMed Central

Daempfling, Lea

2013-01-01

Fibroblast growth factor (FGF)-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types and underlies several genetic skeletal disorders caused by activating FGF receptor (FGFR) mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the retinoblastoma protein (Rb) family member p107 by protein phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report, we show that the PP2A-B55α holoenzyme (PP2A containing the B55α subunit) is responsible for this phenomenon. Only the B55α (55-kDa regulatory subunit, alpha isoform) regulatory subunit of PP2A was able to bind p107, and this interaction was induced by FGF in chondrocytes but not in other cell types. Small interfering RNA (siRNA)-mediated knockdown of B55α prevented p107 dephosphorylation and FGF-induced growth arrest of RCS (rat chondrosarcoma) chondrocytes. Importantly, the B55α subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55α is also the site of cyclin-dependent kinase (CDK) binding, B55α association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55α subunit itself on several serine residues that drastically increases the affinity of B55α for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55α dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals. PMID:23716589

Mechanisms of ring chromosome formation, ring instability and clinical consequences.

PubMed

Guilherme, Roberta S; Meloni, Vera F Ayres; Kim, Chong A; Pellegrino, Renata; Takeno, Sylvia S; Spinner, Nancy B; Conlin, Laura K; Christofolini, Denise M; Kulikowski, Leslie D; Melaragno, Maria I

2011-12-21

The breakpoints and mechanisms of ring chromosome formation were studied and mapped in 14 patients. Several techniques were performed such as genome-wide array, MLPA (Multiplex Ligation-Dependent Probe Amplification) and FISH (Fluorescent in situ Hybridization). The ring chromosomes of patients I to XIV were determined to be, respectively: r(3)(p26.1q29), r(4)(p16.3q35.2), r(10)(p15.3q26.2), r(10)(p15.3q26.13), r(13)(p13q31.1), r(13)(p13q34), r(14)(p13q32.33), r(15)(p13q26.2), r(18)(p11.32q22.2), r(18)(p11.32q21.33), r(18)(p11.21q23), r(22)(p13q13.33), r(22)(p13q13.2), and r(22)(p13q13.2). These rings were found to have been formed by different mechanisms, such as: breaks in both chromosome arms followed by end-to-end reunion (patients IV, VIII, IX, XI, XIII and XIV); a break in one chromosome arm followed by fusion with the subtelomeric region of the other (patients I and II); a break in one chromosome arm followed by fusion with the opposite telomeric region (patients III and X); fusion of two subtelomeric regions (patient VII); and telomere-telomere fusion (patient XII). Thus, the r(14) and one r(22) can be considered complete rings, since there was no loss of relevant genetic material. Two patients (V and VI) with r(13) showed duplication along with terminal deletion of 13q, one of them proved to be inverted, a mechanism known as inv-dup-del. Ring instability was detected by ring loss and secondary aberrations in all but three patients, who presented stable ring chromosomes (II, XIII and XIV). We concluded that the clinical phenotype of patients with ring chromosomes may be related with different factors, including gene haploinsufficiency, gene duplications and ring instability. Epigenetic factors due to the circular architecture of ring chromosomes must also be considered, since even complete ring chromosomes can result in phenotypic alterations, as observed in our patients with complete r(14) and r(22).

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10 mg/kg b.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) andmore » Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits–NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. - Highlights: • Aluminium decreases the mRNA levels of mitochondrial and nuclear encoded subunits. • It decreases the mtDNA copy number and mitochondrial content in rat brain. • It down-regulates the mRNA and protein levels of PGC-1α, NRF-1, NRF-2 and Tfam. • It also disturbs the mitochondrial or nuclear architecture of neurons. • Finally it also decreases mitochondrial number in HC and CS regions of rat brain.« less

40 CFR Appendix Xiii to Part 86 - State Requirements Incorporated by Reference in Part 86 of the Code of Federal Regulations

Code of Federal Regulations, 2010 CFR

2010-07-01

... Specifications, adopted March 1, 1978, amended June 24, 1996, excluding paragraphs 2(b), 3.5, and 10. [62 FR... Vehicles, adopted November 24, 1981, amended June 24, 1996. (b) State of California; Air Resources Board... and Medium-Duty Vehicles, adopted June 24, 1996. (c) California Code of Regulations, Title 13...

Bridging Cultures and Traditions for Educational and International Development: Comparative Research, Dialogue and Difference

ERIC Educational Resources Information Center

Crossley, Michael

2008-01-01

Addressing the central theme of the XIII World Congress, the paper explores a number of contemporary theoretical, methodological and organisational developments in the field of comparative education. In doing so it draws upon the author's recent work and a selection of studies carried out in the South Pacific, the Caribbean and Africa. It is…

APOLLO XIII CREW - MISSION OPERATIONS CONTROL ROOM (MOCR) - APOLLO XII - LUNAR EXTRAVEHICULAR ACTIVITY (EVA) - MSC

NASA Image and Video Library

1969-11-21

S69-59525 (19 Nov. 1969) --- Overall view of activity in the Mission Operations Control Room (MOCR) in the Mission Control Center (MCC), Building 30, during the Apollo 12 lunar landing mission. When this picture was made the first Apollo 12 extravehicular activity (EVA) was being televised from the surface of the moon. Photo credit: NASA

Identification of Decision Support Concepts for the Planning of Air Force Immediate Contingencies Operations

DTIC Science & Technology

2010-03-01

that is becoming the norm in military operations, especial rapid response. Significance : The context of the work for this project focused on one...Time constraint variance ................................................................... 37 DRDC Valcartier CR 2010-353 xiii 3.5.8.1...employment activities that are normally recurring in nature and fall within the delegated authority of an appointed standing operational Commander

Employing Replay Connectors for SIEM Operator Education

DTIC Science & Technology

2013-09-01

BLANK xiii LIST OF ACRONYMS AND ABBREVIATIONS CORR Correlation Optimized Retention and Retrieval CII Critical Information Infrastructure GLBA...vast distances is now quicker and easier with the advancement in mobile computing devices and more ubiquitous connectivity and bandwidth. As a result...breakdown of the Critical Information Infrastructure (CII) is one of the core risks facing the international economy. The World Economic Forum

Wind-Tunnel Research Comparing Lateral Control Devices Particularly at High Angles of Attack XIII : Auxiliary Airfoils Used as External Ailerons

NASA Technical Reports Server (NTRS)

Weick, Fred E; Noyes, Richard W

1936-01-01

This is the thirteenth report on a series of systematic tests comparing lateral control devices with particular reference to their effectiveness at high angles of attack. The present wind tunnel tests were made to determine the most feasible locations for lateral control surfaces mounted externally to a rectangular Clark y wing.

Chippewa Indians: A Native American Curriculum Unit for the Third Grade. NATAM XIII.

ERIC Educational Resources Information Center

Kocur, Darlene

The document reports on an extension course taken in the spring of 1970 by public school teachers in the Columbia Heights Public School System via the University of Minnesota. The course, on American Indian education, included the usual on-campus requirements, as well as several lectures by guest Indians. Additionally, each teacher who enrolled in…

Water Quality Instructional Resources Information System (IRIS). A Compilation of Abstracts to Water Quality and Water Resources Materials. Supplement XIII.

ERIC Educational Resources Information Center

Ohio State Univ., Columbus, OH. Information Reference Center for Science, Mathematics, and Environmental Education.

Compiled are abstracts and indexes to selected print and non-print materials related to wastewater treatment and water quality education and instruction, as well as materials related to pesticides, hazardous wastes, and public participation. Sources of abstracted/indexed materials include all levels of government, private concerns, and educational…

Waiving a requirement of clause 6(a) of rule XIII with respect to consideration of certain resolutions reported from the Committee on Rules, and providing for consideration of motions to suspend the rules.

THOMAS, 111th Congress

Rep. Hastings, Alcee L. [D-FL-23

2010-05-25

House - 05/28/2010 Pursuant to the provisions of H. Res. 1403, H. Res. 1392 is laid on the table. (All Actions) Tracker: This bill has the status IntroducedHere are the steps for Status of Legislation:

Test Plan Framework for Cross Domain Solution (CDS) Devices

DTIC Science & Technology

2010-06-01

modification to the CASTER software to reflect the latest changes to RDAC is being discussed by the developers with input by the independent labs and NSA...7 xii THIS PAGE INTENTIONALLY LEFT BLANK xiii LIST OF ABBREVIATIONS AND ACRONYMS CASTER Certification...protecting information and information systems from unauthorized access, use, disclosure, disruption, modification , or destruction. While this is a

Naval Arms Control: A Post-Cold War Reappraisal

DTIC Science & Technology

1991-06-01

94 A . BACKGRO UN D ......................................................................................... 94 B. WHY ...control, but that an appropriate time may come to exist in the future. For reasons why naval arms control may make more sense in the future, but not...34. Current Research on Peace And Violence. Tampere Peace Research Institute, Tampere Finland, Vol XIII, No. 2, 1990, pp. 65-86. For reasons why naval

Defining the Role and Responsibility of the Fire Service Within Homeland Security

DTIC Science & Technology

2010-03-01

Occupational Safety and Health NPS Naval Postgraduate School NRF National Response Framework OSHA Occupational Safety and Health Administration ...Kingdom USAR Urban Search and Rescue USFA United States Fire Administration USMA United States Military Academy xiii ACKNOWLEDGMENTS I want...Association (NFPA), the Occupational Safety and Health Administration (OSHA), the United States Fire Administration (USFA), and the National Institute for

Developing a Resilient Green Cellular Network

DTIC Science & Technology

2013-12-01

to provide BS autonomy from grid power through alternative energy, such as: fuel cells and xiii renewable photovoltaic (PV), wind energy...stations with adequate backup power or utilizing alternative/renewable energy technology such as photovoltaic or wind power to allow them to...mitigating strategies with the consensus view on BSs migrating away from grid power , to renewable energy ( photovoltaic ), and alternative fuels. 40

40 CFR 721.5185 - Morpholine, 4-(1-oxo-2-propenyl)-.

Code of Federal Regulations, 2010 CFR

2010-07-01

...), (a)(5)(i), (a)(5)(ii), (a)(5)(iii), (a)(5)(xii), (a)(5)(xiii), (a)(5)(xiv), (a)(5)(xv), (a)(6)(v), (b) (concentration set at 0. 1 percent), and (c). The following material has been tested in accordance with the American Society for Testing Materials (ASTM) F739 method and found by EPA to satisfy the consent order's...

Influence of drying time and temperature on bond strength of contemporary adhesives to dentine.

PubMed

Garcia, Fernanda C P; Almeida, Júlio C F; Osorio, Raquel; Carvalho, Ricardo M; Toledano, Manuel

2009-04-01

To evaluate the bond strength (microTBS) of self-etching adhesives in different solvent evaporation conditions. Flat dentine surfaces from extracted human third molars were bonded with: (1) 2 two-steps self-etching adhesives (Clearfil SE Bond-CSEB); (Protect Bond-PB) and (2) 2 one-step self-etch systems (Adper Prompt L Pop-ADPLP); (Xeno III-XIII). Bonded dentine surfaces were air-dried for 5s, 20s, 30s or 40s at either 21 degrees C or 38 degrees C. Composite build-ups were constructed incrementally. After storage in water for 24h at 37 degrees C, the specimens were prepared for microtensile bond strength testing. Data were analyzed by two-way ANOVA and Student-Newman-Keuls at alpha=0.05. CSEB and PB performed better at warm temperature with only 20s of air-blowing. The bond strength increased when XIII was performed at warm temperature at 40s air-blowing. Extended air-blowing not affect the performance of ADPLP, except at 30s air-blowing time at warm temperature. The use of a warm air-dry stream seems to be a clinical tool to improve the bond strength to self-etching adhesives.

Progress report on new antiepileptic drugs: A summary of the Thirteenth Eilat Conference on New Antiepileptic Drugs and Devices (EILAT XIII).

PubMed

Bialer, Meir; Johannessen, Svein I; Levy, René H; Perucca, Emilio; Tomson, Torbjörn; White, H Steve

2017-02-01

The Thirteenth Eilat Conference on New Antiepileptic Drugs and Devices (EILAT XIII) took place in Madrid, Spain, on June 26-29, 2016, and was attended by >200 delegates from 31 countries. The present Progress Report provides an update on experimental and clinical results for drugs presented at the Conference. Compounds for which summary data are presented include an AED approved in 2016 (brivaracetam), 12 drugs in phase I-III clinical development (adenosine, allopregnanolone, bumetanide, cannabidiol, cannabidivarin, 2-deoxy-d-glucose, everolimus, fenfluramine, huperzine A, minocycline, SAGE-217, and valnoctamide) and 6 compounds or classes of compounds for which only preclinical data are available (bumetanide derivatives, sec-butylpropylacetamide, FV-082, 1OP-2198, NAX 810-2, and SAGE-689). Overall, the results presented at the Conference show that considerable efforts are ongoing into discovery and development of AEDs with potentially improved therapeutic profiles compared with existing agents. Many of the drugs discussed in this report show innovative mechanisms of action and many have shown promising results in patients with pharmacoresistant epilepsies, including previously neglected rare and severe epilepsy syndromes. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

Nucleotide Excision Repair and Transcriptional Regulation: TFIIH and Beyond.

PubMed

Compe, Emmanuel; Egly, Jean-Marc

2016-06-02

Transcription factor IIH (TFIIH) is a multiprotein complex involved in both transcription and DNA repair, revealing a striking functional link between these two processes. Some of its subunits also belong to complexes involved in other cellular processes, such as chromosome segregation and cell cycle regulation, emphasizing the multitasking capabilities of this factor. This review aims to depict the structure of TFIIH and to dissect the roles of its subunits in different cellular mechanisms. Our understanding of the biochemistry of TFIIH has greatly benefited from studies focused on diseases related to TFIIH mutations. We address the etiology of these disorders and underline the fact that TFIIH can be considered a promising target for therapeutic strategies.

Selective Activation of Transcription by a Novel CCAAT Binding Factor

NASA Astrophysics Data System (ADS)

Maity, Sankar N.; Golumbek, Paul T.; Karsenty, Gerard; de Crombrugghe, Benoit

1988-07-01

A novel CCAAT binding factor (CBF) composed of two different subunits has been extensively purified from rat liver. Both subunits are needed for specific binding to DNA. Addition of this purified protein to nuclear extracts of NIH 3T3 fibroblasts stimulates transcription from several promoters including the α 2(I) collagen, the α 1(I) collagen, the Rous sarcoma virus long terminal repeat (RSV-LTR), and the adenovirus major late promoter. Point mutations in the CCAAT motif that show either no binding or a decreased binding of CBF likewise abolish or reduce activation of transcription by CBF. Activation of transcription requires, therefore, the specific binding of CBF to its recognition sites.

The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mattiroli, Francesca; Gu, Yajie; Balsbaugh, Jeremy L.

Nucleosome assembly following DNA replication controls epigenome maintenance and genome integrity. Chromatin assembly factor 1 (CAF-1) is the histone chaperone responsible for histone (H3-H4)2 deposition following DNA synthesis. Structural and functional details for this chaperone complex and its interaction with histones are slowly emerging. Using hydrogen-deuterium exchange coupled to mass spectrometry, combined with in vitro and in vivo mutagenesis studies, we identified the regions involved in the direct interaction between the yeast CAF-1 subunits, and mapped the CAF-1 domains responsible for H3-H4 binding. The large subunit, Cac1 organizes the assembly of CAF-1. Strikingly, H3-H4 binding is mediated by a compositemore » interface, shaped by Cac1-bound Cac2 and the Cac1 acidic region. Cac2 is indispensable for productive histone binding, while deletion of Cac3 has only moderate effects on H3-H4 binding and nucleosome assembly. These results define direct structural roles for yeast CAF-1 subunits and uncover a previously unknown critical function of the middle subunit in CAF-1.« less

P‐TEFb goes viral

PubMed Central

Zaborowska, Justyna; Isa, Nur F.

2015-01-01

Positive transcription elongation factor b (P‐TEFb), which comprises cyclin‐dependent kinase 9 (CDK9) kinase and cyclin T subunits, is an essential kinase complex in human cells. Phosphorylation of the negative elongation factors by P‐TEFb is required for productive elongation of transcription of protein‐coding genes by RNA polymerase II (pol II). In addition, P‐TEFb‐mediated phosphorylation of the carboxyl‐terminal domain (CTD) of the largest subunit of pol II mediates the recruitment of transcription and RNA processing factors during the transcription cycle. CDK9 also phosphorylates p53, a tumor suppressor that plays a central role in cellular responses to a range of stress factors. Many viral factors affect transcription by recruiting or modulating the activity of CDK9. In this review, we will focus on how the function of CDK9 is regulated by viral gene products. The central role of CDK9 in viral life cycles suggests that drugs targeting the interaction between viral products and P‐TEFb could be effective anti‐viral agents. PMID:27398404

Identification of Telomerase Components and Telomerase Regulating Factors in Yeast

DTIC Science & Technology

1998-07-01

subunit of telomerase in S. cerevisiae is encoded by TLCJ (7). Recently , through sequence comparison with the telomerase catalytic 6 subunit from Euplotes...length maintenance has been unclear, although very recent data has shown that Ku80p can be found specifically associated with telomeric DNA in vivo...chromatin structure. It has been recently observed that loss of either YKU80 or HDF1 results in altered telomere end structure, such that there appears to

Ribulose-1,5-Bis-Phosphate Carboxylase/Oxygenase Accumulation Factor1 Is Required for Holoenzyme Assembly in Maize[C][W

PubMed Central

Feiz, Leila; Williams-Carrier, Rosalind; Wostrikoff, Katia; Belcher, Susan; Barkan, Alice; Stern, David B.

2012-01-01

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process. PMID:22942379

Identification of a new adapter protein that may link the common beta subunit of the receptor for granulocyte/macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 to phosphatidylinositol 3-kinase.

PubMed

Jücker, M; Feldman, R A

1995-11-17

Binding of human granulocyte/macrophage colony-stimulating factor (hGM-CSF) to its receptor induces the rapid activation of phosphatidylinositol-3 kinase (PI 3-kinase). As hGM-CSF receptor (hGMR) does not contain a consensus sequence for binding of PI 3-kinase, hGMR must use a distinct mechanism for its association with and activation of PI 3-kinase. Here, we describe the identification of a tyrosine-phosphorylated protein of 76-85 kDa (p80) that associates with the common beta subunit of hGMR and with the SH2 domains of the p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells. Src/Yes and Lyn were tightly associated with the p80.PI 3-kinase complex, suggesting that p80 and other phosphotyrosyl proteins present in the complex were phosphorylated by Src family kinases. Tyrosine phosphorylation of p80 was only detected in hGM-CSF or human interleukin-3-stimulated cells, suggesting that activation of p80 might be specific for signaling via the common beta subunit. We postulate that p80 functions as an adapter protein that may participate in linking the hGM-CSF receptor to the PI 3-kinase signaling pathway.

NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.

PubMed

Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

2011-07-01

Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus.

Heat-shock inactivation of the TFIIH-associated kinase and change in the phosphorylation sites on the C-terminal domain of RNA polymerase II.

PubMed

Dubois, M F; Vincent, M; Vigneron, M; Adamczewski, J; Egly, J M; Bensaude, O

1997-02-15

The C-terminal domain (CTD) of the RNA polymerase II largest subunit (RPB1) plays a central role in transcription. The CTD is unphosphorylated when the polymerase assembles into a preinitiation complex of transcription and becomes heavily phosphorylated during promoter clearance and entry into elongation of transcription. A kinase associated to the general transcription factor TFIIH, in the preinitiation complex, phosphorylates the CTD. The TFIIH-associated CTD kinase activity was found to decrease in extracts from heat-shocked HeLa cells compared to unstressed cells. This loss of activity correlated with a decreased solubility of the TFIIH factor. The TFIIH-kinase impairment during heat-shock was accompanied by the disappearance of a particular phosphoepitope (CC-3) on the RPB1 subunit. The CC-3 epitope was localized on the C-terminal end of the CTD and generated in vitro when the RPB1 subunit was phosphorylated by the TFIIH-associated kinase but not by another CTD kinase such as MAP kinase. In apparent discrepancy, the overall RPB1 subunit phosphorylation increased during heat-shock. The decreased activity in vivo of the TFIIH kinase might be compensated by a stress-activated CTD kinase such as MAP kinase. These results also suggest that heat-shock gene transcription may have a weak requirement for TFIIH kinase activity.

Heat-shock inactivation of the TFIIH-associated kinase and change in the phosphorylation sites on the C-terminal domain of RNA polymerase II.

PubMed Central

Dubois, M F; Vincent, M; Vigneron, M; Adamczewski, J; Egly, J M; Bensaude, O

1997-01-01

The C-terminal domain (CTD) of the RNA polymerase II largest subunit (RPB1) plays a central role in transcription. The CTD is unphosphorylated when the polymerase assembles into a preinitiation complex of transcription and becomes heavily phosphorylated during promoter clearance and entry into elongation of transcription. A kinase associated to the general transcription factor TFIIH, in the preinitiation complex, phosphorylates the CTD. The TFIIH-associated CTD kinase activity was found to decrease in extracts from heat-shocked HeLa cells compared to unstressed cells. This loss of activity correlated with a decreased solubility of the TFIIH factor. The TFIIH-kinase impairment during heat-shock was accompanied by the disappearance of a particular phosphoepitope (CC-3) on the RPB1 subunit. The CC-3 epitope was localized on the C-terminal end of the CTD and generated in vitro when the RPB1 subunit was phosphorylated by the TFIIH-associated kinase but not by another CTD kinase such as MAP kinase. In apparent discrepancy, the overall RPB1 subunit phosphorylation increased during heat-shock. The decreased activity in vivo of the TFIIH kinase might be compensated by a stress-activated CTD kinase such as MAP kinase. These results also suggest that heat-shock gene transcription may have a weak requirement for TFIIH kinase activity. PMID:9016617

Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian

2009-11-06

Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3more » in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.« less

Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.

PubMed

Ghospurkar, Padmaja L; Wilson, Timothy M; Liu, Shengqin; Herauf, Anna; Steffes, Jenna; Mueller, Erica N; Oakley, Gregory G; Haring, Stuart J

2015-02-01

Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Interactions of the α-subunits of heterotrimeric G-proteins with GPCRs, effectors and RGS proteins: a critical review and analysis of interacting surfaces, conformational shifts, structural diversity and electrostatic potentials.

PubMed

Baltoumas, Fotis A; Theodoropoulou, Margarita C; Hamodrakas, Stavros J

2013-06-01

G-protein coupled receptors (GPCRs) are one of the largest families of membrane receptors in eukaryotes. Heterotrimeric G-proteins, composed of α, β and γ subunits, are important molecular switches in the mediation of GPCR signaling. Receptor stimulation after the binding of a suitable ligand leads to G-protein heterotrimer activation and dissociation into the Gα subunit and Gβγ heterodimer. These subunits then interact with a large number of effectors, leading to several cell responses. We studied the interactions between Gα subunits and their binding partners, using information from structural, mutagenesis and Bioinformatics studies, and conducted a series of comparisons of sequence, structure, electrostatic properties and intermolecular energies among different Gα families and subfamilies. We identified a number of Gα surfaces that may, in several occasions, participate in interactions with receptors as well as effectors. The study of Gα interacting surfaces in terms of sequence, structure and electrostatic potential reveals features that may account for the Gα subunit's behavior towards its interacting partners. The electrostatic properties of the Gα subunits, which in some cases differ greatly not only between families but also between subfamilies, as well as the G-protein interacting surfaces of effectors and regulators of G-protein signaling (RGS) suggest that electrostatic complementarity may be an important factor in G-protein interactions. Energy calculations also support this notion. This information may be useful in future studies of G-protein interactions with GPCRs and effectors. Copyright © 2013 Elsevier Inc. All rights reserved.

ADP binding to TF1 and its subunits induces ultraviolet spectral changes.

PubMed

Hisabori, T; Yoshida, M; Sakurai, H

1986-09-01

Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.

TFIIH Subunit Alterations Causing Xeroderma Pigmentosum and Trichothiodystrophy Specifically Disturb Several Steps during Transcription

PubMed Central

Singh, Amita; Compe, Emanuel; Le May, Nicolas; Egly, Jean-Marc

2015-01-01

Mutations in genes encoding the ERCC3 (XPB), ERCC2 (XPD), and GTF2H5 (p8 or TTD-A) subunits of the transcription and DNA-repair factor TFIIH lead to three autosomal-recessive disorders: xeroderma pigmentosum (XP), XP associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTD). Although these diseases were originally associated with defects in DNA repair, transcription deficiencies might be also implicated. By using retinoic acid receptor beta isoform 2 (RARB2) as a model in several cells bearing mutations in genes encoding TFIIH subunits, we observed that (1) the recruitment of the TFIIH complex was altered at the activated RARB2 promoter, (2) TFIIH participated in the recruitment of nucleotide excision repair (NER) factors during transcription in a manner different from that observed during NER, and (3) the different TFIIH variants disturbed transcription by having distinct consequences on post-translational modifications of histones, DNA-break induction, DNA demethylation, and gene-loop formation. The transition from heterochromatin to euchromatin was disrupted depending on the variant, illustrating the fact that TFIIH, by contributing to NER factor recruitment, orchestrates chromatin remodeling. The subtle transcriptional differences found between various TFIIH variants thus participate in the phenotypic variability observed among XP, XP/CS, and TTD individuals. PMID:25620205

Reduced Chrna7 expression in C3H mice is associated with increases in hippocampal parvalbumin and glutamate decarboxylase-67 (GAD67) as well as altered levels of GABAA receptor subunits

PubMed Central

Bates, Ryan C.; Stith, Bradley J.; Stevens, Karen E.; Adams, Catherine E.

2014-01-01

Decreased expression of CHRNA7, the gene encoding the α7* subtype of nicotinic receptor, may contribute to the cognitive dysfunction observed in schizophrenia by disrupting the inhibitory/excitatory balance in the hippocampus. C3H mice with reduced Chrna7 expression have significant reductions in hippocampal α7* receptor density, deficits in hippocampal auditory gating, increased hippocampal activity as well as significant decreases in hippocampal glutamate decarboxylase-65 (GAD65) and γ-aminobutyric acid-A (GABAA) receptor levels. The current study investigated whether altered Chrna7 expression is associated with changes in the levels of parvalbumin, GAD67 and/or GABAA receptor subunits in hippocampus from male and female C3H Chrna7 wildtype, C3H Chrna7 heterozygous and C3H Chrna7 knockout mice using quantitative western immunoblotting. Reduced Chrna7 expression was associated with significant increases in hippocampal parvalbumin and GAD67 and with complex alterations in GABAA receptor subunits. A decrease in α3 subunit protein was seen in both female C3H Chrna7 Het and KO mice while a decrease in α4 subunit protein was also detected in C3H Chrna7 KO mice with no sex difference. In contrast, an increase in δ subunit protein was observed in C3H Chrna7 Het mice while a decrease in this subunit was observed in C3H Chrna7 KO mice, with δ subunit protein levels being greater in males than in females. Finally, an increase in γ2 subunit protein was found in C3H Chrna7 KO mice with the levels of this subunit again being greater in males than in females. The increases in hippocampal parvalbumin and GAD67 observed in C3H Chrna7 mice are contrary to reports of reductions in these proteins in postmortem hippocampus from schizophrenic individuals. We hypothesize that the disparate results may occur because of the influence of factors other than CHRNA7 that have been found to be abnormal in schizophrenia. PMID:24836856

Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin

PubMed Central

Chulluncuy, Roberto; Espiche, Carlos; Nakamoto, Jose Alberto; Fabbretti, Attilio; Milón, Pohl

2016-01-01

Aminoglycoside antibiotics are widely used to treat infectious diseases. Among them, streptomycin and kanamycin (and derivatives) are of importance to battle multidrug-resistant (MDR) Mycobacterium tuberculosis. Both drugs bind the small ribosomal subunit (30S) and inhibit protein synthesis. Genetic, structural, and biochemical studies indicate that local and long-range conformational rearrangements of the 30S subunit account for this inhibition. Here, we use intramolecular FRET between the C- and N-terminus domains of the flexible IF3 to monitor real-time perturbations of their binding sites on the 30S platform. Steady and pre-steady state binding experiments show that both aminoglycosides bring IF3 domains apart, promoting an elongated state of the factor. Binding of Initiation Factor IF1 triggers closure of IF3 bound to the 30S complex, while both aminoglycosides revert the IF1-dependent conformation. Our results uncover dynamic perturbations across the 30S subunit, from the A-site to the platform, and suggest that both aminoglycosides could interfere with prokaryotic translation initiation by modulating the interaction between IF3 domains with the 30S platform. PMID:27983590

Structural integration in hypoxia-inducible factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Dalei; Potluri, Nalini; Lu, Jingping

The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-alpha and ARNT (also called HIF-1 beta) subunits. Here we describe crystal structures for each of mouse HIF-2 alpha-ARNT and HIF-1 alpha-ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2 alpha-ARNT and HIF-1 alpha-ARNT, wherein ARNT spirals around the outside of each HIF-alpha subunit. Five distinctmore » pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-alpha mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.« less

Structure of Ribosomal Silencing Factor Bound to Mycobacterium tuberculosis Ribosome.

PubMed

Li, Xiaojun; Sun, Qingan; Jiang, Cai; Yang, Kailu; Hung, Li-Wei; Zhang, Junjie; Sacchettini, James C

2015-10-06

The ribosomal silencing factor RsfS slows cell growth by inhibiting protein synthesis during periods of diminished nutrient availability. The crystal structure of Mycobacterium tuberculosis (Mtb) RsfS, together with the cryo-electron microscopy (EM) structure of the large subunit 50S of Mtb ribosome, reveals how inhibition of protein synthesis by RsfS occurs. RsfS binds to the 50S at L14, which, when occupied, blocks the association of the small subunit 30S. Although Mtb RsfS is a dimer in solution, only a single subunit binds to 50S. The overlap between the dimer interface and the L14 binding interface confirms that the RsfS dimer must first dissociate to a monomer in order to bind to L14. RsfS interacts primarily through electrostatic and hydrogen bonding to L14. The EM structure shows extended rRNA density that it is not found in the Escherichia coli ribosome, the most striking of these being the extended RNA helix of H54a. Copyright © 2015 Elsevier Ltd. All rights reserved.

Paradoxical resistance of multiple myeloma to proteasome inhibitors by decreased levels of 19S proteasomal subunits

PubMed Central

Acosta-Alvear, Diego; Cho, Min Y; Wild, Thomas; Buchholz, Tonia J; Lerner, Alana G; Simakova, Olga; Hahn, Jamie; Korde, Neha; Landgren, Ola; Maric, Irina; Choudhary, Chunaram; Walter, Peter; Weissman, Jonathan S; Kampmann, Martin

2015-01-01

Hallmarks of cancer, including rapid growth and aneuploidy, can result in non-oncogene addiction to the proteostasis network that can be exploited clinically. The defining example is the exquisite sensitivity of multiple myeloma (MM) to 20S proteasome inhibitors, such as carfilzomib. However, MM patients invariably acquire resistance to these drugs. Using a next-generation shRNA platform, we found that proteostasis factors, including chaperones and stress-response regulators, controlled the response to carfilzomib. Paradoxically, 19S proteasome regulator knockdown induced resistance to carfilzomib in MM and non-MM cells. 19S subunit knockdown did not affect the activity of the 20S subunits targeted by carfilzomib nor their inhibition by the drug, suggesting an alternative mechanism, such as the selective accumulation of protective factors. In MM patients, lower 19S levels predicted a diminished response to carfilzomib-based therapies. Together, our findings suggest that an understanding of network rewiring can inform development of new combination therapies to overcome drug resistance. DOI: http://dx.doi.org/10.7554/eLife.08153.001 PMID:26327694

Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the alpha6-integrin subunit.

PubMed

Kashimata, M; Gresik, E W

1997-02-01

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) regulate branching morphogenesis of fetal mouse submandibular gland (SMG) rudiments in vitro. The EGF system (EGF, TGF-alpha, and their shared receptor, EGFR) also regulates expression of integrins and their ligands in the extracellular matrix. We show here that inhibition of EGFR tyrosine-kinase activity by a tyrphostin retards in vitro development of SMGs. Using total RNA isolated from pooled SMGs taken from intact mouse fetuses, mRNA transcripts for EGF, TGF-alpha, and EGFR were detected by reverse transcription-polymerase chain reaction (RT-PCR), and age-dependent variations in the levels of these mRNA were quantitatively determined by nuclease protection assays. These findings suggest that the EGF system is operative in the in vivo development of this gland. alpha6-Integrin subunit was localized by immunofluorescence at the basal surface of epithelial cells. Branching morphogenesis of cultured SMG rudiments was inhibited by anti-alpha6 antibodies. Synthesis of alpha6-subunit in cultured SMGs, detected by metabolic labeling and immunoprecipitation, was increased by EGF and drastically reduced by tyrphostin. RT-PCR revealed that mRNAs for alpha6- and beta1- and beta4-integrin subunits are expressed at all ages between embryonic day 13 and postnatal day 7. These findings suggest that 1) the EGF system is a physiologic regulator of development of fetal mouse SMG, and 2) one mechanism by which it acts may be by regulating expression of integrins, which in turn control interaction of epithelial cells with the extracellular matrix.

A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

PubMed Central

Méthot, N; Song, M S; Sonenberg, N

1996-01-01

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3. PMID:8816444

The atypical two-subunit σ factor from Bacillus subtilis is regulated by an integral membrane protein and acid stress.

PubMed

Davis, Maria C; Smith, Logan K; MacLellan, Shawn R

2016-02-01

Extracytoplasmic function (ECF) σ factors constitute a major component of the physicochemical sensory apparatus in bacteria. Most ECF σ factors are co-expressed with a negative regulator called an anti-σ factor that binds to its cognate σ factor and sequesters it from productive association with core RNA polymerase (RNAP). Anti-σ factors constitute an important element of signal transduction pathways that mediate an appropriate transcriptional response to changing environmental conditions. The Bacillus subtilis genome encodes seven canonical ECF σ factors and six of these are co-expressed with experimentally verified anti-σ factors. B. subtilis also expresses an ECF-like atypical two-subunit σ factor composed of subunits SigO and RsoA that becomes active after exposure to certain cell-wall-acting antibiotics and to growth under acidic conditions. This work describes the identification and preliminary characterization of a protein (RsiO, formerly YvrL) that constitutes the anti-σ factor cognate to SigO-RsoA. Synthesis of RsiO represses SigO-RsoA-dependent transcription initiation by binding the N-terminus of SigO under neutral (pH 7) conditions. Reconstitution of the SigO-RsoA-RsiO regulatory system into a heterologous host reveals that the imposition of acid stress (pH 5.4) abolishes the ability of RsiO to repress SigO-RsoA-dependent transcription and this correlates with loss of RsiO binding affinity for SigO. A current model for RsiO function indicates that RsiO responds, either directly or indirectly, to increased extracytoplasmic hydrogen ion concentration and becomes inactivated. This results in the release of SigO into the cytoplasm, where it productively associates with RsoA and core RNAP to initiate transcription from target promoters in the cell.

Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone alpha subunit gene expression.

PubMed

Sato, Takanobu; Kitahara, Kousuke; Susa, Takao; Kato, Takako; Kato, Yukio

2006-10-01

Recently, we have reported that a Prophet of Pit-1 homeodomain factor, Prop-1, is a novel transcription factor for the porcine follicle-stimulating hormone beta subunit (FSHbeta) gene. This study subsequently aimed to examine the role of Prop-1 in the gene expression of two other porcine gonadotropin subunits, pituitary glycoprotein hormone alpha subunit (alphaGSU), and luteinizing hormone beta subunit (LHbeta). A series of deletion mutants of the porcine alphaGSU (up to -1059 bp) and LHbeta (up to -1277 bp) promoters were constructed in the reporter vector, fused with the secreted alkaline phosphatase gene (pSEAP2-Basic). Transient transfection studies using GH3 cells were carried out to estimate the activation of the porcine alphaGSU and LHbeta promoters by Prop-1, which was found to activate the alphaGSU promoter of -1059/+12 bp up to 11.7-fold but not the LHbeta promoter. Electrophoretic mobility shift assay and DNase I footprinting analysis revealed that Prop-1 binds to six positions, -1038/-1026, -942/-928, -495/-479, -338/-326, -153/-146, and -131/-124 bp, that comprise the A/T cluster. Oligonucleotides of six Prop-1 binding sites were directly connected to the minimum promoter of alphaGSU, fused in the pSEAP2-Basic vector, followed by transfecting GH3 cells to determine the cis-acting activity. Finally, we concluded that at least five Prop-1 binding sites are the cis-acting elements for alphaGSU gene expression. The present results revealed a notable feature of the proximal region, where three Prop-1-binding sites are close to and/or overlap the pituitary glycoprotein hormone basal element, GATA-binding element, and junctional regulatory element. To our knowledge, this is the first demonstration of the role of Prop-1 in the regulation of alphaGSU gene expression. These results, taken together with our previous finding that Prop-1 is a transcription factor for FSHbeta gene, confirm that Prop-1 modulates the synthesis of FSH at the transcriptional level. On the other hand, the defects of Prop-1 are known to cause dwarfism and combined pituitary hormone deficiency accompanying hypogonadism. Accordingly, the present observations provide a novel view to understand the hypogonadism caused by Prop-1 defects at the molecular level through the regulatory mechanism of alphaGSU and FSHbeta gene expressions.

[Structure and function of eukaryotic nuclear DNA-dependent RNA polymerase I].

PubMed

Shematorova, E K; Shpakovskiĭ, G V

2002-01-01

In the eukaryotic cell, normal protein biosynthesis is sustained by several million ribosomes, which contain rRNA as an essential component. The high-molecular-weight precursor of large and 5.8S rRNAs is synthesized by DNA-dependent RNA polymerase I (Pol I) in the nucleolus. Data on DNA regulatory elements, protein factors involved in rDNA transcription by Pol I, subunit composition of Pol I, and on the interactions and possible functions of individual subunits are summarized.

Solute removal capacity of high cut-off membrane plasma separators.

PubMed

Ohkubo, Atsushi; Kurashima, Naoki; Nakamura, Ayako; Miyamoto, Satoko; Iimori, Soichiro; Rai, Tatemitsu

2013-10-01

In vitro blood filtration was performed by a closed circuit using high cut-off membrane plasma separators, EVACURE EC-2A10 (EC-2A) and EVACURE EC-4A10 (EC-4A). Samples were obtained from sampling sites before the plasma separator, after each plasma separator, and from the ultrafiltrate of each separator. The sieving coefficient (S.C.) of total protein (TP), albumin (Alb), IgG, interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), fibrinogen (Fib), antithrombin III (AT-III), and coagulation factor XIII (FXIII) were calculated. The S.C. of each solute using EC-2A and EC-A4 were as follows; TP: 0.25 and 0.56, Alb: 0.32 and 0.73, IgG: 0.16 and 0.50, IL-6:0.73 and 0.95, IL-8:0.85 and 0.82, TNF-α: 1.07 and 0.99, Fib: 0 and 0, FXIII: 0.07 and 0.17, respectively. When compared with the conventional type of membrane plasma separators, EVACURE could efficiently remove cytokines while retaining coagulation factors such as fibrinogen. Moreover, EC-2A prevented protein loss, whereas EC-4A could remove approximately 50% of IgG. © 2013 The Authors. Therapeutic Apheresis and Dialysis © 2013 International Society for Apheresis.

Rrp5p, Noc1p and Noc2p form a protein module which is part of early large ribosomal subunit precursors in S. cerevisiae

PubMed Central

Hierlmeier, Thomas; Merl, Juliane; Sauert, Martina; Perez-Fernandez, Jorge; Schultz, Patrick; Bruckmann, Astrid; Hamperl, Stephan; Ohmayer, Uli; Rachel, Reinhard; Jacob, Anja; Hergert, Kristin; Deutzmann, Rainer; Griesenbeck, Joachim; Hurt, Ed; Milkereit, Philipp; Baßler, Jochen; Tschochner, Herbert

2013-01-01

Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p–Noc1p–Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity. PMID:23209026

Deceptive Tactics for Protecting Cities Against Vehicle Borne Improvised Explosive Devices

DTIC Science & Technology

2008-03-01

burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instruction, searching...INTENTIONALLY LEFT BLANK xiii LIST OF ABBREVIATIONS ABS Agent Based Simulation ANA Agent Network Attack DVF Detection Value Function GIS Geographic...any other behavior (than perceptive) may be advantageous to the attacker. - A communicative behavior proves particularly effective over time for the

Utopia, University and Architecture: A Journey that Changed the Design of Contemporary Universities

ERIC Educational Resources Information Center

Calvo-Sotelo, Pablo Campos

2006-01-01

In 1927, a group of advisors to King Alfonso XIII of Spain, led by the architect Modesto Lopez-Otero, set out for the United States and Canada. Previously, they had visited a number of European cities where they examined the medieval architectural form of some famous universities. Inspired by a Utopian vision, the journey to the New World studied…

The Federal Role in Improving Elementary and Secondary Education

DTIC Science & Technology

1993-05-01

programs of teacher educa- to consider the desirability and feasibility of S. ,,,,,,, ,,,,,, ,, .,. . , .,.. . SUMMARY Xiii national academic standards and...of a volun- ceived increased scrutiny during the 1980s be- tary system of assessments to measure the cause many people believed that the academic ...critics charging that they attain, as measured by standardized tests of have little empirical validity, that they may academic knowledge. These outcomes

Foundry Manual

DTIC Science & Technology

1958-01-01

ramming main part of mold or loose mold- ing sand used to support green cores while baking. Bail. Hoop or connection between the crane hook ...Crystallization 3 Heat Transfer 4 Gases in Metals 5 Summary 5 Chapter II. Designing a Casting 15 Strength Requirements 15 Stress Concentrations 15... Stress -Relief Anneal 180 Reasons for Heat Treatment 179 Aluminum 181 Iron and Steel 181 Monel 183 Summary 183 Chapter XIII. Composition of

Catholic Labor Education and the Association of Catholic Trade Unionists. Instructing Workers to Christianize the Workplace

ERIC Educational Resources Information Center

Lubienecki, Paul

2015-01-01

This article analyzes the effect of the American Catholic Church, through its program of specialized labor education, on the growth and development of organized labor in the twentieth century. With the proclamation of Pope Pius XI's encyclical Quadragesimo Anno (1931), he requested that the Church complete the work began by Pope Leo XIII in 1891…

Implementing and Evaluating an Innovative Approach to Simulation Training Acquisitions

DTIC Science & Technology

2006-01-01

busi- ness model, compares it with other approaches for buying simulations and simulation training, reviews economic theories relevant to the model, and...Points in Common with Other Approaches but Also Some Distinctive Characteristics ........................... 53 Contents vii CHAPTER FOUR The Economic ...Appropriate? .................... 65 4.3. Summary of Key Findings from Economic Theory .............. 72 xiii Summary In the wake of the failure of the Joint

Comparative Analysis of a High Bypass Turbofan Using a Pulsed Detonation Combustor

DTIC Science & Technology

2007-03-01

Thrust Specific Fuel Consumption . . . . . . . . . . . . . 67 xiii List of Abbreviations Abbreviation Page PDE Pulsed Detonation Engine...past ten years to develop pulsed det- onation engines ( PDE ) as a means of aircraft propulsion. Detonation combustion holds the promise of a more...aviation engine, and detonation creates more of it than previous aircraft engines. It is hoped that a marriage of the PDE with traditional

Tourism in Peru: The Missing Strategy for Economic and Social Development

DTIC Science & Technology

2006-06-01

Lima , 1970-1996.......................................12 xii THIS PAGE INTENTIONALLY LEFT BLANK xiii...time. Not two miles outside of Lima did things not only change, but they changed drastically. High rise buildings, upscale hotels, and very chic...natural resources. This is the case for Peru. Not only does the country hold a wealth of minerals, but it also possesses a plethora of beaches

Full-Authority Fault-Tolerant Electronic Engine Control System for Variable Cycle Engines.

DTIC Science & Technology

1982-04-01

single internally self-checked VLSI micro - processor . The selected configuration is an externally checked pair of com- mercially available...Electronic Engine Control FPMH Failures per Million Hours FTMP Fault Tolerant Multi- Processor FTSC Fault Tolerant Spaceborn Computer GRAMP Generalized...Removal * MTBR Mean Time Between Repair MTTF Mean Time to Failure xiii List of Abbreviations (continued) - NH High Pressure Rotor Speed O&S Operating

Understanding the Dynamic System of Terrorist-Government Interaction

DTIC Science & Technology

2003-03-01

organizations out of support or for services rendered (Department of State, 2002:65; Wallace, 2003:2). However, financial support does not have to be in...America’s critical infrastructure encompasses a large number of sectors[:]” agriculture, food, water, public health, emergency services , government...military, international law enforcement community, or the U.S. or international intelligence services (Department of State, 2002:vii-xiii). In fact

Proceedings of the Annual Meeting of the Association for Education in Journalism and Mass Communication (74th, Boston, Massachusetts, August 7-10, 1991). Part XIII: Studies on Television.

ERIC Educational Resources Information Center

Association for Education in Journalism and Mass Communication.

The Studies on Television section of the proceedings contains the following 16 papers: "Television and the Cultivation of Perceptions about Racial Integration" (Paul Delva and others); "Breaking the News: The Ideology of Television News" (Paul Belgrade); "Whittle's Channel One and CNN's Newsroom: A Systematic Pilot Study…

Analysis of Selected Enhancements for Soil Vapor Extraction

DTIC Science & Technology

1997-09-01

Inches per second ACRONYMS AND ABBREVIATIONS (Continued) xiii ISB In situ bioremediation JFK John F. Kennedy Airport K Hydraulic conductivity KAI KAI...wells by an applied vacuum. RFH is effective for treating VOCs in low-permeability soil in the vadose zone. Electrical Resistance Heating : This... applied vacuum. However, application of steam injection/stripping systems is limited to medium- to high-permeability soils. ER heating is more

Proceedings of the Annual Meeting of the Association for Education in Journalism and Mass Communication (76th, Kansas City, Missouri, August 11-14, 1993). Part XIII: Miscellaneous Studies.

ERIC Educational Resources Information Center

Association for Education in Journalism and Mass Communication.

The Miscellaneous Studies section of this collection of conference presentations contains the following 12 papers: "Revelatory Silences: A Critical Analysis of the Structuring Absences in Sydney Pollack's 'Out of Africa'" (Brenda Cooper and David Descutner); "The Cox Fight with the FCC: Gamesmanship, Hidden Agendas, and Personal…

Stereo photo series for quantifying natural fuels.Volume XIII: grasslands, shrublands, oak-bay woodlands, and eucalyptus forests in the East Bay of California.

Treesearch

Clinton S. Wright; Robert E. Vihnanek

2014-01-01

Four series of photographs display a range of natural conditions and fuel loadings for grassland, shrubland, oak-bay woodland, and eucalyptus forest ecosystems on the eastern slopes of the San Francisco Bay area of California. Each group of photos includes inventory information summarizing vegetation composition, structure, and loading; woody material loading and...

Design and Implementation of a Motor Incremental Shaft Encoder

DTIC Science & Technology

2008-09-01

SDC Student Design Center VHDL Verilog Hardware Description Language VSC Voltage Source Converters ZCE Zero Crossing Event xiii EXECUTIVE...student to make accurate predictions of voltage source converters ( VSC ) behavior via software simulation; these simulated results could also be... VSC ), and several other off-the-shelf components, a circuit board interface between FPGA and the power source, and a desktop computer [1]. Now, the

With Agriculture Knowledge and Wisdom (Me Ka Ike Ame Ka Na'auao). Proceedings of the Annual Western Region Agricultural Education Research Meeting (Honolulu, Hawaii, April 13-16, 1994). Volume XIII, Number 1.

ERIC Educational Resources Information Center

Cox, David E., Ed.; Walton, Frank C., Ed.

This proceedings includes the following papers: "Examining Learning Styles of Students in College of Agriculture" (Torres, Cano); "Developing a Scale to Research and Evaluate Youth Leadership Life Skills Development" (Seevers, Dormody, Clason); "Predicting Youth Leadership Life Skills Development among FFA (Future Farmers…

Technical Modification Within the Healthcare Industry: Improving Both the Efficacy of the National Drug Code Carrier and the Accessibility of Electronic Health Records to Reduce Adverse Drug Events

DTIC Science & Technology

2013-06-01

with an EHR .................................................. 97 C. SWOT ANALYSIS OF USING QR CODES WITH THE NDC AND WITH EHRS...96 Figure 41. SWOT analysis ................................................................................... 99 xiii LIST OF...Coordinator for Health Information Technology OTC Over-the-Counter PHI Personal Health Information QR Quick Response SWOT Strengths, Weaknesses

Proceedings of the Annual Meeting of the Association for Education in Journalism and Mass Communication (75th, Montreal, Quebec, Canada, August 5-8, 1992). Part XIII: Magazines.

ERIC Educational Resources Information Center

Association for Education in Journalism and Mass Communication.

The Magazine section of these proceedings contains the following six papers: "An Analysis of 'Magazine Type': Toward an Empirically Based Typology of Magazines and Non-Newspaper Periodicals" (Marcia R. Prior-Miller); "'Redbook': Changes in Attitude and Advice 1965-1990" (Jennifer Harbour); "A Quantitative Analysis of U.S.…

AUTOMOTIVE DIESEL MAINTENANCE 1. UNIT XIII, I--MAINTAINING THE FUEL SYSTEM (PART III), CUMMINS DIESEL ENGINES, II--RADIATOR SHUTTER SYSTEM.

ERIC Educational Resources Information Center

Human Engineering Inst., Cleveland, OH.

THIS MODULE OF A 30-MODULE COURSE IS DESIGNED TO DEVELOP AN UNDERSTANDING OF THE CONSTRUCTION, OPERATION, AND MAINTENANCE OF THE DIESEL ENGINE FUEL AND RADIATOR SHUTTER SYSTEMS. TOPICS ARE (1) MORE ABOUT THE CUMMINS FUEL SYSTEM, (2) CALIBRATING THE PT FUEL PUMP, (3) CALIBRATING THE FUEL INJECTORS, (4) UNDERSTANDING THE SHUTTER SYSTEM, (5) THE…

Scaling Bulk Data Analysis with Mapreduce

DTIC Science & Technology

2017-09-01

Submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN COMPUTER SCIENCE from the NAVAL POSTGRADUATE SCHOOL September...2017 Approved by: Michael McCarrin Thesis Co-Advisor Marcus S. Stefanou Thesis Co-Advisor Peter J. Denning Chair, Department of Computer Science iii...98 xiii THIS PAGE INTENTIONALLY LEFT BLANK xiv List of Acronyms and Abbreviations CART Computer Analysis and Response Team DELV Distributed Environment

The AMP-activated protein kinase beta 1 subunit modulates erythrocyte integrity.

PubMed

Cambridge, Emma L; McIntyre, Zoe; Clare, Simon; Arends, Mark J; Goulding, David; Isherwood, Christopher; Caetano, Susana S; Reviriego, Carmen Ballesteros; Swiatkowska, Agnieszka; Kane, Leanne; Harcourt, Katherine; Adams, David J; White, Jacqueline K; Speak, Anneliese O

2017-01-01

Failure to maintain a normal in vivo erythrocyte half-life results in the development of hemolytic anemia. Half-life is affected by numerous factors, including energy balance, electrolyte gradients, reactive oxygen species, and membrane plasticity. The heterotrimeric AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that acts as a critical regulator of cellular energy balance. Previous roles for the alpha 1 and gamma 1 subunits in the control of erythrocyte survival have been reported. In the work described here, we studied the role of the beta 1 subunit in erythrocytes and observed microcytic anemia with compensatory extramedullary hematopoiesis together with splenomegaly and increased osmotic resistance. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy

PubMed Central

Kuschal, Christiane; Botta, Elena; Orioli, Donata; Digiovanna, John J.; Seneca, Sara; Keymolen, Kathelijn; Tamura, Deborah; Heller, Elizabeth; Khan, Sikandar G.; Caligiuri, Giuseppina; Lanzafame, Manuela; Nardo, Tiziana; Ricotti, Roberta; Peverali, Fiorenzo A.; Stephens, Robert; Zhao, Yongmei; Lehmann, Alan R.; Baranello, Laura; Levens, David; Kraemer, Kenneth H.; Stefanini, Miria

2016-01-01

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP. PMID:26996949

Xanthomonas TAL effectors hijack host basal transcription factor IIA α and γ subunits for invasion.

PubMed

Ma, Ling; Wang, Qiang; Yuan, Meng; Zou, Tingting; Yin, Ping; Wang, Shiping

2018-02-05

The Xanthomonas genus includes Gram-negative plant-pathogenic bacteria, which infect a broad range of crops and wild plant species, cause symptoms with leaf blights, streaks, spots, stripes, necrosis, wilt, cankers and gummosis on leaves, stems and fruits in a wide variety of plants via injecting their effector proteins into the host cell during infection. Among these virulent effectors, transcription activator-like effectors (TALEs) interact with the γ subunit of host transcription factor IIA (TFIIAγ) to activate the transcription of host disease susceptibility genes. Functional TFIIA is a ternary complex comprising α, β and γ subunits. However, whether TALEs recruit TFIIAα, TFIIAβ, or both remains unknown. The underlying molecular mechanisms by which TALEs mediate host susceptibility gene activation require full elucidation. Here, we show that TALEs interact with the α+γ binary subcomplex but not the α+β+γ ternary complex of rice TFIIA (holo-OsTFIIA). The transcription factor binding (TFB) regions of TALEs, which are highly conserved in Xanthomonas species, have a dominant role in these interactions. Furthermore, the interaction between TALEs and the α+γ complex exhibits robust DNA binding activity in vitro. These results collectively demonstrate that TALE-carrying pathogens hijack the host basal transcription factors TFIIAα and TFIIAγ, but not TFIIAβ, to enhance host susceptibility during pathogen infection. The uncovered mechanism widens new insights on host-microbe interaction and provide an applicable strategy to breed high-resistance crop varieties. Copyright © 2018 Elsevier Inc. All rights reserved.

NOS1 mediates AP1 nuclear translocation and inflammatory response.

PubMed

Srivastava, Mansi; Baig, Mirza S

2018-06-01

A hallmark of the AP1 functioning is its nuclear translocation, which induces proinflammatory cytokine expression and hence the inflammatory response. After endotoxin shock AP1 transcription factor, which comprises Jun, ATF2, and Fos family of proteins, translocates into the nucleus and induces proinflammatory cytokine expression. In the current study, we found, NOS1 inhibition prevents nuclear translocation of the AP1 transcription factor subunits. Pharmacological inhibition of NOS1 impedes translocation of subunits into the nucleus, suppressing the transcription of inflammatory genes causing a diminished inflammatory response. In conclusion, the study shows the novel mechanism of NOS1- mediated AP1 nuclear translocation, which needs to be further explored. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

AP-1 subunits: quarrel and harmony among siblings.

PubMed

Hess, Jochen; Angel, Peter; Schorpp-Kistner, Marina

2004-12-01

The AP-1 transcription factor is mainly composed of Jun, Fos and ATF protein dimers. It mediates gene regulation in response to a plethora of physiological and pathological stimuli, including cytokines, growth factors, stress signals, bacterial and viral infections, as well as oncogenic stimuli. Studies in genetically modified mice and cells have highlighted a crucial role for AP-1 in a variety of cellular events involved in normal development or neoplastic transformation causing cancer. However, emerging evidence indicates that the contribution of AP-1 to determination of cell fates critically depends on the relative abundance of AP-1 subunits, the composition of AP-1 dimers, the quality of stimulus, the cell type and the cellular environment. Therefore, AP-1-mediated regulation of processes such as proliferation, differentiation, apoptosis and transformation should be considered within the context of a complex dynamic network of signalling pathways and other nuclear factors that respond simultaneously.

Mechanism of repression of the inhibin alpha-subunit gene by inducible 3',5'-cyclic adenosine monophosphate early repressor.

PubMed

Burkart, Anna D; Mukherjee, Abir; Mayo, Kelly E

2006-03-01

The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.

The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis.

PubMed

Aoubala, M; Holt, J; Clegg, R A; Rowlands, D J; Harris, M

2001-07-01

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

RNA Silencing of Exocyst Genes in the Stigma Impairs the Acceptance of Compatible Pollen in Arabidopsis1[OPEN

PubMed Central

Safavian, Darya; Indriolo, Emily; Chapman, Laura; Ahmed, Abdalla

2015-01-01

Initial pollen-pistil interactions in the Brassicaceae are regulated by rapid communication between pollen grains and stigmatic papillae and are fundamentally important, as they are the first step toward successful fertilization. The goal of this study was to examine the requirement of exocyst subunits, which function in docking secretory vesicles to sites of polarized secretion, in the context of pollen-pistil interactions. One of the exocyst subunit genes, EXO70A1, was previously identified as an essential factor in the stigma for the acceptance of compatible pollen in Arabidopsis (Arabidopsis thaliana) and Brassica napus. We hypothesized that EXO70A1, along with other exocyst subunits, functions in the Brassicaceae dry stigma to deliver cargo-bearing secretory vesicles to the stigmatic papillar plasma membrane, under the pollen attachment site, for pollen hydration and pollen tube entry. Here, we investigated the functions of exocyst complex genes encoding the remaining seven subunits, SECRETORY3 (SEC3), SEC5, SEC6, SEC8, SEC10, SEC15, and EXO84, in Arabidopsis stigmas following compatible pollinations. Stigma-specific RNA-silencing constructs were used to suppress the expression of each exocyst subunit individually. The early postpollination stages of pollen grain adhesion, pollen hydration, pollen tube penetration, seed set, and overall fertility were analyzed in the transgenic lines to evaluate the requirement of each exocyst subunit. Our findings provide comprehensive evidence that all eight exocyst subunits are necessary in the stigma for the acceptance of compatible pollen. Thus, this work implicates a fully functional exocyst complex as a component of the compatible pollen response pathway to promote pollen acceptance. PMID:26443677

Bovine papillomavirus type 2 (BPV-2) E5 oncoprotein binds to the subunit D of the V₁-ATPase proton pump in naturally occurring urothelial tumors of the urinary bladder of cattle.

PubMed

Roperto, Sante; Russo, Valeria; Borzacchiello, Giuseppe; Urraro, Chiara; Lucà, Roberta; Esposito, Iolanda; Riccardi, Marita Georgia; Raso, Cinzia; Gaspari, Marco; Ceccarelli, Dora Maria; Galasso, Rocco; Roperto, Franco

2014-01-01

Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers. In all cancer samples, PCR analysis revealed a BPV-2-specific 503 bp DNA fragment. E5 protein, the major oncoprotein of the virus, was shown both by immunoprecipitation and immunohistochemical analysis. E5 was found to bind to the activated (phosphorylated) form of the platelet derived growth factor β receptor. PDGFβR immunoprecipitation from bladder tumor samples and from normal bladder tissue used as control revealed a protein band which was present in the pull-down from bladder cancer samples only. The protein was identified with mass spectrometry as "V₁-ATPase subunit D", a component of the central stalk of the V₁-ATPase vacuolar pump. The subunit D was confirmed in this complex by coimmunoprecipitation investigations and it was found to colocalize with the receptor. The subunit D was also shown to be overexpressed by Western blot, RT-PCR and immunofluorescence analyses. Immunoprecipitation and immunofluorescence also revealed that E5 oncoprotein was bound to the subunit D. For the first time, a tri-component complex composed of E5/PDGFβR/subunit D has been documented in vivo. Previous in vitro studies have shown that the BPV-2 E5 oncoprotein binds to the proteolipid c ring of the V₀-ATPase sector. We suggest that the E5/PDGFβR/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses.

The gamma subunit of transducin is farnesylated.

PubMed Central

Lai, R K; Perez-Sala, D; Cañada, F J; Rando, R R

1990-01-01

Protein prenylation with farnesyl or geranylgeranyl moieties is an important posttranslational modification that affects the activity of such diverse proteins as the nuclear lamins, the yeast mating factor mata, and the ras oncogene products. In this article, we show that whole retinal cultures incorporate radioactive mevalonic acid into proteins of 23-26 kDa and one of 8 kDa. The former proteins are probably the "small" guanine nucleotide-binding regulatory proteins (G proteins) and the 8-kDa protein is the gamma subunit of the well-studied retinal heterotrimeric G protein (transducin). After deprenylating purified transducin and its subunits with Raney nickel or methyl iodide/base, the adducted prenyl group can be identified as an all-trans-farnesyl moiety covalently linked to a cysteine residue. Thus far, prenylation reactions have been found to occur at cysteine in a carboxyl-terminal consensus CAAX sequence, where C is the cysteine, A is an aliphatic amino acid, and X is undefined. Both the alpha and gamma subunits of transducin have this consensus sequence, but only the gamma subunit is prenylated. Therefore, the CAAX motif is not necessary and sufficient to direct prenylation. Finally, since transducin is the best understood G protein, both structurally and mechanistically, the discovery that it is farnesylated should allow for a quantitative understanding of this post-translational modification. Images PMID:2217200

Molecular oxygen migration through the xenon docking sites of human hemoglobin in the R-state.

PubMed

Lepeshkevich, Sergei V; Gilevich, Syargey N; Parkhats, Marina V; Dzhagarov, Boris M

2016-09-01

A nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O2) rebinding to tetrameric human hemoglobin and its isolated α and β chains in buffer solutions equilibrated with 1atm of air and up to 25atm of xenon. Xenon binding to the isolated α chains and to the α subunits within tetrameric hemoglobin was found to cause a decrease in the efficiency of O2 escape by a factor of ~1.30 and 3.3, respectively. A kinetic model for O2 dissociation, rebinding, and migration through two alternative pathways in the hemoglobin subunits was introduced and discussed. It was shown that, in the isolated α chains and α subunits within tetrameric hemoglobin, nearly one- and two-third escaping molecules of O2 leave the protein via xenon docking sites, respectively. The present experimental data support the idea that O2 molecule escapes from the β subunits mainly through the His(E7) gate, and show unambiguously that, in the α subunits, in addition to the direct E7 channel, there is at least one alternative escape route leading to the exterior via the xenon docking sites. Copyright © 2016 Elsevier B.V. All rights reserved.

MC EMiNEM maps the interaction landscape of the Mediator.

PubMed

Niederberger, Theresa; Etzold, Stefanie; Lidschreiber, Michael; Maier, Kerstin C; Martin, Dietmar E; Fröhlich, Holger; Cramer, Patrick; Tresch, Achim

2012-01-01

The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs), a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC) sampling with an Expectation-Maximization (EM) algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors.

NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62▿

PubMed Central

Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

2011-01-01

Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus. PMID:21543505

The ESCRT-III Subunit hVps24 Is Required for Degradation but Not Silencing of the Epidermal Growth Factor Receptor

PubMed Central

Bache, Kristi G.; Stuffers, Susanne; Malerød, Lene; Slagsvold, Thomas; Raiborg, Camilla; Lechardeur, Delphine; Wälchli, Sébastien; Lukacs, Gergely L.; Brech, Andreas; Stenmark, Harald

2006-01-01

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling. PMID:16554368

The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor.

PubMed

Bache, Kristi G; Stuffers, Susanne; Malerød, Lene; Slagsvold, Thomas; Raiborg, Camilla; Lechardeur, Delphine; Wälchli, Sébastien; Lukacs, Gergely L; Brech, Andreas; Stenmark, Harald

2006-06-01

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.

Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts.

PubMed

Taylor, David M; Kabashi, Edor; Agar, Jeffrey N; Minotti, Sandra; Durham, Heather D

2005-01-01

Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes.

Ribosomal protein L14 contributes to the early assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.

PubMed

Espinar-Marchena, Francisco; Rodríguez-Galán, Olga; Fernández-Fernández, José; Linnemann, Jan; de la Cruz, Jesús

2018-05-18

The contribution of most ribosomal proteins to ribosome synthesis has been quite well analysed in Saccharomyces cerevisiae. However, few yeast ribosomal proteins still await characterization. Herein, we show that L14, an essential 60S ribosomal protein, assembles in the nucleolus at an early stage into pre-60S particles. Depletion of L14 results in a deficit in 60S subunits and defective processing of 27SA2 and 27SA3 to 27SB pre-rRNAs. As a result, 27S pre-rRNAs are subjected to turnover and export of pre-60S particles is blocked. These phenotypes likely appear as the direct consequence of the reduced pre-60S particle association not only of L14 upon its depletion but also of a set of neighboring ribosomal proteins located at the solvent interface of 60S subunits and the adjacent region surrounding the polypeptide exit tunnel. These pre-60S intermediates also lack some essential trans-acting factors required for 27SB pre-rRNA processing but accumulate practically all factors required for processing of 27SA3 pre-rRNA. We have also analysed the functional interaction between the eukaryote-specific carboxy-terminal extensions of the neighboring L14 and L16 proteins. Our results indicate that removal of the most distal parts of these extensions cause slight translation alterations in mature 60S subunits.

Ribosomal protein L14 contributes to the early assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

PubMed Central

Espinar-Marchena, Francisco; Rodríguez-Galán, Olga; Fernández-Fernández, José; Linnemann, Jan; de la Cruz, Jesús

2018-01-01

Abstract The contribution of most ribosomal proteins to ribosome synthesis has been quite well analysed in Saccharomyces cerevisiae. However, few yeast ribosomal proteins still await characterization. Herein, we show that L14, an essential 60S ribosomal protein, assembles in the nucleolus at an early stage into pre-60S particles. Depletion of L14 results in a deficit in 60S subunits and defective processing of 27SA2 and 27SA3 to 27SB pre-rRNAs. As a result, 27S pre-rRNAs are subjected to turnover and export of pre-60S particles is blocked. These phenotypes likely appear as the direct consequence of the reduced pre-60S particle association not only of L14 upon its depletion but also of a set of neighboring ribosomal proteins located at the solvent interface of 60S subunits and the adjacent region surrounding the polypeptide exit tunnel. These pre-60S intermediates also lack some essential trans-acting factors required for 27SB pre-rRNA processing but accumulate practically all factors required for processing of 27SA3 pre-rRNA. We have also analysed the functional interaction between the eukaryote-specific carboxy-terminal extensions of the neighboring L14 and L16 proteins. Our results indicate that removal of the most distal parts of these extensions cause slight translation alterations in mature 60S subunits. PMID:29788267

Analysis of the SWI/SNF chromatin-remodeling complex during early heart development and BAF250a repression cardiac gene transcription during P19 cell differentiation

PubMed Central

Singh, Ajeet Pratap; Archer, Trevor K.

2014-01-01

The regulatory networks of differentiation programs and the molecular mechanisms of lineage-specific gene regulation in mammalian embryos remain only partially defined. We document differential expression and temporal switching of BRG1-associated factor (BAF) subunits, core pluripotency factors and cardiac-specific genes during post-implantation development and subsequent early organogenesis. Using affinity purification of BRG1 ATPase coupled to mass spectrometry, we characterized the cardiac-enriched remodeling complexes present in E8.5 mouse embryos. The relative abundance and combinatorial assembly of the BAF subunits provides functional specificity to Switch/Sucrose NonFermentable (SWI/SNF) complexes resulting in a unique gene expression profile in the developing heart. Remarkably, the specific depletion of the BAF250a subunit demonstrated differential effects on cardiac-specific gene expression and resulted in arrhythmic contracting cardiomyocytes in vitro. Indeed, the BAF250a physically interacts and functionally cooperates with Nucleosome Remodeling and Histone Deacetylase (NURD) complex subunits to repressively regulate chromatin structure of the cardiac genes by switching open and poised chromatin marks associated with active and repressed gene expression. Finally, BAF250a expression modulates BRG1 occupancy at the loci of cardiac genes regulatory regions in P19 cell differentiation. These findings reveal specialized and novel cardiac-enriched SWI/SNF chromatin-remodeling complexes, which are required for heart formation and critical for cardiac gene expression regulation at the early stages of heart development. PMID:24335282

Biogenesis of the bacterial cbb3 cytochrome c oxidase: Active subcomplexes support a sequential assembly model.

PubMed

Durand, Anne; Bourbon, Marie-Line; Steunou, Anne-Soisig; Khalfaoui-Hassani, Bahia; Legrand, Camille; Guitton, Audrey; Astier, Chantal; Ouchane, Soufian

2018-01-19

The cbb 3 oxidase has a high affinity for oxygen and is required for growth of bacteria, including pathogens, in oxygen-limited environments. However, the assembly of this oxidase is poorly understood. Most cbb 3 are composed of four subunits: the catalytic CcoN subunit, the two cytochrome c subunits (CcoO and CcoP) involved in electron transfer, and the small CcoQ subunit with an unclear function. Here, we address the role of these four subunits in cbb 3 biogenesis in the purple bacterium Rubrivivax gelatinosus Analyses of membrane proteins from different mutants revealed the presence of active CcoNQO and CcoNO subcomplexes and also showed that the CcoP subunit is not essential for their assembly. However, CcoP was required for the oxygen reduction activity in the absence of CcoQ. We also found that CcoQ is dispensable for forming an active CcoNOP subcomplex in membranes. CcoNOP exhibited oxygen reductase activity, indicating that the cofactors (hemes b and copper for CcoN and cytochromes c for CcoO and CcoP) were present within the subunits. Finally, we discovered the presence of a CcoNQ subcomplex and showed that CcoN is the required anchor for the assembly of the full CcoNQOP complex. On the basis of these findings, we propose a sequential assembly model in which the CcoQ subunit is required for the early maturation step: CcoQ first associates with CcoN before the CcoNQ-CcoO interaction. CcoP associates to CcoNQO subcomplex in the late maturation step, and once the CcoNQOP complex is fully formed, CcoQ is released for degradation by the FtsH protease. This model could be conserved in other bacteria, including the pathogenic bacteria lacking the assembly factor CcoH as in R. gelatinosus . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

The extreme halophyte Salicornia veneta is depleted of the extrinsic PsbQ and PsbP proteins of the oxygen-evolving complex without loss of functional activity

PubMed Central

Pagliano, Cristina; La Rocca, Nicoletta; Andreucci, Flora; Deák, Zsuzsanna; Vass, Imre; Rascio, Nicoletta; Barbato, Roberto

2009-01-01

Background and Aims Photosystem II of oxygenic organisms is a multi-subunit protein complex made up of at least 20 subunits and requires Ca2+ and Cl− as essential co-factors. While most subunits form the catalytic core responsible for water oxidation, PsbO, PsbP and PsbQ form an extrinsic domain exposed to the luminal side of the membrane. In vitro studies have shown that these subunits have a role in modulating the function of Cl− and Ca2+, but their role(s) in vivo remains to be elucidated, as the relationships between ion concentrations and extrinsic polypeptides are not clear. With the aim of understanding these relationships, the photosynthetic apparatus of the extreme halophyte Salicornia veneta has been compared with that of spinach. Compared to glycophytes, halophytes have a different ionic composition, which could be expected to modulate the role of extrinsic polypeptides. Methods Structure and function of in vivo and in vitro PSII in S. veneta were investigated and compared to spinach. Light and electron microscopy, oxygen evolution, gel electrophoresis, immunoblotting, DNA sequencing, RT–PCR and time-resolved chlorophyll fluorescence were used. Key Results Thylakoids of S. veneta did not contain PsbQ protein and its mRNA was absent. When compared to spinach, PsbP was partly depleted (30 %), as was its mRNA. All other thylakoid subunits were present in similar amounts in both species. PSII electron transfer was not affected. Fluorescence was strongly quenched upon irradiation of plants with high light, and relaxed only after prolonged dark incubation. Quenching of fluorescence was not linked to degradation of D1 protein. Conclusions In S. veneta the PsbQ protein is not necessary for photosynthesis in vivo. As the amount of PsbP is sub-stoichiometric with other PSII subunits, this protein too is largely dispensable from a catalytic standpoint. One possibility is that PsbP acts as an assembly factor for PSII. PMID:19033288

Fission yeast translation initiation factor 3 subunit eIF3h is not essential for global translation initiation, but deletion of eif3h+ affects spore formation.

PubMed

Ray, Anirban; Bandyopadhyay, Amitabha; Matsumoto, Tomohiro; Deng, Haiteng; Maitra, Umadas

2008-11-01

The fission yeast Schizosaccharomyces pombe homologue of the p40/eIF3h subunit of mammalian translation initiation factor eIF3 has been characterized in this study. We show that this protein physically associates with the 40S ribosomal particles as a constituent of the multimeric eIF3 protein complex, which consists of all five known eIF3 core subunits (eIF3a, eIF3b, eIF3c, eIF3g and eIF3i) as well as the five non-core subunits (eIF3d, eIF3e, eIF3f, eIF3h and eIF3m) that constitute an eIF3 holocomplex in fission yeast. However, affinity purification of eIF3 from fission yeast cells expressing TAP-tagged eIF3h suggests the presence of distinct forms of eIF3 that differ in their composition of the non-core subunits. Further characterization of eIF3h shows that strains lacking eif3h(+) (eif3hDelta) are viable and show no gross defects, either in vegetative growth or in the rate of in vivo protein synthesis. Polysome profile analysis shows no apparent defects in translation initiation. Furthermore, deletion of eif3h(+) does not affect the ability of the other eIF3 subunits to remain associated with one another in a tight protein complex similar to the situation in wild-type cells. Additionally, we show that human eIF3h can functionally substitute fission yeast eIF3h in complementing in vivo a genetic deletion of eif3h(+). Interestingly, mutant eif3hDelta cells show several prominent phenotypic properties. They are hypersensitive to caffeine and highly defective in meiosis, producing either no spores or incomplete tetrads with a very high frequency. The implications of these results in relation to the functions of eIF3h in Sz. pombe are discussed. (c) 2008 John Wiley & Sons, Ltd.

Intracranial hemorrhage in congenital bleeding disorders.

PubMed

Tabibian, Shadi; Motlagh, Hoda; Naderi, Majid; Dorgalaleh, Akbar

2018-01-01

: Intracranial hemorrhage (ICH), as a life-threatening bleeding among all kinds of congenital bleeding disorders (CBDs), is a rare manifestation except in factor XIII (FXIII) deficiency, which is accompanied by ICH, early in life, in about one-third of patients. Most inherited platelet function disorders (IPFDs) are mild to moderate bleeding disorders that can never experience a severe bleeding as in ICH; however, Glanzmann's thrombasthenia, a common and severe inherited platelet function disorder, can lead to ICH and occasional death. This bleeding feature can also be observed in grey platelet syndrome, though less frequently than in Glanzmann's thrombasthenia. In hemophilia, intracerebral hemorrhage is affected by various risk factors one of which is the severity of the disease. The precise prevalence of ICH in these patients is not clear but an estimated incidence of 3.5-4% among newborns with hemophilia is largely ascertained. Although ICH is a rare phenomenon in CBDs, it can be experienced by every patient with severe hemophilia A and B, FXIII deficiency (FXIIID), FVIID, FXD, FVD, FIID, and afibrinogenemia. Upon observing the general signs and symptoms of ICH such as vomiting, seizure, unconsciousness, and headache, appropriate replacement therapies and cranial ultrasound scans must be done to decrease ICH-related morbidity and mortality.

Evolution of a tissue-specific splicing network

PubMed Central

Taliaferro, J. Matthew; Alvarez, Nehemiah; Green, Richard E.; Blanchette, Marco; Rio, Donald C.

2011-01-01

Alternative splicing of precursor mRNA (pre-mRNA) is a strategy employed by most eukaryotes to increase transcript and proteomic diversity. Many metazoan splicing factors are members of multigene families, with each member having different functions. How these highly related proteins evolve unique properties has been unclear. Here we characterize the evolution and function of a new Drosophila splicing factor, termed LS2 (Large Subunit 2), that arose from a gene duplication event of dU2AF50, the large subunit of the highly conserved heterodimeric general splicing factor U2AF (U2-associated factor). The quickly evolving LS2 gene has diverged from the splicing-promoting, ubiquitously expressed dU2AF50 such that it binds a markedly different RNA sequence, acts as a splicing repressor, and is preferentially expressed in testes. Target transcripts of LS2 are also enriched for performing testes-related functions. We therefore propose a path for the evolution of a new splicing factor in Drosophila that regulates specific pre-mRNAs and contributes to transcript diversity in a tissue-specific manner. PMID:21406555

Oxidative stress-induced miR-27a targets the redox gene nuclear factor erythroid 2-related factor 2 in diabetic embryopathy.

PubMed

Zhao, Yang; Dong, Daoyin; Reece, E Albert; Wang, Ashley R; Yang, Peixin

2018-01-01

Maternal diabetes induces neural tube defects, and oxidative stress is a causal factor for maternal diabetes-induced neural tube defects. The redox gene nuclear factor erythroid 2-related factor 2 is the master regulator of the cellular antioxidant system. In this study, we aimed to determine whether maternal diabetes inhibits nuclear factor erythroid 2-related factor 2 expression and nuclear factor erythroid 2-related factor 2-controlled antioxidant genes through the redox-sensitive miR-27a. We used a well-established type 1 diabetic embryopathy mouse model induced by streptozotocin for our in vivo studies. Embryos at embryonic day 8.5 were harvested for analysis of nuclear factor erythroid 2-related factor 2, nuclear factor erythroid 2-related factor 2-controlled antioxidant genes, and miR-27a expression. To determine if mitigating oxidative stress inhibits the increase of miR-27a and the decrease of nuclear factor erythroid 2-related factor 2 expression, we induced diabetic embryopathy in superoxide dismutase 2 (mitochondrial-associated antioxidant gene)-overexpressing mice. This model exhibits reduced mitochondria reactive oxygen species even in the presence of hyperglycemia. To investigate the causal relationship between miR-27a and nuclear factor erythroid 2-related factor 2 in vitro, we examined C17.2 neural stem cells under normal and high-glucose conditions. We observed that the messenger RNA and protein levels of nuclear factor erythroid 2-related factor 2 were significantly decreased in embryos on embryonic day 8.5 from diabetic dams compared to those from nondiabetic dams. High-glucose also significantly decreased nuclear factor erythroid 2-related factor 2 expression in a dose- and time-dependent manner in cultured neural stem cells. Our data revealed that miR-27a was up-regulated in embryos on embryonic day 8.5 exposed to diabetes, and that high glucose increased miR-27a levels in a dose- and time-dependent manner in cultured neural stem cells. In addition, we found that a miR-27a inhibitor abrogated the inhibitory effect of high glucose on nuclear factor erythroid 2-related factor 2 expression, and a miR-27a mimic suppressed nuclear factor erythroid 2-related factor 2 expression in cultured neural stem cells. Furthermore, our data indicated that the nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1 were down-regulated by maternal diabetes in embryos on embryonic day 8.5 and high glucose in cultured neural stem cells. Inhibiting miR-27a restored expression of glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1. Overexpressing superoxide dismutase 2 reversed the maternal diabetes-induced increase of miR-27a and suppression of nuclear factor erythroid 2-related factor 2 and nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes. Our study demonstrates that maternal diabetes-induced oxidative stress increases miR-27a, which, in turn, suppresses nuclear factor erythroid 2-related factor 2 and its responsive antioxidant enzymes, resulting in diabetic embryopathy. Copyright © 2017 Elsevier Inc. All rights reserved.

Phylogeny, divergence time and historical biogeography of Laetiporus (Basidiomycota, Polyporales).

PubMed

Song, Jie; Cui, Bao-Kai

2017-04-20

The aim of this study was to characterize the molecular relationship, origin and historical biogeography of the species in important brown rot fungal genus Laetiporus from East Asia, Europe, Pan-America, Hawaii and South Africa. We used six genetic markers to estimate a genus-level phylogeny including (1) the internal transcribed spacer (ITS), (2) nuclear large subunit rDNA (nrLSU), (3) nuclear small subunit rDNA (nrSSU), (4) translation elongation factor 1-α (EF-1α), (5) DNA-directed RNA polymerase II subunit 2 (RPB2), and (6) mitochondrial small subunit rDNA (mtSSU). Results of multi-locus phylogenetic analyses show clade support for at least seventeen species-level lineages including two new Laetiporus in China. Molecular dating using BEAST estimated the present crown group diverged approximately 20.16 million years ago (Mya) in the early Miocene. Biogeographic analyses using RASP indicated that Laetiporus most likely originated in temperate zones with East Asia and North America having the highest probability (48%) of being the ancestral area. Four intercontinental dispersal routes and a possible concealed dispersal route were established for the first time.

PsB multiprotein complex of Dictyostelium discoideum. Demonstration of cellulose binding activity and order of protein subunit assembly.

PubMed

McGuire, V; Alexander, S

1996-06-14

The differentiated spores of Dictyostelium are surrounded by an extracellular matrix, the spore coat, which protects them from environmental factors allowing them to remain viable for extended periods of time. This presumably is a major evolutionary advantage. This unique extracellular matrix is composed of cellulose and glycoproteins. Previous work has shown that some of these spore coat glycoproteins exist as a preassembled multiprotein complex (the PsB multiprotein complex) which is stored in the prespore vesicles (Watson, N., McGuire, V., and Alexander, S (1994) J. Cell Sci. 107, 2567-2579). Later in development, the complex is synchronously secreted from the prespore vesicles and incorporated into the spore coat. We now have shown that the PsB complex has a specific in vitro cellulose binding activity. The analysis of mutants lacking individual subunits of the PsB complex revealed the relative order of assembly of the subunit proteins and demonstrated that the protein subunits must be assembled for cellulose binding activity. These results provide a biochemical explanation for the localization of this multiprotein complex in the spore coat.

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits.

PubMed

Valkova, Christina; Albrizio, Marina; Röder, Ira V; Schwake, Michael; Betto, Romeo; Rudolf, Rüdiger; Kaether, Christoph

2011-01-11

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.

Structural characterization of ribosome recruitment and translocation by type IV IRES.

PubMed

Murray, Jason; Savva, Christos G; Shin, Byung-Sik; Dever, Thomas E; Ramakrishnan, V; Fernández, Israel S

2016-05-09

Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts tvhe otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (Fernández et al., 2014; Koh et al., 2014), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation.

TFIIH subunit alterations causing xeroderma pigmentosum and trichothiodystrophy specifically disturb several steps during transcription.

PubMed

Singh, Amita; Compe, Emanuel; Le May, Nicolas; Egly, Jean-Marc

2015-02-05

Mutations in genes encoding the ERCC3 (XPB), ERCC2 (XPD), and GTF2H5 (p8 or TTD-A) subunits of the transcription and DNA-repair factor TFIIH lead to three autosomal-recessive disorders: xeroderma pigmentosum (XP), XP associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTD). Although these diseases were originally associated with defects in DNA repair, transcription deficiencies might be also implicated. By using retinoic acid receptor beta isoform 2 (RARB2) as a model in several cells bearing mutations in genes encoding TFIIH subunits, we observed that (1) the recruitment of the TFIIH complex was altered at the activated RARB2 promoter, (2) TFIIH participated in the recruitment of nucleotide excision repair (NER) factors during transcription in a manner different from that observed during NER, and (3) the different TFIIH variants disturbed transcription by having distinct consequences on post-translational modifications of histones, DNA-break induction, DNA demethylation, and gene-loop formation. The transition from heterochromatin to euchromatin was disrupted depending on the variant, illustrating the fact that TFIIH, by contributing to NER factor recruitment, orchestrates chromatin remodeling. The subtle transcriptional differences found between various TFIIH variants thus participate in the phenotypic variability observed among XP, XP/CS, and TTD individuals. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

77 FR 29633 - Alta Wind VII, LLC, Alta Wind IX, LLC, Alta Wind X, LLC, Alta Wind XI, LLC, Alta Wind XII, LLC...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-18

... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. EL12-68-000] Alta Wind VII, LLC, Alta Wind IX, LLC, Alta Wind X, LLC, Alta Wind XI, LLC, Alta Wind XII, LLC, Alta Wind XIII, LLC, Alta Wind XIV, LLC, Alta Wind XV, LLC, Alta Windpower Development, LLC, TGP Development Company, LLC...

Macroeconomic Consequences of Job Discrimination Against Women in Russia

DTIC Science & Technology

2010-06-01

operator engaged in hot dismantling of resistance furnaces in the production of abrasives 208. Caster of abrasive materials 209. Furnace bottom...are the first fired .125 Additionally, despite more education than their male counterparts, women stay unemployed twice as long as men.126 As a result...and networks (Nos. 204-205) XII. Production of abrasives (Nos. 206-210) XIII. Electrical-engineering production (Nos. 211-219) XIV. Radio

Analysis of Contract Source Selection Strategy

DTIC Science & Technology

2015-07-07

Logistic xiii ACKNOWLEDGMENTS We would like to express our gratitude to our advisors, Professor Rene Rendon and Professor Karen Landale, for their...like to dedicate this project to my beautiful and supportive wife, Amy, and my daughters, Peyton and Claire. Thank you for all your unconditional love...accomplish this milestone due to his unconditional love. I would like to thank my mom, Saraswati, and my dad, Khilendra, for their support and patience

Beyond Consolidation: U.S. Government International Broadcasting in the Post-Cold War Era

DTIC Science & Technology

1994-01-01

vii A c kowledgments .......................................... xiii 1. INTRODUCTION ....................................... 1 2. THE UNITED STATES IN...military power more generally, is diminishing in relevance, paced by the decline of U.S. economic preponderance. The difference between military and...economic power on the one hand, and political and moral authority on the other hand, has been made starkly clear at the beginning of the post-Cold War era

Rapid Training of Information Extraction with Local and Global Data Views

DTIC Science & Technology

2012-05-01

56 xiii 4.1 An example of words and their bit string representations. Bold ones are transliterated Arabic words...Natural Language Processing ( NLP ) community faces new tasks and new domains all the time. Without enough labeled data of a new task or a new domain to...conduct supervised learning, semi-supervised learning is particularly attractive to NLP researchers since it only requires a handful of labeled examples

An Approach to the Management of Hazardous Materials.

DTIC Science & Technology

1981-09-01

fluoride carbon disulfide carbon monoxide hydrochloric acid hydrogen sulfide acetone cyanohydrin phosgene Group XII: Poison B - Meeflaneous a) Gases...b) Liquids sulfur dioxide bromine chlorine boron trifluoride Group XIII: Poison C Group XIV: Poison D Liquid Gas tetraethyl lead fluorine Group XV...with a high and o Nitric acid leaks from a 5,000 gallon storage tank In a growing concentration of electronics industries. Over plant one night and

Application of the Sterility Principle for Tsetse Fly Eradication or Control. Revision.

DTIC Science & Technology

1981-08-14

1977. Digestive processes of haematophagous insects . XIII. Evidence for the digestive function of midgut proleinases in Glossina morsitans morsitans...FOOD AND AGRICULTURAL DEVELOPMENT ABSTRACT This review deals with the conditions required for the successful application of the sterile insect ...numbers of sterile male insects are released into a wild population so that they can disperse and compete with the fertile males of the natural population

Integrated Fusion, Performance Prediction, and Sensor Management for Automatic Target Exploitation

DTIC Science & Technology

2007-05-30

with large region of attraction about the true minimum. The physical optics models provide features for high confidence identification of stationary...the detection test are used to estimate 3D object scattering; multiple images can be noncoherently combined to reconstruct a more complete object...Proc. SPIE Algorithms for Synthetic Aper- ture Radar Imagery XIII, The International Society for Optical Engineering, April 2006. [40] K. Varshney, M. C

Modern Advances to the Modular Fly-Away Kit (MFLAK) to Support Maritime Interdiction Operations

DTIC Science & Technology

2007-09-01

combined Indonesia- Malaysia -Singapore-Thailand-U.S. R&D effort to investigate commercial-off-the- shelf (COTS) Command and Control, Communications...Operations and Applied Science & Technology Studies (COASTS). COASTS is a combined Indonesia- Malaysia -Singapore-Thailand-U.S. R&D effort to investigate...Message Authentication Code MALSINDO Malaysia , Indonesia and Singapore xiii Mbps Megabits per Second MCP Mission Capability Package MCSC Marine

Getting ready to translate: cytoplasmic maturation of eukaryotic ribosomes.

PubMed

Panse, Vikram Govind

2011-01-01

The ribosome is the 'universal ribozyme' that is responsible for the final step of decoding genetic information into proteins. While the function of the ribosome is being elucidated at the atomic level, in comparison, little is known regarding its assembly in vivo and intracellular transport. In contrast to prokaryotic ribosomes, the construction of eukaryotic ribosomes, which begins in the nucleolus, requires >200 evolutionary conserved non-ribosomal trans-acting factors, which transiently associate with pre-ribosomal subunits at distinct assembly stages and perform specific maturation steps. Notably, pre-ribosomal subunits are transported to the cytoplasm in a functionally inactive state where they undergo maturation prior to entering translation. In this review, I will summarize our current knowledge of the eukaryotic ribosome assembly pathway with emphasis on cytoplasmic maturation events that render pre-ribosomal subunits translation competent.

Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast

PubMed Central

Konikkat, Salini; Woolford, John L.

2017-01-01

Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ~76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae. We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly. PMID:28062837

Localization of a gene for a glutamate binding subunit of a NMDA receptor (GRINA) to 8q24

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewis, T.B.; DuPont, B.R.; Leach, R.

1996-02-15

This article reports on the localization of a gene for a glutamate binding subunit of an N-methyl-D-aspartate (NMDA) receptor, called GRINA, to human chromosome 8q24 using fluorescence in situ hybridization and radiation hybridization mapping. This gene mapped outside the critical region for benign familial neonatal convulsions (BFNC), a rare form of epilepsy; however, GRINA could be the causative genetic factor inducing idiopathic generalized epilepsy. Further studies need to be conducted. 15 refs., 2 figs.

Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes.

PubMed

Biedka, Stephanie; Micic, Jelena; Wilson, Daniel; Brown, Hailey; Diorio-Toth, Luke; Woolford, John L

2018-04-24

Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C 2 site of 27SB pre-rRNA. C 2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C 2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C 2 cleavage and interpreted these results using cryo-electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C 2 cleavage. Interestingly, when C 2 cleavage is directly blocked by depleting or inactivating the C 2 endonuclease, assembly progresses through all other subsequent steps. © 2018 Biedka et al.

Central nervous system bleeding in pediatric patients with factor XIII deficiency: a study on 23 new cases.

PubMed

Naderi, Majid; Alizadeh, Shaban; Kazemi, Ahmad; Tabibian, Shadi; Zaker, Farhad; Bamedi, Taregh; Kashani Khatib, Zahra; Dorgalaleh, Akbar

2015-03-01

Factor XIII (FXIII) deficiency is an extremely rare bleeding disorder, which has the highest incidence in Sistan and Baluchistan Province in Iran, compared to its overall incidence around the world. This disorder has different clinical manifestations ranging from mild bleeding tendency to lethal bleeding episodes including central nervous system (CNS) hemorrhage. The aim of this study was to evaluate the demographic data, pattern of CNS bleeding, and the role of plasminogen activator inhibitor-1 (PAI-1) (PAI-1) 4G/5G and thrombin activatable fibrinolysis inhibitor (TAFI) Thr325Ile polymorphisms in intracranial and extracranial hemorrhages in 23 new cases of FXIII-deficient subjects. This case-control study was conducted on 23 FXIII-deficient patients with CNS bleeding episodes and 23 patients as the control group with FXIII deficiency but without any history of CNS bleeding. Initially, to confirm the molecular defect, both groups were evaluated for the most frequently reported mutation of FXIII (Trp187Arg mutation) in a previous study in Sistan and Baluchistan Province. Then, demographic data, clinical manifestations, and pattern of CNS bleeding were determined. Eventually, the patients were assessed for PAI-14G/5G and TAFI Thr325Ile polymorphisms. The results of this study revealed that all the subjects (including the case and control groups) were homozygous for Trp187Arg mutation. Nineteen patients (82.6%) had intracranial hemorrhage (ICH) and four patients (17.4%) had extracranial hemorrhage (ECH). Intraparenchymal hemorrhage was the most common form of ICH (89.5%), and epidural hemorrhage was observed in two patients (10.5%). Anatomic regions in patients with intraparenchymal hemorrhage were temporal in six (35.3%), occipital in four (23.5%), diffused intraparenchymal in four (23.5%), temporal-occipital in two (11.8%), and subdural with temporal in one (5.9%) patient. We found that in the case group, 14 patients (60.8%) were homozygous for TAFI Thr325Ile polymorphism and 8 cases (34.7%) were heterozygous. In the control group, 4 (17.4%) and 13 (56.5%) patients were homozygous and heterozygous, respectively (P < 0.001 vs. P < 0.01).We also found that an equal number of patients (two individuals) in the case and control groups (8.7% in each group) were heterozygous for PAI-14G/5G polymorphism. It seems that PAI-14G/5G polymorphism does not have any effect on occurrence of ICH and ECH in patients with FXIII deficiency, while TAFI Thr325Ile is a strong genetic risk factor (odds ratio:14.9, 95% confidence interval: 7.4-31.1).

Structural and biochemical analyses reveal insights into covalent flavinylation of the Escherichia coli Complex II homolog quinol:fumarate reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Starbird, C. A.; Maklashina, Elena; Sharma, Pankaj

The Escherichia coli Complex II homolog quinol:fumarate reductase (QFR, FrdABCD) catalyzes the interconversion of fumarate and succinate at a covalently attached FAD within the FrdA subunit. The SdhE assembly factor enhances covalent flavinylation of Complex II homologs, but the mechanisms underlying the covalent attachment of FAD remain to be fully elucidated. Here, we explored the mechanisms of covalent flavinylation of the E. coli QFR FrdA subunit. Using a ΔsdhE E. coli strain, we show that the requirement for the assembly factor depends on the cellular redox environment. We next identified residues important for the covalent attachment and selected the FrdAE245more » residue, which contributes to proton shuttling during fumarate reduction, for detailed biophysical and structural characterization. We found that QFR complexes containing FrdAE245Q have a structure similar to that of the WT flavoprotein, but lack detectable substrate binding and turnover. In the context of the isolated FrdA subunit, the anticipated assembly intermediate during covalent flavinylation, FrdAE245 variants had stability similar to that of WT FrdA, contained noncovalent FAD, and displayed a reduced capacity to interact with SdhE. However, small-angle X-ray scattering (SAXS) analysis of WT FrdA cross-linked to SdhE suggested that the FrdAE245 residue is unlikely to contribute directly to the FrdA-SdhE protein-protein interface. We also found that no auxiliary factor is absolutely required for flavinylation, indicating that the covalent flavinylation is autocatalytic. We propose that multiple factors, including the SdhE assembly factor and bound dicarboxylates, stimulate covalent flavinylation by preorganizing the active site to stabilize the quinone-methide intermediate.« less

Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate-ubiquinone oxidoreductase.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Amino, H; Tomitsuka, E; Kita, K

1999-08-04

We have mapped large (cybL) and small (cybS) subunits of cytochrome b in the succinate-ubiquinone oxidoreductase (complex II) of human mitochondria to chromosome 1q21 and 11q23, respectively (H. Hirawake et al., Cytogenet. Cell Genet. 79 (1997) 132-138). In the present study, the human SDHD gene encoding cybS was cloned and characterized. The gene comprises four exons and three introns extending over 19 kb. Sequence analysis of the 5' promoter region showed several motifs for the binding of transcription factors including nuclear respiratory factors NRF-1 and NRF-2 at positions -137 and -104, respectively. In addition to this gene, six pseudogenes of cybS were isolated and mapped on the chromosome.

MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana

PubMed Central

Stiller, Jiri; Davoine, Celine; Björklund, Stefan; Manners, John M.; Kazan, Kemal; Schenk, Peer M.

2017-01-01

The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display down-regulation of genes associated with jasmonate signaling and biosynthesis while up-regulation of salicylic acid associated pathogenesis related genes and reactive oxygen producing and scavenging genes. We propose that MED18 and MED20 form a sub-domain within Mediator that controls the balance of salicylic acid and jasmonate associated defense pathways. PMID:28441405

Atomic resolution model of the antibody Fc interaction with the complement C1q component.

PubMed

Schneider, Sebastian; Zacharias, Martin

2012-05-01

The globular C1q heterotrimer is a subunit of the C1 complement factor. Binding of the C1q subunit to the constant (Fc) part of antibody molecules is a first step and key event of complement activation. Although three-dimensional structures of C1q and antibody Fc subunits have been determined experimentally no atomic resolution structure of the C1q-Fc complex is known so far. Based on systematic protein-protein docking searches and Molecular Dynamics simulations a structural model of the C1q-IgG1-Fc-binding geometry has been obtained. The structural model is compatible with available experimental data on the interaction between the two partner proteins. It predicts a binding geometry that involves mainly the B-subunit of the C1q-trimer and both subunits of the IgG1-Fc-dimer with small conformational adjustments with respect to the unbound partners to achieve high surface complementarity. In addition to several charge-charge and polar contacts in the rim region of the interface it also involves nonpolar contacts between the two proteins and is compatible with the carbohydrate moiety of the Fc subunit. The model for the complex structure provides a working model for rationalizing available biochemical data on this important interaction and can form the basis for the design of Fc variants with a greater capacity to activate the complement system for example on binding to cancer cells or other target structures. Copyright © 2012 Elsevier Ltd. All rights reserved.

In silico predictions of LH2 ring sizes from the crystal structure of a single subunit using molecular dynamics simulations.

PubMed

Janosi, Lorant; Keer, Harindar; Cogdell, Richard J; Ritz, Thorsten; Kosztin, Ioan

2011-07-01

Most of the currently known light-harvesting complexes 2 (LH2) rings are formed by 8 or 9 subunits. As of now, questions like "what factors govern the LH2 ring size?" and "are there other ring sizes possible?" remain largely unanswered. Here, we investigate by means of molecular dynamics (MD) simulations and stochastic modeling the possibility of predicting the size of an LH2 ring from the sole knowledge of the high resolution crystal structure of a single subunit. Starting with single subunits of two LH2 rings with known size, that is, an 8-ring from Rs. moliscianum (MOLI) and a 9-ring from Rps. acidophila (ACI), and one with unknown size (referred to as X), we build atomic models of subunit dimers corresponding to assumed 8-, 9-, and 10-ring geometries. After inserting each of the dimers into a lipid-water environment, we determine the preferred angle between the corresponding subunits by three methods: (1) energy minimization, (2) free MD simulations, and (3) potential of mean force calculations. We find that the results from all three methods are consistent with each other, and when taken together, it allows one to predict with reasonable level of confidence the sizes of the corresponding ring structures. One finds that X and ACI very likely form a 9-ring, while MOLI is more likely to form an 8-ring than a 9-ring. Finally, we discuss both the merits and limitations of all three prediction methods. Copyright © 2011 Wiley-Liss, Inc.

The Na, K-ATPase β-Subunit Isoforms Expression in Glioblastoma Multiforme: Moonlighting Roles

PubMed Central

Rotoli, Deborah; Cejas, Mariana-Mayela; Maeso, María-del-Carmen; Pérez-Rodríguez, Natalia-Dolores; Morales, Manuel; Ávila, Julio

2017-01-01

Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Recent studies point out that gliomas exploit ion channels and transporters, including Na, K-ATPase, to sustain their singular growth and invasion as they invade the brain parenchyma. Moreover, the different isoforms of the β-subunit of Na, K-ATPase have been implicated in regulating cellular dynamics, particularly during cancer progression. The aim of this study was to determine the Na, K-ATPase β subunit isoform subcellular expression patterns in all cell types responsible for microenvironment heterogeneity of GBM using immunohistochemical analysis. All three isoforms, β1, β2/AMOG (Adhesion Molecule On Glia) and β3, were found to be expressed in GBM samples. Generally, β1 isoform was not expressed by astrocytes, in both primary and secondary GBM, although other cell types (endothelial cells, pericytes, telocytes, macrophages) did express this isoform. β2/AMOG and β3 positive expression was observed in the cytoplasm, membrane and nuclear envelope of astrocytes and GFAP (Glial Fibrillary Acidic Protein) negative cells. Interestingly, differences in isoforms expression have been observed between primary and secondary GBM: in secondary GBM, β2 isoform expression in astrocytes was lower than that observed in primary GBM, while the expression of the β3 subunit was more intense. These changes in β subunit isoforms expression in GBM could be related to a different ionic handling, to a different relationship between astrocyte and neuron (β2/AMOG) and to changes in the moonlighting roles of Na, K-ATPase β subunits as adaptor proteins and transcription factors. PMID:29117147

DEFECTIVE TRAFFICKING OF CONE PHOTORECEPTOR CNG CHANNELS INDUCES THE UNFOLDED PROTEIN RESPONSE AND ER STRESS-ASSOCIATED CELL DEATH

PubMed Central

Duricka, Deborah L.; Brown, R. Lane; Varnum, Michael D.

2011-01-01

SYNOPSIS Mutations that perturb the function of photoreceptor cyclic nucleotide-gated (CNG) channels are associated with several human retinal disorders, but the molecular and cellular mechanisms leading to photoreceptor dysfunction and degeneration remain unclear. Many loss-of-function mutations result in intracellular accumulation of CNG channel subunits. Accumulation of proteins in the endoplasmic reticulum (ER) is known to cause ER stress and trigger the unfolded protein response (UPR), an evolutionarily conserved cellular program that results in either adaptation via increased protein processing capacity or apoptotic cell death. We hypothesize that defective trafficking of cone photoreceptor CNG channels can induce UPR-mediated cell death. To test this idea, CNGA3 subunits bearing the R563H and Q655X mutations were expressed in photoreceptor-derived 661W cells with CNGB3 subunits. Compared to wild type, R563H and Q655X subunits displayed altered degradation rates and/or were retained in the ER. ER retention was associated with increased expression of UPR-related markers of ER stress and with decreased cell viability. Chemical and pharmacological chaperones (TUDCA, 4PBA, and the cGMP analog CPT-cGMP) differentially reduced degradation and/or promoted plasma-membrane localization of defective subunits. Improved subunit maturation was concordant with reduced expression of ER stress markers and improved viability of cells expressing localization-defective channels. These results indicate that ER stress can arise from expression of localization defective CNG channels, and may represent a contributing factor for photoreceptor degeneration. PMID:21992067

Na, K-ATPase subunits as markers for epithelial-mesenchymal transition in cancer and fibrosis

PubMed Central

Rajasekaran, Sigrid A.; Huynh, Thu P.; Wolle, Daniel G.; Espineda, Cromwell E.; Inge, Landon J.; Skay, Anna; Lassman, Charles; Nicholas, Susanne B.; Harper, Jeffrey F.; Reeves, Anna E.; Ahmed, Mansoor M.; Leatherman, James M; Mullin, James M.; Rajasekaran, Ayyappan K.

2010-01-01

Epithelial-to-mesenchymal transition (EMT) is an important developmental process, participates in tissue repair and occurs during pathological processes of tumor invasiveness, metastasis and tissue fibrosis. The molecular mechanisms leading to EMT are poorly understood. While it is well documented that transforming growth factor (TGF)-β plays a central role in the induction of EMT, the targets of TGF-β signaling are poorly defined. We have shown earlier that Na,K-ATPase β1-subunit levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients’ tumor samples. In this study, we provide evidence that Na,K-ATPase is a new target of TGF-β1-mediated EMT in renal epithelial cells, a model system used in studies of both cancer progression and fibrosis. We show that following treatment with TGF-β1 the surface expression of the β1-subunit of Na,K-ATPase is reduced, prior to well-characterized EMT markers and is associated with the acquisition of a mesenchymal phenotype. RNAi mediated knockdown confirmed the specific involvement of the Na,K-ATPase β1-subunit in the loss of the epithelial phenotype and exogenous over-expression of the Na,K-ATPase β1-subunit attenuated TGF-β1-mediated EMT. We further show that both Na,K-ATPase α- and β-subunit levels are highly reduced in renal fibrotic tissues. These findings for the first time reveal that Na,K-ATPase is a target of TGF-β1-mediated EMT and is associated with the progression of EMT in both cancer and fibrosis. PMID:20501797

Regulatory Subunit B′γ of Protein Phosphatase 2A Prevents Unnecessary Defense Reactions under Low Light in Arabidopsis1[W][OA

PubMed Central

Trotta, Andrea; Wrzaczek, Michael; Scharte, Judith; Tikkanen, Mikko; Konert, Grzegorz; Rahikainen, Moona; Holmström, Maija; Hiltunen, Hanna-Maija; Rips, Stephan; Sipari, Nina; Mulo, Paula; Weis, Engelbert; von Schaewen, Antje; Aro, Eva-Mari; Kangasjärvi, Saijaliisa

2011-01-01

Light is an important environmental factor that modulates acclimation strategies and defense responses in plants. We explored the functional role of the regulatory subunit B′γ (B′γ) of protein phosphatase 2A (PP2A) in light-dependent stress responses of Arabidopsis (Arabidopsis thaliana). The predominant form of PP2A consists of catalytic subunit C, scaffold subunit A, and highly variable regulatory subunit B, which determines the substrate specificity of PP2A holoenzymes. Mutant leaves of knockdown pp2a-b′γ plants show disintegration of chloroplasts and premature yellowing conditionally under moderate light intensity. The cell-death phenotype is accompanied by the accumulation of hydrogen peroxide through a pathway that requires CONSTITUTIVE EXPRESSION OF PR GENES5 (CPR5). Moreover, the pp2a-b′γ cpr5 double mutant additionally displays growth suppression and malformed trichomes. Similar to cpr5, the pp2a-b′γ mutant shows constitutive activation of both salicylic acid- and jasmonic acid-dependent defense pathways. In contrast to cpr5, however, pp2a-b′γ leaves do not contain increased levels of salicylic acid or jasmonic acid. Rather, the constitutive defense response associates with hypomethylation of DNA and increased levels of methionine-salvage pathway components in pp2a-b′γ leaves. We suggest that the specific B′γ subunit of PP2A is functionally connected to CPR5 and operates in the basal repression of defense responses under low irradiance. PMID:21571669

Structure–function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring

PubMed Central

Schwer, Beate; Kruchten, Joshua; Shuman, Stewart

2016-01-01

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure–function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein–RNA and protein–protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not. PMID:27417296

Defective trafficking of cone photoreceptor CNG channels induces the unfolded protein response and ER-stress-associated cell death.

PubMed

Duricka, Deborah L; Brown, R Lane; Varnum, Michael D

2012-01-15

Mutations that perturb the function of photoreceptor CNG (cyclic nucleotide-gated) channels are associated with several human retinal disorders, but the molecular and cellular mechanisms leading to photoreceptor dysfunction and degeneration remain unclear. Many loss-of-function mutations result in intracellular accumulation of CNG channel subunits. Accumulation of proteins in the ER (endoplasmic reticulum) is known to cause ER stress and trigger the UPR (unfolded protein response), an evolutionarily conserved cellular programme that results in either adaptation via increased protein processing capacity or apoptotic cell death. We hypothesize that defective trafficking of cone photoreceptor CNG channels can induce UPR-mediated cell death. To test this idea, CNGA3 subunits bearing the R563H and Q655X mutations were expressed in photoreceptor-derived 661W cells with CNGB3 subunits. Compared with wild-type, R563H and Q655X subunits displayed altered degradation rates and/or were retained in the ER. ER retention was associated with increased expression of UPR-related markers of ER stress and with decreased cell viability. Chemical and pharmacological chaperones {TUDCA (tauroursodeoxycholate sodium salt), 4-PBA (sodium 4-phenylbutyrate) and the cGMP analogue CPT-cGMP [8-(4-chlorophenylthio)-cGMP]} differentially reduced degradation and/or promoted plasma-membrane localization of defective subunits. Improved subunit maturation was concordant with reduced expression of ER-stress markers and improved viability of cells expressing localization-defective channels. These results indicate that ER stress can arise from expression of localization-defective CNG channels, and may represent a contributing factor for photoreceptor degeneration.

Sequence and functional characterization of hypoxia-inducible factors, HIF1α, HIF2αa, and HIF3α, from the estuarine fish, Fundulus heteroclitus

PubMed Central

Townley, Ian K.; Karchner, Sibel I.; Skripnikova, Elena; Wiese, Thomas E.; Hahn, Mark E.

2017-01-01

The hypoxia-inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus is found in habitats prone to hypoxia. It responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, HIF2α, and HIF3α independently formed complexes with the β-subunit, aryl hydrocarbon receptor nuclear translocator, to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α—HIF2αb—a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations. PMID:28039194

Sequence and functional characterization of hypoxia-inducible factors, HIF1α, HIF2αa, and HIF3α, from the estuarine fish, Fundulus heteroclitus.

PubMed

Townley, Ian K; Karchner, Sibel I; Skripnikova, Elena; Wiese, Thomas E; Hahn, Mark E; Rees, Bernard B

2017-03-01

The hypoxia-inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus is found in habitats prone to hypoxia. It responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, HIF2α, and HIF3α independently formed complexes with the β-subunit, aryl hydrocarbon receptor nuclear translocator, to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α-HIF2αb-a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations. Copyright © 2017 the American Physiological Society.